NAT10-mediated β-hydroxybutyrylation Affects DNA Replication

NAT10-mediated β-hydroxybutyrylation Affects DNA Replication | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article NAT10-mediated β-hydroxybutyrylation Affects DNA Replication Wenhui Zhao, Siyi Jiang, Yaqi Sui, Rui Zhou, KUN LIU, Zhisong Fu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7110451/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Accurate DNA replication is essential for genome integrity, with dysregulated replication dynamics, replication stress and genomic instability-hallmarks of cancer and aging. Here, we observe NAT10 is a β-hydroxybutyryl-transerase and NAT10-mediated β-hydroxybutyrylation (Kbhb) of histones that appears to affect replication fork progression without significantly impacting origin firing, potentially to reduce replication stress and to help maintain genomic stability. DNA fiber analyses show β-hydroxybutyrate (BHB) treatment enhances replication efficiency while maintaining fork symmetry, effects abolished by NAT10 depletion or inhibition. BrdU/EdU labeling, and EdU-FACS analyses reveal that NAT10-mediated Kbhb accelerates replication fork velocity and shortens S-phase duration. LC-MS/MS profiling shows no significant changes in origin firing following BHB treatment. Assessment of replication stress markers, including γH2AX foci, non-denaturing BrdU incorporation, RPA2 foci, S317-CHK1 phosphorylation, and levels of γH2AX and RPA2 on chromatin, suggests that NAT10-mediated Kbhb reduces replication stress. Evaluation of genomic instability, measured by micronuclei formation, sister chromatid bridges, and chromatid breaks/gaps during mitosis, indicates that NAT10-mediated Kbhb also reduces genomic instability. Mechanistically, NAT10-mediated Kbhb modulates chromatin association, thereby modulating chromatin accessibility to establish a replication-permissive environment. This epigenetic remodeling serves to moderate replication stress markers and genomic instability. Conserved effects in transformed and primary cell models position NAT10 as a metabolic-epigenetic nexus linking nutrient signaling to replication fidelity. Our findings suggest targeting Kbhb signaling as a potential therapeutic strategy against replication stress-associated pathologies. Biological sciences/Biochemistry/Proteins/DNA-binding proteins Biological sciences/Molecular biology/Chromatin/Histone post-translational modifications β-hydroxybutyrylation NAT10 DNA replication replication stress genomic instability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Histone posttranslational modifications (PTM) are critical regulators of DNA replication in eukaryotic cells, influencing chromatin structure and replication timing 1 – 3 . Among these, histone acetylation and methylation are particularly well-studied, with high levels of acetylation accelerating replication in distinct genomic regions 1 – 5 . Open chromatin marks, such as H3K4me1/2/3, H3K9ac, H3K18ac and H3K27ac are consistently enriched in early-replicating regions, highlighting their pivotal role in replication process 1 , 6 – 8 . Lysine β-hydroxybutyrylation (Kbhb), a recently identified histone PTM, may play a role in DNA replication. Kbhb is derived from β-hydroxybutyrate (BHB), a key component of ketone bodies, which is converted into β-hydroxybutyryl-CoA to provide β-hydroxybutyryl group to conjugate to lysine. Kbhb is a conserved PTM observed across species from yeast to human 9 . The identified histone Kbhb sites in mice and human include H3K4, H3K9, H3K18 and H3K27 lysine residues, whose acetylation marks open chromatin structure and early replication genomic regions 9 , 10 . H3K9bhb is associated with upregulation of genes involved in starvation-responsive pathways 9 , 11 . H3K56bhb colocalized at the super-enhancer regions with coactivators such as BRD4 12 . These clues imply that histone Kbhb may affect DNA replication. Until now, there is lacking experimental evidence linking histone Kbhb to DNA replication. N-acetyltransferase 10 (NAT10) is a candidate regulator of Kbhb and DNA replication. NAT10, as a histone acetyltransferases (HATs), catalyzes RNA acetylation (N4-acetylation of Cytidine of RNA, ac4C) and lysine acetylation (acetylation of histones, UBF1, p53, etc.) 13 – 18 . NAT10 has 2-hydroxyisobutyrylation (Khib), a modification with a chemical structure closely resembling to Kbhb 19 . Other HATs, such as CBP/p300, exhibit lysine β-hydroxybutyryl-transferase activity 20 – 22 . Those clues raise the possibility that NAT10 also catalyze Kbhb besides Kac and ac4C. Furthermore, NAT10 is enriched in mitotic chromosome scaffold fraction and play critical roles in genome integrity, bipolar assembly, and chromatin segregation during mitosis 14 , 23 – 26 . NAT10 depletion or inhibition activity promote DNA replication 19 , 20 , 27 – 29 . These clues suggest NAT10 might regulate DNA replication. However, direct evidence linking NAT10 to Kbhb modification and its functional implication in DNA replication has remained elusive. Here, we demonstrate that NAT10 as a β-hydroxybutyryl-transferase catalyzes Kbhb at multiple lysine residues of histones, thereby facilitating DNA replication, lower replication stress and better maintenance of genomic stability. Mechanistically, we show that NAT10-mediated Kbhb enhances replication dynamics by accelerating replication fork speed and shortening the S phase duration. NAT10-mediated Kbhb results in lower replication stress, as evidenced by significant reductions in single-stranded DNA (ssDNA) accumulation, γH2AX foci formation, RPA2 foci localization, and phospho-S317 CHK1 levels. NAT10-mediated Kbhb possibly leads to preserved genomic instability, as indicted by lower frequency of micronuclei, chromosome bridge and DNA breakage occurrence. Notably, we find that NAT10 depletion or NAT10-mediated Kbhb of itself dynamically reduces chromatin-bound NAT10 and chromatin accessibility, highlighting its regulatory role in chromatin structure. Our findings provide direct evidence linking NAT10 to histone Kbhb and uncover its critical role in promoting DNA replication, reducing replication stress, and preserving genomic stability. These insights establish a previously unrecognized regulatory pathway connecting metabolic signaling, histone modification, and DNA replication. Results BHB treatment accelerates DNA replication To evaluate the impacts of sodium β-hydroxybutyrate (BHB) on cellular processes, we first assessed its impact on U2OS cell viability. CCK8 assays revealed a dose-dependent enhancement in cell viability ( Fig. 1 A ) , suggesting that BHB positively influences cell growth. Subsequent flow cytometric analysis of synchronized cells released from G1/S boundary arrest demonstrated accelerated S-phase progression under BHB treatment (Fig. 1 B). To explore the effects of BHB on DNA replication, we conducted DNA fiber analysis to evaluate replication fork progression. Single-molecule DNA fiber assays revealed a significant increase in replication fork velocity with preserved fork symmetry in BHB-treated cell (Fig. 1 C- 1 E). Dual-pulse BrdU/EdU labeling corroborated these findings, showing redistribution of S-phase populations toward early-middle and middle-late replication stages (Fig. 1 F- 1 G). Temporal EdU FACS quantification analysis revealed that 8-hour BHB treatment transiently elevated both the proportion of S-phase cell and EdU incorporation intensity. These effects progressively diminished at 16–24 hours but persisted when cells were released into fresh media post-treatment (Fig. 1 H- 1 M, Supplementary Fig. 1A-1B ). These findings indicate that BHB as a temporal accelerator of S-phase progression, operating within a defined, time-restricted window of EdU labeling. To determine whether BHB impacts replication origin firing—a critical regulatory step in DNA replication dynamics 30 – 34 , we analyzed chromatin-bound replication initiation complexes by intensity-based absolute quantification (iBAQ) mass spectrometry. Quantitative proteomics revealed no significant changes in replication initiation factor abundance following BHB treatment (Supplementary Fig. 1C-1E) , demonstrating that BHB accelerates DNA replication without significantly impacting origin firing. BHB treatment reduces replication stress Accelerated DNA replication fork progression is known to induces replication stress and genomic instability 30 . Therefore, we further evaluated whether the BHB-induced replication acceleration results in replication stress. Initial evaluation of γH2AX foci formation in S-phase cells demonstrated a significant decrease following BHB treatment (Fig. 2 A- 2 B). Similarly, non-denaturing BrdU incorporation assays showed a reduction in ssDNA-positive S-phase populations (Fig. 2 C- 2 D). Concordant reductions in γH2AX and RPA2 signal intensities were observed by immunostaining analysis (Fig. 2 E- 2 F, Supplementary Fig. 2 ). Western blot analysis of S317-CHK1 phosphorylation, γH2AX, and RPA2 levels confirmed these observations, showing marked reductions following BHB treatment (Fig. 2 G- 2 H). Collectively, these data demonstrate that BHB enhances DNA replication efficiency ( Fig. 1 ) while simultaneously alleviating replication stress (Fig. 2 A- 2 H). BHB treatment reduces genomic instability Given that rapid replication fork progression can induce genomic instability 30 , manifesting as micronuclei formation, sister-chromatid bridges, and chromatid breaks or gaps during mitosis 37 – 39 . We assessed whether BHB-induced acceleration exacerbates these defects. We performed immunostaining analysis to measure the frequency of the DAPI-stained micronuclei and anaphase-bridge in BHB-treated U2OS cells. BHB treatment modestly reduced the incidence of micronuclei and anaphase-bridges (Fig. 2 I-K). Furthermore, under genomic instability induced by low-dose (0.2 µM) aphidicolin (APH) 38 , 40 , 41 , BHB-treated cells exhibited fewer chromatid breaks and gaps compared to controls (Fig. 2 L). These findings demonstrate that BHB promotes DNA replication by accelerating replication fork progression ( Fig. 1 ) while concurrently suppressing both replication stress (Fig. 2 A- 2 H) and genomic instability (Fig. 2 I- 2 L), thereby preserving genomic integrity despite accelerated replication dynamics. NAT10 is a β-hydroxybutyryl-transferase β-Hydroxybutyrate (BHB), the main component of ketone body, predominantly synthesized during hepatic fatty acid oxidation, circulates systemically to serve as an energy substrate through conversion to acetyl-CoA and eventually to ATP. Beyond its metabolic role, BHB acts as a signaling metabolite via β-hydroxybutyryl-CoA-mediated β-hydroxybutyryl group to conjugate to lysine residue (Kbhb), a protein posttranslational modification 9 , 42 – 44 . Given that BHB treatment accelerates DNA replication and reduces replication stress and genomic instability, and that similar dosage of BHB induce widespread histone Kbhb in human cells 9 , we hypothesized that BHB affects replication processes through Kbhb modification. We performed LC-MS/MS analysis of protein extracts from BHB-treated U2OS cells, identified NAT10 as a Kbhb-modified protein at lysine 989 (K989), alongside known acetyltransferases CBP, p300, and HAT1 ( Fig. 3 A ) . Given that CBP/p300-mediated Kbhb of histones and p53 is established 20 – 22 , CBP/p300 are known to exhibit diverse of acyl-transferase activities 43 and NAT10 previously linked to lysine 2-hydroxyisobutyrylation (Khib) modification 19 , we predictes that NAT10 is a potential β-hydroxybutyryl-transferase. To validate this, we overexpressed ten Flag-tagged HATs in 293T cells under BHB treatment. The proteins were purified and enriched under very stringent condition by immunoprecipitation (IP) with M2 beads, and detected using a pan anti-β-hydroxybutyrylation antibody (BHB-K). The results confirmed that CBP, p300, HAT1, and NAT10 are Kbhb proteins (Fig. 3 B). We also purified and enriched the Kbhb proteins by IP with BHB-K antibody from BHB treated cells lysate, and detected endogenous NAT10 (Fig. 3 C). We next interrogated NAT10’s catalytic mechanism. Studies implicate G641 as essential for NAT10’s acetyltransferase activity, which is inhibited by remodelin 31 . To verify whether G641 is also critical for β-hydroxybutyryl-transferase activity of NAT10, whether K989 is the key Kbhb site and whether the Khib site K823 affects its Kbhb activity, we performed in vitro β-hydroxybutyrylation assays with the purified NAT10 (Fig. 3 D). The results demonstrated that wild-type NAT10 and NAT10/K823R mutants retained activity, whereas NAT10/K989R and NAT10/G641E mutants abolished Kbhb modification ( Fig. 3 D ) . Remodelin treatment similarly suppressed β-hydroxybutyryl-transferase activity ( Fig. 3 D ) . To corroborate these findings, we performed in vivo β-hydroxybutyrylation assays by expressing Flag-tagged NAT10 wild-type and mutants in U2OS cells. Cells were treated with BHB and remodelin, and proteins were purified and enriched under very stringent condition by IP with M2 beads, and detected using BHB-K (Fig. 3 E- 3 F). The in vivo results were consistent with the in vitro findings (Fig. 3 E- 3 F). Together, these results indicated that NAT10 is a β-hydroxybutyryl-transferase. NAT10 β-hydroxybutyrylates histones at multiple lysine sites NAT10, predominantly localized in the nucleolar 13 , 17 , 18 , was investigated for its potential role in histone β-hydroxybutyrylation (Kbhb). We depleted NAT10 in U2OS cells treated with BHB using a pool of siRNA oligonucleotides. Immunoblotting analysis of extracted histones using an BHB-K antibody revealed a significant reduction in total levels of Kbhb histones in NAT10-depleted cells compared to control cells (Fig. 4 A). To further verify the role of NAT10 in histone Kbhb, we conducted additional NAT10 knockdown experiments with distinct siRNA oligonucleotides in U2OS cells. Immunoblotting with site-specific BHB-K antibodies demonstrated reduction in Kbhb levels at multiple lysine sites, except H3K14bhb and H4K8bhb no changed, in NAT10-depleted cells (Fig. 4 B). We performed in vitro β-hydroxybutyrylation assay. The purified recombinant human NAT10 expressed in 293T cells was incubated with mononucleosome in the presence of BHB-CoA in an in vitro Kbhb assay. With wildtype NAT10, histone Kbhb was detected at multiple lysine residues, whereas addition of remodelin in the assay completely inhibited histone Kbhb. Furthermore, with NAT10/K989R mutant, the levels of Kbhb histone were reduced. Finally, with NAT10/G641E inactive mutant, Kbhb histones was completely abolished (Fig. 4 C). The in vitro assay confirmed the previous findings on NAT10 activity (Fig. 3 ). To validate the β-hydroxybutyryl-transferase activity of endogenous NAT10, we employed a CRISPR-Cas9 system to knock out NAT10 in HeLa cells transiently, as previously described 13 . Histones extracted from the pool of NAT10 knockout cells showed significantly lower Kbhb levels at multiple lysine residues, except H3K14bhb and H4K8bhb no changed, compared to histones from parental HeLa cells (Fig. 4 D). To further confirm this activity, rescue experiments were conducted by re-expressing wild-type NAT10, NAT10/G641E, or NAT10/K989R in NAT10 knockout cells. Only wild-type NAT10 restored histone Kbhb levels in the presence of BHB, while neither the G641E nor K989R mutants were able to recover histone Kbhb (Fig. 4 E). We analyzed the global patterns of acetylated and β-hydroxybutyrylated proteins in whole-cell lysates using pan-acetylation and pan-Kbhb antibodies, respectively. With increasing durations of BHB treatment, total Kbhb levels in whole-cell lysates were significantly elevated, while global protein acetylation levels remained largely unchanged (Fig. 4 F). NAT10 depletion selectively reduced histone Kbhb, whereas CBP/p300/HAT1 triple knockdown showed minimal effect. Neither perturbation influenced acetylation (Fig. 4 G). Quantitative histone mass spectrometry analysis further established NAT10’s essential role in regulating Kbhb. Notably, NAT10 does not modulate H3K24ac, H4K6ac, H4K13ac, or H4K17ac upon BHB treatment. Instead, CBP/p300/HAT1 are the key mediators of H4K6ac, H4K13ac, and H4K17ac in response to BHB (Fig. 4 H, Supplementary Fig. 3 ). NAT10-mediated β-hydroxybutyrylation dynamically reduces chromatin-bound NAT10 and alteres chromatin accessibility Given that NAT10 mediates histone Kbhb at multiple lysine sites, we investigated the mechanism deeply of this modification on NAT10 and chromatin interaction and chromatin accessibility. To determine whether Kbhb affects NAT10 binding to chromatin, we performed GST pull-down assays. The results demonstrated that NAT10 directly interacts with nucleosomes, and both Kbhb NAT10 and Kbhb histones exhibited significantly reduces interactions with nucleosomes (Fig. 5 A- 5 E). Subcellular fractionation showed BHB treatment selectively reduced chromatin-associated NAT10 levels while leaving whole-cell lysates and soluble nuclear pools unchanged (Fig. 5 F). Immunostaining of nuclei pre-extracted to eliminate RNA-bound and free NAT10 confirmed diminished chromatin occupancy of NAT10 post-BHB treatment, despite preserved nucleolar localization (Fig. 5 G– 5 H). ChIP-seq analysis demonstrated NAT10 enrichment in intergenic regions under basal conditions, with a two-thirds reduction in binding peaks following BHB treatment (Fig. 5 I– 5 M). These finding consistent with the reduction of chromatin-bound NAT10 observed in immunoblotting and immunostaining experiments (Fig. 5 F– 5 M). To explore whether the BHB-induced reduction of chromatin-bound NAT10 is caused by NAT10-mediated β-hydroxybutyrylation, we used remodelin to inhibit NAT10 activity. Subcellular fractionation and immunostaining of chromatin-bound NAT10 in nuclei pre-extracted with CSK buffer showed that BHB treatment and NAT10 inhibition individually caused a modest reduction in chromatin-bound NAT10. However, the reduction observed in NAT10-inhibited cells treated with BHB was not significantly greater than in control cells (Fig. 5 N– 5 P). Importantly, the BHB-induced reduction in chromatin-bound NAT10 was reversed by pre-treatment with remodelin (Fig. 5 N– 5 P). These results indicate that both NAT10 depletion and NAT10-mediated β-hydroxybutyrylation negatively regulate NAT10 binding to chromatin in vivo . To further explore the effects of BHB treatment on chromatin architecture, we conducted ATAC-seq to assess chromatin accessibility. Differential chromatin accessibility regions were identified by comparing the following groups: group A, cells treated with control siRNA; group B, cells treated with control siRNA and BHB; group C, cells treated with NAT10 siRNA; and group D, cells treated with NAT10 siRNA and BHB (p 2). Among these regions, 1043 were up-regulated in group B compared to group A, of which 398 (38.2%) exhibited a normalized average count of 0 in group B. Additionally, 1282 regions were down-regulated in group B compared to group A, with 445 (44.9%) showing a normalized average count of 0 in group A (p 2) (Fig. 5 Q- 5 T, Supplementary Fig. 4A-4H ). Importantly, the differential chromatin accessibility regions observed between groups B and A (1043 up-regulated and 1282 down-regulated) were not detected between groups D and C, indicating that the BHB-induced changes in chromatin accessibility are dependent on NAT10 (Fig. 5 W- 5 X, Supplementary Fig. 4A-4H ). To investigate the association between chromatin accessibility dynamics and protein-DNA interactions, we performed integrated analysis of ATAC-seq and ChIP-seq datasets. Technical divergence in sequencing read lengths between these methodologies (ATAC-seq vs ChIP-seq) necessitated length-normalized comparison, restricting direct analysis to regions with equivalent peak widths. Cross-platform evaluation identified 288 overlapping genomic loci from 2,325 chromatin accessibility changes and 5,466 protein binding alterations, representing a 12.4% spatial concordance (Fig. 5 S-T; Supplementary Fig. 4A-4H ). These findings suggest coordinated chromatin restructuring and replication complex recruitment during BHB exposure. Further analysis demonstrated that NAT10-mediated Kbhb modulates chromatin occupancy patterns, with reduced NAT10 binding correlating spatially with differential ATAC-seq accessibility regions. NAT10-mediated β-hydroxybutyryltion accelerates DNA replication Previous results demonstrated that BHB treatment accelerates DNA replication, induces NAT10-mediated Kbhb at multiple lysine sites on histones, concurrently reducing chromatin-bound NAT10 and altering chromatin accessibility. We further investigated whether the effects of BHB-induced β-hydroxybutyrylation on DNA replication require NAT10 activity. BHB increased cell viability in control U2OS cells, consistent with replication enhancement. However, NAT10 depletion or remodelin treatment abolished this effect (Fig. 6 A– 6 B). Immunostaining revealed that BHB-induced increases the proportion of EdU-positive cells, indicative of enhanced DNA synthesis. This increase was absent in NAT10-knockdown or remodelin-treated cells, further implicating NAT10 in mediating this response (Fig. 6 C– 6 D, Supplementary Fig. 5 ). Flow cytometry-based DNA content analysis showed BHB-treated controls completed replication earlier than untreated cells, whereas NAT10-depleted or remodelin-treated cells exhibited no such acceleration (Fig. 6 E– 6 F). Consistently, DNA fiber assays confirmed BHB increased replication fork speed and symmetrical fork progression in controls, effects attenuated by NAT10 inhibition (Fig. 6 G– 6 K). Dual-pulse BrdU/EdU labeling demonstrated BHB promoted early-to-late S-phase transition in controls, but NAT10 depletion or remodelin treatment failed to recapitulate this progression ( Fig. 6 L-M ) . Similarly, BHB enhanced both S-phase cell proportion and EdU incorporation intensity in controls, with no changes observed in NAT10-deficient cells ( Fig. 6 N-O, Supplementary Fig. 6) . These findings establish NAT10-mediated β-hydroxybutyrylation plays a pivotal role in BHB-driven replication acceleration. Increased fork velocity, S-phase progression, and replication timing advance are strictly dependent on NAT10 activity, mechanistically linking β-hydroxybutyrylation to replication dynamics. NAT10-mediated β-hydroxybutyrylation reduces replication stress and genomic instability To investigate whether NAT10 mediates BHB-induced suppression of replication stress and genomic instability, we analyzed these phenotypes in NAT10-depleted or remodelin-treated U2OS cells under BHB treatment. BHB treatment reduced the proportion of γH2AX foci-positive S-phase cells—a replication stress marker—in control cells, whereas NAT10-depleted cells showed no such reduction (Fig. 7 A). Similarly, BrdU-based quantification of ssDNA-positive S-phase cells revealed decreased replication stress in BHB-treated controls but not in NAT10-depleted cells (Fig. 7 B). Western blot analysis further revealed that replication stress markers, including phosphorylated S317-CHK1, γH2AX, and RPA2, were reduced in control cells following BHB treatment. These effects were abolished in NAT10-depleted cells regardless of BHB treatment (Fig. 7 C– 7 D). Notably, NAT10 depletion alone increased replication stress, as evidenced by unchanged or mildly elevated levels of these markers compared to controls (Fig. 7 C– 7 D). Consistent with these, immunostaining showed diminished RPA2 and γH2AX signals in BHB-treated control cells but not in NAT10-depleted cells (Fig. 7 E- 7 F, Supplementary Fig. 7 ). Strikingly, NAT10 depletion alone further increased RPA2 and γH2AX signals, while remodelin plus BHB treatment had no effect compared to controls, which reinforce its role in promoting replication stress (Fig. 7 E– 7 F). Together, these results demonstrate that NAT10-mediated β-hydroxybutyrylation reduces replication stress, but NAT10 depletion alone enhances it. To evaluate genomic stability, we examined chromosomal abnormalities. BHB treatment reduced the incidence of micronuclei and anaphase bridges in control cells, whereas NAT10 depletion or remodelin treatment abrogated these protective effects (Fig. 7 G– 7 H). BHB treatment also decreased chromosomal aberrations induced by low-dose aphidicolin (APH). In control cells, the frequency of chromatid breaks and gaps was significantly reduced following BHB treatment. This protection was lost in NAT10-depleted or remodelin-treatment-treated cell (Fig. 7 I). EdU incorporationon metaphase chromosome spreads, reduced by BHB in control cells, remained unchanged in NAT10-depleted or remodelin-treated cells (Fig. 7 J– 7 K). These findings demonstrate that NAT10-mediated β-hydroxybutyrylation is essential for BHB’s suppression of replication stress and genomic instability. Genetic or pharmacological inhibition of NAT10 abolishes these protective effects of BHB treatment, highlighting the essential function of NAT10 in mediating these responses. NAT10-mediated β-hydroxybutylation accelerates replication, reduces replication stress and genomic instability in WI-38 cells and in 2BS cells. We further explored the role of NAT10-mediated β-hydroxybutyrylation in the primary human cell lines WI-38 and 2BS, in addition to the previously studied U2OS cells, a human osteosarcoma cell line. In WI-38 cells, BHB treatment significantly increased replication fork speed and maintained replication fork symmetry, as demonstrated by DNA fiber assays. BrdU incorporation analysis under non-denaturing conditions revealed a reduction in ssDNA levels, indicating decreased replication stress. Similarly, NAT10 knockdown or inhibition by remodelin abolished the protective effects of BHB, as evidenced by increased micronuclei formation, anaphase bridges, and chromosomal aberrations, including broken chromosomes and EdU incorporation events on metaphase chromosome spreads (Fig. 8 A– 8 I, Supplementary Fig. 8 ). In 2BS cell, consistent results were observed. BHB treatment accelerated replication fork progression and preserved fork symmetry, while ssDNA levels were reduced, indicating alleviation of replication stress. NAT10 depletion or inhibition negated these effects, leading to increased genomic instability marker, such as micronuclei, mitotic chromosome bridges, and chromatid breaks. Additionally, EdU incorporation analysis revealed that the BHB-induced reduction in replication-associated abnormalities was dependent on NAT10 activity (Fig. 9 A– 9 I, Supplementary Fig. 9 ). Collectively, these findings demonstrate that NAT10-mediated β-hydroxybutyrylation plays a critical role in reducing replication stress and genomic instability. Knockdown of NAT10 or its inhibition by remodelin negates the protective effects of BHB treatment, highlighting the essential function of NAT10 in mediating these responses. DISCUSSION The faithful execution of DNA replication is fundamental to cellular homeostasis, and disruptions in replication dynamics can lead to replication stress and genomic instability, hallmarks of cancer, aging, and various metabolic disorders 45 , 46 . In this study, we observe that NAT10-mediated β-hydroxybutyrylation appears to function as an epigenetic mechanism that supports DNA replication, while potentially reducing replication stress and genomic instability. These effects seem to occur not only in cancer cells (U2OS) but also in primary human fibroblasts (WI-38 and 2BS), suggesting NAT10 may play a role in replication dynamics across different cell types. Our data indicate that NAT10 may modify histones through β-hydroxybutyrylation, possibly influencing chromatin accessibility to facilitate DNA synthesis and help manage replication stress. When NAT10 is depleted or inhibited, these effects appear to be diminished, which may point to its involvement in replication homeostasis. These findings could contribute to our understanding of how metabolic signaling, chromatin structure, and genome stability interact, with NAT10 potentially being one factor in replication regulation and cellular stress responses. NAT10-mediated β-hydroxybutyrylation appears to be a potential regulator of replication efficiency and genome stability. Our findings suggest that NAT10-mediated β-hydroxybutyrylation may represent a previously unexplored chromatin modification that could influence replication efficiency. DNA fiber assays indicated that BHB-induced β-hydroxybutyrylation might be associated with faster replication fork progression while preserving fork symmetry, which is often linked to regulated replication dynamics. This effect appears to rely on NAT10 activity, as both genetic depletion and chemical inhibition (e.g., remodelin treatment) reduced the observed replication acceleration. In contrast to histone acetylation, which is primarily known to open chromatin for transcription, β-hydroxybutyrylation might modulate chromatin accessibility in a way that supports replication origin licensing and firing. These differences could imply that β-hydroxybutyrylation contributes to a distinct form of epigenetic regulation, possibly specialized for replication-associated chromatin remodeling. Furthermore, our results suggest that NAT10-mediated β-hydroxybutyrylation may help reduce replication stress by stabilizing replication forks and decreasing ssDNA accumulation. In BHB-treated cells, we observed lower levels of replication stress markers such as γH2AX, phosphorylated CHK1, and RPA2, though these effects were not seen when NAT10 was knocked down or inhibited. The observed decrease in ssDNA, along with preserved replication fork symmetry, could indicate that β-hydroxybutyrylation influences chromatin structure in ways that might limit excessive fork stalling or collapse. Since replication stress contributes to genomic instability, these findings raise the possibility that NAT10 could play a role in maintaining genomic stability during replication. NAT10 as a potential chromatin organizer: Implications for genome stability and human disease. Chromatin structure is dynamically regulated during replication, ensuring that DNA synthesis proceeds efficiently while maintaining accessibility for replication and repair factors. Our results imply that NAT10 may contribute to this process by modifying histones, which could affect chromatin-bound protein interactions and replication-associated chromatin remodeling. ChIP-seq and ATAC-seq data indicate that NAT10-mediated β-hydroxybutyrylation might influence chromatin accessibility in response to BHB treatment, potentially creating conditions favorable for replication progression. It's worth noting that NAT10 depletion appeared to reduce chromatin-bound NAT10 levels, possibly pointing to an autoregulatory mechanism where β-hydroxybutyrylation helps regulate NAT10-chromatin interactions to maintain balanced replication activity. These findings could have implications for human diseases involving chromatin dysregulation and replication stress. In aging cells, replication stress tends to accumulate, possibly due to less efficient origin activation, fork stalling, and compromised repair pathways. Since WI-38 and 2BS cells are commonly used as models of cellular senescence, our observation that NAT10 appears to reduce replication stress and genomic instability in these cells raises the possibility that β-hydroxybutyrylation might play a role in mitigating age-related replication defects. If further validated, modulating NAT10 function could be explored as a potential strategy to address replication stress in aging and age-related conditions. These findings appear to align with prior studies suggesting NAT10's involvement in chromatin structure during mitosis, particularly in Hutchinson-Gilford Progeria Syndrome (HGPS). NAT10 may contribute to maintaining genome integrity during mitosis, possibly helping preserve genetic fidelity, though this effect has been primarily linked to NAT10's acetylation of CCDC84 and Eg5, which are involved in centrosome duplication 25 , 26 . Additionally, some studies have reported that depleting NAT10 reduces DNA damage (γH2AX) accumulation in Lmna G609G HGPS mouse models and HGPS patient-derived cells, which often exhibit various genetic abnormalities and nuclear deformities 28 , 47 . In the context of cancer, replication stress is a double-edged sword. While excessive replication stress drives genomic instability, which fuels tumorigenesis, controlled modulation of replication stress can be exploited for cancer therapy. Our observations raise the possibility that NAT10 overexpression could potentially support cancer cell proliferation by alleviating replication stress. On the other hand, inhibiting NAT10 might preferentially elevate replication stress in malignant cells, possibly making them more vulnerable to agents like ATR or CHK1 inhibitors. These findings could open avenues for exploring therapeutic approaches that leverage NAT10 activity to differentially target cancer cells while maintaining normal cellular replication. Beyond aging and cancer, metabolic disorders such as diabetes and obesity have been linked to chromatin modifications that alter gene expression and replication dynamics. Our findings position NAT10 as a potential metabolic sensor that integrates ketone body availability with chromatin regulation. The interplay between metabolic states and chromatin accessibility remains poorly understood, and our study provides a framework for exploring how metabolic signals influence epigenetic modifications to maintain genome stability. Lethality of NAT10 depletion: Limitations and future directions. A key limitation in targeting NAT10 is its essential role in cellular viability. Our observations indicate that NAT10 depletion appears to lead to significant replication stress, elevated genomic instability, and possible cell cycle arrest, highlighting its potentially unique role in replication regulation. The essential nature of NAT10 for cell viability suggests it may play a critical role in maintaining replication fork stability and origin activation. However, it remains unclear whether these effects are directly mediated by its β-hydroxybutyrylation activity or involve other epigenetic mechanisms. One possible explanation for the essential nature of NAT10 for cell viability is its role in stabilizing replication forks and coordinating replication-transcription conflicts. The loss of NAT10 may lead to uncoordinated origin firing, increased replication-transcription collisions, or failure to resolve stalled forks, ultimately triggering replication catastrophe. Additionally, NAT10 has been implicated in RNA modification and ribosomal biogenesis, raising the possibility that its essential function extends beyond chromatin remodeling. Future studies should focus on dissecting whether NAT10 replication-associated functions are distinct from its roles in RNA metabolism or whether these pathways are interdependent. Another key question is whether NAT10 functions independently or as part of a larger chromatin remodeling complex. Since its dynamic association with chromatin, NAT10 may interact with histone acetyltransferases, chromatin remodelers, or replication-associated factors to coordinate replication and repair. Identifying these interacting partners will provide deeper mechanistic insights into how NAT10 regulates chromatin structure and genome stability. From a therapeutic perspective, the essential nature of NAT10 for cell viability presents both challenges and opportunities. While complete loss of NAT10 is detrimental, partial inhibition or selective modulation of its β-hydroxybutyrylation activity may offer a way to fine-tune replication stress responses without inducing cytotoxicity. Developing small-molecule inhibitors that target specific enzymatic activities of NAT10, while preserving its essential cellular functions, will be crucial for future translational applications. In summary, this study suggests that NAT10-mediated β-hydroxybutyrylation may represent an important mechanism connecting metabolic signaling with chromatin remodeling, replication efficiency, and genomic stability. Our data indicate that NAT10 appears to contribute to maintaining a chromatin environment that ensures efficient and faithful DNA synthesis. These observations offer potential new perspectives on how chromatin modifications might interact with replication control, which could have relevance for understanding aging, cancer, and metabolic disorders. Further investigation of NAT10's regulatory network may help evaluate its possible utility as a therapeutic target in replication stress-associated conditions. MATERIALS AND METHODS Plasmid Construction and Cloning The cDNA encoding wild-type NAT10 was amplified via PCR and inserted into the pcDNA3.1(+) vector, including an N-terminal FLAG or HA tags. Site-directed mutagenesis was employed to generate NAT10 mutants (G641E, K989R and K823R) using the Fast Mutagenesis System. Full-length NAT10 and its truncated variants were cloned into the pGEX-4T-3 vector to generate GST-fusion proteins. Cell Culture, siRNA and Plasmid Transfections U2OS, HeLa, and 293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS, Gibco, A5670401), essential amino acids, and 1% streptomycin/penicillin (100 U/ml) at 37°C under 5% CO 2 . WI-38 and 2BS cells were maintained in RPMI 1640 medium with 15% FBS, essential amino acids, and 1% streptomycin/penicillin. To knock down NAT10 expression, U2OS cells (30–40% confluence) were transfected with 120 pmol siRNA oligonucleotides per well in 6-well plates using Entranster™-in vivo Transfection Reagent (Engreen Biosystem, 18668-11-1). The medium was refreshed after 24 hours, and a second transfection was performed. Cells were treated 48 hours later with chemical agents, including (±)-sodium 3-hydroxybutyrate (BHB, Sigma-Aldrich, 54965), aphidicolin (APH, Santa Cruz, sc-201535), RO-3306 (Sigma-Aldrich, SML0569), or nocodazole (Selleck, S2775). The siRNA sequences targeting NAT10 were: Homo siNAT10 #1: 5’-GAGCAUGGACCUCUCUGAAUACAUA-3’ Homo siNAT10 #2: 5’-CAAACAUUCGCUACUGCUACUACAA-3’ Homo siNAT10 #3: 5’-CAGGCUGAACUAGUUGUGAUUGAUG-3’ Plasmid transfection in 293T and U2OS cells were performed using the NEOFECT™ DNA Transfection Reagent (Neofect Biotech) following the manufacturer’s protocol. siRNA transfections in WI-38 and 2BS cells utilized RFectPM (Baidai Bio, 11014). Antibodies The primary antibodies utilized included: anti-NAT10 (Santa Cruz, B-4, sc-271770; C-10, sc-271141), anti-53BP1 (Cell Signaling Technology, 4937S), anti-Phospho H2A.X S139 (Cell Signaling Technology, 9718), anti-Cyclin A (B-8) (Santa Cruz, sc-271682), anti-β-actin (Sigma-Aldrich, A2228), anti-Flag M2 (Sigma-Aldrich, F1804), anti-H2B (V119) (Cell Signaling Technology, 8135S), anti-HA-Tag (6E2) (Cell Signaling Technology, 2367) and anti-COX-IV (Cell Signaling Technology, 4844), anti-phosphorylation S317-CHK1(Proteintech, 28807-1-AP), anti-RPA2(Cell Signaling Technology, 35869S), anti-BrdU (Serotec, OBT0030) and anti-BrdU (Becton Dickinson, 347580). The β-hydroxybutyrylation-lysine site specific antibodies are from Jingjie PTM BioLab, Co. Ltd, Hangzhou, China: anti-pan β-hydroxybutyrylation-lysine (BHB-K) antibody (PTM-1201), anti-H2AK5BHB (PTM-1220), anti-H2AK118BHB (PTM-1224), anti-H2BK5BHB (PTM-1230), anti-H2BK11BHB (PTM-1231), anti- H2BK16 BHB (PTM-1234), anti- H2BK20BHB (PTM-1235), anti-H2BK23BHB (PTM-1236), anti- H2BK34 BHB (PTM-1238), anti-H3K9BHB (PTM-1250), anti-H3K14BHB (PTM-1251), anti-H3K23BHB (PTM-1300), anti-H4K5BHB (PTM-1205), anti-H4K8BHB (PTM-1253), anti-H4K9BHB (PTM-1210), anti-H4K12BHB (PTM-1206). Secondary antibodies used were Goat anti-Rabbit IgG Secondary Antibody HRP conjugated (Jackson ImmunoResearch, 111-035-003) and Goat anti-Mouse IgG Secondary Antibody HRP conjugated (Jackson ImmunoResearch, 115-035-003). Cell Proliferation Assay Cell proliferation was evaluated using the Cell Counting Kit-8 (Selleck, B34302). U2OS cells were plated in 96-well plates at appropriate densities. After 24 hours of attachment, 10 µl of CCK-8 reagent was added per well, and absorbance at 450 nm was measured after a 2-hour incubation using a microplate reader. Cell viability was monitored at 0, 12, 24, 36, 48, and 72 hours post-seeding. Immunofluorescence Assays Cells plated on coverslips with siRNA transfected or treated with drugs were fixed in 4% PFA/PBS for 15 minutes and neutralized with Glycine. After washing twice with PBS, the cells were permeabilized with 0.3% Triton-X/PBS for 10 minutes. After blocked with 5% BSA/PBS for 30 minutes, the cells were incubated with the indicated primary antibody for 60 minutes at room temperature, following by incubation with the Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H + L) antibody (Jackson ImmunoResearch, 111-545-003) or Alexa Fluor® 594 AffiniPure Goat Anti-Mouse IgG (H + L) antibody (Jackson ImmunoResearch, 111-585-003) for 45 minutes. After staining nuclei with 2 µg/ml diamidinophenylindole (DAPI) (Roche, 28718-90-3) for 5 minutes, the coverslips were mounted with Vectashield medium. Images were obtained on a ZEISS LSM880 confocal system with a 63x/1.40 (oil) or 100x/1.40 (oil) objective lens or a high content screening system (Operetta™, Perkin Elmer) with a 60×, 0.95-NA objective at the perfect focus. Confocal and super-resolution images were analyzed with ZEN (Blue edition) software from Carl Zeiss Microscopy, and fluorescence intensity was calculated with Harmony 3.5 software. Note that for immunostaining of pre-extracted cells, cells grown on coverslips were incubated in CSK buffer containing 0.3 mg/ml RNase A for 15 minutes at room temperature before fixed as descripted above. EdU Labeling and Detection in Mitotic Cells Cells were incubated with 9 mM RO-3306 and 0.4 µM APH (Santa Cruz, sc-201535) for 16 hours to synchronize in late G2 phase, subsequently washed three times with PBS for 5 minutes at room temperature, and then released into pre-warmed fresh medium containing 20 mM 5-ethynyl-2’-deoxyuridine (EdU, Sigma-Aldrich, 61135-33-9) and 0.1 mg/ml Colcemid (Thermo Fisher Scientific, 15210040) for 60 minutes. Cells were harvested by centrifuged at 2,000 g for 5 minutes at 4°C, and resuspended and incubated in 8 ml pre-warmed 75 mM KCl for 15 minutes at 37°C. Swollen mitotic cells were subsequently fixed in Carnoy’s buffer (75% methanol, 25% glacial acetic acid) three times for 15 minutes, spread on pre-hydrated slides (Thermo Fisher Scientific) and dried overnight at room temperature followed by EdU detection with Click-IT Plus EdU Alexa fluor 488 Imaging Kit (Thermo Fisher Scientific, C10337) according to the manual. After staining chromosomes with 2 µg/ml DAPI (Roche, 28718-90-3) for 5 minutes and mounting slides with vectashield mounting medium, images were captured on a ZEISS LSM880 confocal system with a 100×/1.40 (oil) objective. At least 150 metaphase chromosomes were analyzed in three independent experiments. Flow Cytometer For DNA content analysis, cells were synchronized at the G1/S boundary with 2 mM thymidine for 18 hours, followed by release every two hours. Cells were harvested with trypsin and fixed overnight in pre-chilled 100% methanol at -20°C. Subsequently, cells were stained with 40 µg/ml propidium iodide (PI, Sigma-Aldrich, 25535-16-4) in PBS, containing 100 µg/ml RNase A, for 15 minutes at room temperature in the dark. Flow cytometric analysis was performed immediately using a FACSCalibur system (Becton Dickinson). DNA content was analyzed using CellQuest or FlowJo VX software. EdU FACS Asynchronously growing U2OS cells were labeled with 20 uM EdU for 20 minutes. After labeling, cells were harvested. Cells were washed once with PBS and fixed in 70% ethanol over night at 4°C. Cells were incubated in 2 N HCl at room temperature for 20 mins, washed in PBS. EdU incorporation was detected using the YF @ 648A Click-iT EdU Flow Cytometry Assay Kit (UE, C6046S) following the manufacturer’s protocol. Nuclei were stained with 40 µg/ml propidium iodide (PI, Sigma-Aldrich, 25535-16-4) in PBS, containing 100 µg/ml RNase A, for 15 minutes at room temperature in the dark. Flow cytometric analysis was conducted using a FACSCalibur system, and data were processed with FlowJo VX software to assess cell cycle progression and EdU-positive populations. DNA fiber analysis DNA fiber analysis was conducted as described previously 30 , 31 . In summary, cells were either directly treated with 5 mM or 10 mM BHB (Sigma-Aldrich, 54965) for 24 hours, or transfected with siRNA or treated with 10 µM remodelin (Selleck, S7641) with or without 10 mM BHB. Following treatment, cells were pulse-labeled with 25 µM chlorodeoxyuridine (CldU, Sigma-Aldrich, 50-90-8) for 20 minutes, washed, and labeled with 250 µM iododeoxyuridine (IdU, Sigma-Aldrich, 54-42-2) for another 20 minutes. CldU and IdU were detected using a rat anti-BrdU (Serotec, OBT0030) and mouse anti-BrdU (Becton Dickinson, 347580) primary antibodies, respectively, followed by DyLight 550 anti-rat (Thermo Fisher, SA5-10019) and Alexa Fluor 488 anti-mouse (Thermo Fisher, A-10680) secondary antibodies.DNA fibers were imaged with a ZEISS LSM880 confocal microscope equipped with a 100×/1.40 oil objective. Images were acquired using autofocus and tile-array methods, and double-labeled replication forks were analyzed manually with ZEN software. For each slide, 50–100 forks were evaluated, with data aggregated from three independent experiments. ssDNA Visualization Single-stranded DNA (ssDNA) in S-phase cells was visualized by detecting BrdU foci without DNA denaturation. Cells labeled with 10 µM BrdU for 48 hours were fixed in 4% formaldehyde for 15 minutes, permeabilized with 0.3% Triton X-100 for 10 minutes, and blocked with 5% BSA for 30 minutes. Cells were incubated with primary anti-BrdU (1:400) and anti-cyclin A (1:400) antibodies (Santa Cruz, sc-271682) for 60 minutes, followed by Alexa Fluor® 488 (1:200, Jackson ImmunoResearch, 111-545-003) or Alexa Fluor® 594 (1:200, Jackson ImmunoResearch, 111-585-003) secondary antibody for 45 minutes. Nuclei were stained with 2 µg/ml DAPI for 5 minutes, and images were acquired using a high-content screening system (Perkin Elmer) with a 60×, 0.95-NA objective. Images were obtained on a ZEISS LSM880 confocal system with a 63x/1.40 (oil) or 100x/1.40 (oil) objective lens or a high content screening system (Operetta™, Perkin Elmer) with a 60×, 0.95-NA objective at the perfect focus. Confocal and super-resolution images were analyzed with ZEN (Blue edition) software from Carl Zeiss Microscopy, and fluorescence intensity was calculated with Harmony 3.5 software. Single-stranded DNA (ssDNA) in S-phase cells was also visualized by detecting RPA2 foci in pre-extracted nuclei. Cells grown on coverslips were incubated in CSK buffer containing 0.3 mg/ml RNase A for 15 minutes at room temperature before fixed. Cells were immunostained by RPA2 antibody (1:400), and imagines were obtained as described above. Mass Spectrometry Analysis Protein extraction, digestion, and mass spectrometry analysis were outsourced to (Beijing Proteome Research Center). Proteins were subjected to intensity-based absolute quantification (iBAQ) mass spectrometry to analyze. Origin Firing Origin firing analysis was conducted as described previously 32 , 33 . Briefly, chromatin-binding proteins were enriched, and were analysis by label-free quantitative proteomic analysis. U2OS cells were lysed with cytoplasmic lysis buffer (10 mM HEPES pH 7.9, 340 mM sucrose, 3 mM CaCl 2 , 2 mM MgOAc, 0.1 mM EDTA, 1 mM DTT, 0.5% Triton X-100 and protease inhibitors) for 20 min on ice. Intact nuclei were pelleted by centrifugation at 3,500 × g for 15 min. Nuclei were washed with cytoplasmic lysis buffer without Triton X-100. Nuclei were lysed with nuclear lysis buffer (20 mM HEPES pH 7.9, 150 mM K 2 OAc, 1.5 mM MgCl 2 , 3 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 10% glycerol, and protease inhibitors) for 30 min on ice. The nucleoplasmic fraction was cleared by centrifugation at 15,000 × g for 30 min. The chromatin-enriched pellet was resuspended in 50 µl denaturation buffer (8 M Urea; 100 mM Tris pH 8, 5 mM DTT) and incubated at 22°C for 30 min. The sample was cleared by centrifugation at 20,000 × g for 30 min, and the supernatant containing the solubilized denature chromatin proteins was collected. The chromatin-bound proteins were subjected to intensity-based absolute quantification (iBAQ) mass spectrometry to analyze replication initiation factors. Replication Program U2OS cells in logarithmic phase were pulse-labeled with 10 µM BrdU (Sigma-Aldrich, 59-14-3) for 45 minutes, washed twice with warm medium and chased in fresh medium for 4 hours. Cells were then labeled with 10 µM EdU for another 45 minutes. Fixed cells (4% formaldehyde,15 minutes) were permeabilized with 0.3% Triton X-100 for 10 minutes, and DNA was denatured with 2N HCl for 30 minutes at room temperature, followed by neutralization with 0.1M sodium borate buffer (pH 8.5). EdU was detected using the Cell-Light EdU Apollo567 In Vitro Kit (Ribobio, C10310), and BrdU was visualized with an anti-BrdU antibody (1:400) followed by Alexa Fluor® 488 secondary antibody (Jackson ImmunoResearch, 111-545-003), after blocking with 5% BSA for 1 hour. Nuclei were counterstained with 2 µg/ml DAPI, and images were captured using a high-content screening system (Perkin Elmer) with a 60×, 0.95-NA objective, and BrdU or EdU intensity per nucleus was quantified using Harmony 3.5 software. Replication patterns and S phase progression were analyzed as previously described 34 . The data obtained from > 250 nuclei across three independent experiments. Anaphase Bridge Analysis Cells were synchronized with 9 mM RO-3306 for 16 hours, washed with PBS, and released into DMEM for 1.5 hours at 37°C. Cells were then cross-linked and permeabilized in PTEMF buffer (20 mM PIPES, pH 6.8, 10 mM EGTA, 1 mM MgCl₂, 0.2% Triton X-100, 4% formaldehyde) and stained with 2 µg/ml DAPI (Roche, 28718-90-3). Anaphase chromatin structures were imaged using a ZEISS LSM880 confocal microscope with a 63×/1.40 oil objective. At least 150 anaphase cells were analyzed per experiment. Micronuclei Analysis Cells treated with siRNA or drugs were cultured in medium containing 2 mg/ml cytochalasin B (Sigma-Aldrich, C6762) for 16 hours to arrest cytokinesis. Fixed cells were stained with 4% formaldehyde and 2 µg/ml DAPI (Roche, 28718-90-3) for nuclear visualization. Micronuclei were quantified using a ZEISS LSM880 confocal microscope. At least 150 binucleated cells per condition were scored, considering only distinct micronuclei adjacent to DAPI-stained nuclei. Preparation and Analysis of Chromosome Spreads Cells were treated with 0.2 µM aphidicolin (Santa Cruz, sc-201535) for 24 hours, followed by 200 ng/ml nocodazole (Selleck, S2775) for the final 5 hours. Mitotic cells were collected by shake-off, centrifuged at 2,000 g for 5 minutes at 4°C. The pellet was resuspended in 1 ml PBS, followed by the addition of 8 ml pre-warmed 75 mM KCl, and incubated at 37°C for 15 minutes. After this incubation, 5 ml freshly prepared Carnoy’s buffer (75% methanol, 25% glacial acetic acid) was added for 15 minutes. Cells were fixed with Carnoy’s buffer (three times, 15 minutes each), spread onto slides and dried overnight at room temperature. Chromosomes were stained with 2 µg/ml DAPI and imaged using a ZEISS LSM880 confocal microscope with a 100×/1.40 oil objective. At least 150 spreads were analyzed for chromatid breaks and gaps. Protein Expression, Purification, and Pull-Down Assays To purify GST-NAT10 (full length or various fragments), bacterial expression constructs were transformed into E. coli Rosetta (DE3) (Novagen, 70956). Selected clones were cultured in 500 ml LB medium at 37°C until the OD 600 reached ~ 0.6, at which point protein expression was induced with 1 mM IPTG (AppliChem, A1008) at 20°C overnight. Harvested cell were resuspended in BC500 buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 1.5 mM MgCl 2 , 10 mM KCl, 0.5% Triton X-100, 20% glycerol) supplemented with 1 mM PMSF and protease inhibitors. The suspension was lysed by sonication (70% amplitude, 10 seconds on/10 seconds off for 20 minutes) using a Hielscher-Ultrasound Technology Sonicator. Clarified lysates, obtained by centrifugation at 20,000 g for 30 minutes at 4°C, were incubated overnight at 4°C with pre-equilibrated glutathione Sepharose 4B beads (GE Healthcare, 17527902). Beads were washed once and resuspended in BC100 buffer (20 mM Tris-HCl, pH 7.9, 100 mM NaCl, 1.5 mM MgCl₂, 10 mM KCl, 0.1% Triton X-100, 20% glycerol) containing 50 U/ml Benzonase Nuclease (Sigma, 9025-65-4) on ice for 30 minutes, followed by four additional washes with BC100 buffer. The GST-fusion proteins were eluted with 20 mM glutathione (GSH) in BC100 buffer, dialyzed overnight against BC100 buffer, and stored for pull-down assays. Protein concentrations were determined using the Bradford assay. For FLAG-tagged proteins, 293T cells overexpressing the indicated constructs were lysed in BC500 buffer supplemented with 1 mM PMSF and protease inhibitors, followed by sonication. Lysates (2 mg total protein) were incubated with 40 µl anti-FLAG M2 agarose beads (Sigma-Aldrich). Beads were washed once with BC500 buffer and resuspended in BC100 buffer containing 50 U/ml Benzonase Nuclease (Sigma, 9025-65-4) on ice for 30 minutes, followed by four times washes with BC100 buffer. FLAG-tagged proteins were eluted using 0.5 µg/µl FLAG peptide in BC100 buffer. GST pull-down assays were performed as previously described 35 . Briefly, 5 µg GST-fusion protein was incubated with 2 µg eukaryotic purified protein or 100 µg purified mononucleosomes in 1 ml BC100 buffer or various binding buffers containing different NaCl concentrations (100 mM, 150 mM, 200 mM, 300 mM, 500 mM). The mixture was incubated overnight at 4°C with gentle shaking, followed by 4-hour incubation with glutathione Sepharose (GE Healthcare, 17527902). After five washes with the respective buffer, bound proteins were eluted with 1× SDS loading buffer and analyzed by SDS-PAGE. Mononucleosome Extraction Mononucleosomes for use in in vitro pull-down and β-hydroxybutyrylation assays were isolated using micrococcal nuclease 36 , 37 . Briefly, U2OS cells, treated with or without BHB, were lysed in BC300 buffer on ice for 30 minutes. Chromatin pellet, obtained by centrifugation at 1,000 g for 3 minutes, were washed and digested with 60 U/ml micrococcal nuclease (MNase) in digestive buffer (10 mM Tris-Cl pH 8.0, 10 mM CaCl 2 , 5 mM MgCl 2 , 0.05 mM DTT, and 0.01 mM PMSF) at 30°C for 30 minutes, and the reaction was stopped with 10 mM EDTA. The first supernatant (S1) was collected after centrifugation, and mononucleosomes were purified using gel filtration chromatography on the SMART™ FPLC system (Amersham Biosciences). Purity was confirmed by agarose gel electrophoresis (140 bp band) and SDS-PAGE (histones only). β-hydroxybutyrylation assay To evaluate NAT10 as a β-hydroxybutyryltransferase, 293T cells transfected with Flag-NAT10-expressing plasmids were treated with 10 mM BHB for 24 hours. Cell was resuspended in BC500 buffer with 10 mM BHB, sonicated, and the lysate was subjected to immunoprecipitation and western blot analysis. Alternatively, β-hydroxybutyrylated proteins were enriched using pan-β-hydroxybutyrylated lysine antibodies in the BC100 buffer. To confirm NAT10-mediated histone β-hydroxybutyrylation in vivo , NAT10 (wild type or G641E, K989R and K823R mutants) was overexpressed or knocked down in cells, followed by 10 mM BHB treatment for 6 hours. In parallel, cells transfected with NAT10 sgRNA and Cas9 plasmids were screened with puromycin (3 µg/ml) and hygromycin (1 µg/ml) for 72 hours prior to BHB treatment. Histones were purified using standard acid extraction protocols 36 . Briefly, intact nuclei were isolated from cells cracked with pre-cold hypotonic HB buffer, resuspended in 2N HCl and incubated overnight on a rotator. Nuclear debris was removed by centrifugation at 10,000 g for 10 minutes at 4°C, and the supernatant was neutralized with Tris (pH 8.0). Extracted histones were analyzed by SDS-PAGE and immunoblotting with specific antibodies. For in vitro assay, purified NAT10 (wild type or G641E, K989R and K823R) was co-incubated with 25 µM DL-β-Hydroxybutyryl coenzyme A (BHB-CoA, Sigma, H0261) with or without 10 µM remodelin (Selleck, S7641) in 30 µl reaction buffer (50 mM HEPES pH 8.0, 10% glycerol, 1 mM DTT, 1 mM PMSF). Alternatively, 1 µg NAT10 was incubated with 2 µg mononucleosome in the same buffer. Reactions were performed at 30°C for 60 minutes, stopped with SDS buffer, and analyzed by Western blotting. Subcellular Fractionation U2OS cells (1 × 10⁷) were homogenized in HB buffer (10 mM Tris-Cl, pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 1 mM DTT, and 1× EDTA-free protease inhibitor cocktail, Sigma-Aldrich, 11873580001) until ~ 90% lysis, confirmed by trypan blue. Lysates were centrifuged at 1,000 g for 15 minutes at 4°C to separate the cytoplasmic fraction (Cyto), which was supplemented with 100 mM NaCl and 0.1% Triton X-100. The nuclear pellet was resuspended in BC300 buffer (20 mM Tris-HCl pH 7.9, 300 mM NaCl, 10 mM KCl, 1.5 mM MgCl 2 , 0.1% Triton X-100, 20% glycerol, and 1× protease inhibitor cocktail), vortexed for 30 minutes and centrifuged. The supernatant was collected as the nuclear soluble fraction (Nucl) and adjusted to 100 mM NaCl. Chromatin fractions were isolated by lysing cells in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100) supplemented with 0.2 mM PMSF and protease inhibitors. The lysates were centrifuged at 20,000 g for 15 minutes, and the supernatant was designated as the soluble fraction (Sup). The insoluble chromatin fraction was sonicated in RIPA buffer containing 1% SDS, centrifuged at 20,000 g for 15 minutes at 4°C, and the supernatant was collected as the chromatin soluble fraction (Chr). Alternatively, cells were lysed in CSK buffer (10 mM HEPES pH 7.9, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl 2 , 0.5% Triton X-100) with 300 µg/ml RNase A. After centrifugation, the supernatant was collected as the soluble fraction (Sup), and the chromatin-containing pellet was sonicated in RIPA buffer with 0.1% SDS to obtain the pellet fraction (Pellet). Chromatin immunoprecipitation and sequencing (ChIP-seq) Chromatin immunoprecipitation was conducted using the ChIP-IT Express Kit (Active Motif, 53008). Briefly, U2OS cells cultured on 15 cm plates were treated with 10 mM BHB or left untreated for 12 hours. Cells were fixed with 1% formaldehyde for 15 minutes at room temperature, and cross-linking was quenched with 125 mM glycine for 5 minutes. After centrifugation at 1,250 g for 3 minutes at 4°C, cells were washed with PBS containing 0.5% Igepal and sonicated (40% amplitude, 4 seconds on/6 seconds off, ~ 60 cycles) to shear chromatin. A 10% aliquot of the supernatant was saved as the input sample. For immunoprecipitation, 50 µg of sonicated chromatin was incubated overnight at 4°C with 2 µg of anti-NAT10 antibody (Santa Cruz, B-4, sc-271770). Immunocomplexes were captured with ChIP Grade Protein A/G Plus Agarose (Thermo Fisher Scientific, 26159), extensive washed, eluted, and de-crosslinking at 65°C overnight. Samples were treated RNase A and proteinase K, and DNA was purified according to the manufacturer’s protocol (Active Motif, 53008). Purified DNA was eluted in 30 µl of distilled water and prepared for sequencing by adding Illumina adapter. Libraries were sequenced on the HiSeq4000 platform (Illumina), generating > 50 million paired-end reads (200–300 bp) per sample. ATAC-seq ATAC sequencing service was provided by Cloud-Seq Biotech (Shanghai, China) with GenSeq® ATAC kit (GenSeq Inc.). Briefly, cells were lysed using cold lysis buffer. After lysis, nuclei were collected by centrifuging at 500 g for 10 min at 4℃. The pellet was transposed with transposome and add tags on the both sides of fragmented genomic DNA according to the manufacturer’s instruction. After tagmentation, the purified tagged DNA was used for PCR amplification with Genseq® 2×HiFi PCR Mix (GenSeq Inc.). The libraries were quantified using Qubit fluorometric assay (ThermoFisher) and then sequenced. in a NovaSeq platform (Illumina). ATAC-Seq high throughput sequencing and subsequent bioinformatics analysis were all done by Cloud-Seq Biotech (Shanghai, China). Briefly, raw data were generated after sequencing, image analysis, base calling and quality filtering on sequencer. Use fastp software (v 0.23.4) to remove joints and low quality reads to obtain high quality clean reads; And clean reads alignment to the reference genome using bowtie2 software (v2.2.4); The reads aligned to the chrM were removed using the samtools software (v1.9), PCR duplicates were removed using getk-picard software (v4); open chromatin regions (Peak Calling) using MACS2 software (v2.1.1); differential enriched regions were identified using diffReps software; and enrichment peaks were annotated using ChIPseeker software (v1.2.6). GO and KEGG pathway analysis were performed for genes associated with differentially enriched peaks. Quantification and Statistical Analysis Data were analyzed using GraphPad InStat software (Version 5.01, GraphPad Prism, GraphPad Software Inc., San Diego, CA) and SPSS 21.0 software. Results are presented as mean ± SD from at least three independent biological replicate experiments. Statistical significance was determined using the Student’s t test or one-way ANOVA, with p-values < 0.05 considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Declarations COMPETING INTERESTS The authors declare no competing interests. FUNDING The project was supported partially by the National Natural Science Foundation of China (Grant No. 32371313) and the Fundamental Research Funds for the Central Universities (2024CDJXY-016) to Wenhui Zhao. AUTHOR CONTRIBUTION Conceptualization, W.Z.; Methodology, Y.S., R.Z., S.J. and W.Z.; Formal Analysis, Y.S., R.Z., S.J. and W.Z.; Investigation, Y.S., R.Z., S.J. and W. Z.; Resources, Y.S., R.Z., S. J., K.L., Z.F., S.J.., S.L., and W.Z.; Data Curation and Visualization, Y.S., R.Z., S.J. and W.Z.; Funding Acquisition, W.Z.; Supervision, W.Z. ACKNOWLEDGMENTS Thanks to Drs. Haibo Wu, Chuan Huang, Zhenghong Lin, Shanshan Pang, Wei Deng, Zhong Luo and Zhengguo Li for critical discussion and critical reading the manuscript. We would like to thank Dr. Yingming Zhao at the University of Chicago, Dr. Wei Gu at Columbia University, Dr. Qinglian Liu at Virginia Commonwealth University and Dr. Liang Ce at Hebei Medical University for technical help. We appreciate Dr. Zhongyi Cheng (Jingjie PTM BioLab, Co. Ltd, Hangzhou, China) for provding β-hydroxybutyrylation histones antibodies. We thank Cloud-Seq Biotech (Shanghai, China) for providing ATAC sequencing and analysis service. Wenhui Zhao was partial supported by National Natural Science Foundation of China (Grant No. 32371313) and the Fundamental Research Funds for the Central Universities (2024CDJXY-016). DATA AVAILABILITY The ChIP-seq data has been deposited in Gene Expression Omnibus (GEO) #GSE175731. The ATAC-seq data has been deposited in Gene Expression Omnibus (GEO) #GSE292952. All data are available on https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE175731 and acc = GSE292952. References Klein, K. N. et al. Replication timing maintains the global epigenetic state in human cells. Science 372, 371–378, doi: 10.1126/science.aba5545 (2021). Van Rechem, C. et al. Collective regulation of chromatin modifications predicts replication timing during cell cycle. Cell Reports 37, doi: 10.1016/j.celrep.2021.109799 (2021). Goren, A., Tabib, A., Hecht, M. & Cedar, H. DNA replication timing of the human β-globin domain is controlled by histone modification at the origin. 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Additional Declarations There is no duality of interest Supplementary Files SupplementaryTable1OriginFiringFactorsMS.xlsx Supplementary Table 1_OriginFiringFactors-MS SupplementaryTable2AcetylatedHistonesMS.xlsx Supplementary Tabl2 2_AcetylatedHistones-MS SupplementaryV96.pptx Supplementary Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7110451","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":488806222,"identity":"0ba66f75-6a39-48e0-b672-860fe8220827","order_by":0,"name":"Wenhui 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Beijing","correspondingAuthor":false,"prefix":"","firstName":"Yanchang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-13 00:20:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7110451/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7110451/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87499063,"identity":"e2554937-c6ff-48a1-9b99-4b230ea17d6a","added_by":"auto","created_at":"2025-07-24 13:33:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147461,"visible":true,"origin":"","legend":"\u003cp\u003eBHB treatment accelerates replication. (\u003cstrong\u003eA\u003c/strong\u003e) β-hydroxybutyrate (BHB) enhanced cell viability, measured by CCK8 assay. U2OS cells were exposed to varying BHB concentrations for different durations. (\u003cstrong\u003eB\u003c/strong\u003e) BHB treatment promotes earlier DNA replication completion. U2OS cells were synchronized at the G1/S boundary with 2 mM thymidine for 18 hours and then released into S-phase in the presence of varying BHB concentrations. DNA content was analyzed via FACS following PI staining. (\u003cstrong\u003eC-E\u003c/strong\u003e) BHB treatment accelerates replication fork speed. (\u003cstrong\u003eC\u003c/strong\u003e) Representative DNA fiber images. U2OS cells were treated with 5 mM or 10 mM BHB, pulse-labeled with 25 µM CldU for 20 minutes, and subsequently with 250 µM IdU for another 20 minutes. DNA fibers were spread and visualized. Scale bar: 10 µm. (\u003cstrong\u003eD\u003c/strong\u003e) Quantitative analysis of replication fork speed from (\u003cstrong\u003eC\u003c/strong\u003e). (\u003cstrong\u003eE\u003c/strong\u003e) BHB unaffected DNA fiber replication fork symmetry. IdU/CldU ratios were derived from data in (\u003cstrong\u003eD\u003c/strong\u003e). (\u003cstrong\u003eF-G\u003c/strong\u003e) BHB treatment alters S-phase progression in U2OS cells. (\u003cstrong\u003eF\u003c/strong\u003e) S-phase progression analysis. Log-phase U2OS cells were pulse-labeled with 10 µM BrdU for 45 minutes, chased in fresh medium for 4 hours, and subsequently labeled with 10 µM EdU for 45 minutes. DNA was denatured and BrdU/EdU intensity per nucleus was quantified. S-phase progression was categorized into early–early, early–mid, mid–late, and early–late stages. (\u003cstrong\u003eG\u003c/strong\u003e) Quantitative analysis of S-phase progression from three independent experiments (n \u0026gt; 250 nuclei/experiment). (\u003cstrong\u003eH-M\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003c/del\u003e) BHB treatment shortens S-phase duration, determined using EdU FACS analysis. \u003cdel\u003e(\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e) EdU FACS analysis of EdU/DNA content. \u003c/del\u003eU2OS cells were treated with 10 mM BHB for different durations, and 20 µM EdU was added during the final 20 minutes of treatment. EdU FACS analysis of EdU/DNA content.\u003cdel\u003e \u003c/del\u003e\u0026nbsp;Gates denote early and late S-phase cells. (\u003cstrong\u003eH\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/del\u003e\u003cstrong\u003e-J\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/del\u003e) Quantitative analysis of S-phase dynamics. (\u003cstrong\u003eH\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/del\u003e) Percentage of S-phase cells, (\u003cstrong\u003eI\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eJ\u003c/strong\u003e\u003c/del\u003e) fold increase in EdU incorporation over time, and (\u003cstrong\u003eJ\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/del\u003e) fold increase in EdU incorporation in early and late S-phases. (\u003cstrong\u003eK\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003c/del\u003e\u003cstrong\u003e-M\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003c/del\u003e) Temporal progression of S-phase analyzed using EdU FACS.\u003cdel\u003e (\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e)\u003c/del\u003e EdU/DNA content following BHB treatment at different time points post-release. (\u003cstrong\u003eK\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003c/del\u003e) S-phase cell percentages over time. (\u003cstrong\u003eL\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/del\u003e) Quantitative analysis of EdU incorporation fold changes. (\u003cstrong\u003eM\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003c/del\u003e) Fold changes in EdU incorporation in early and late S-phases post-release. All data are represented mean ± S.D. from three independent experiments (*p \u0026lt; 0.01; **p \u0026lt; 0.05; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/7fc9cb4919ddbc4080180323.jpg"},{"id":87499069,"identity":"5693d716-19f7-4e57-8b41-6ca803918282","added_by":"auto","created_at":"2025-07-24 13:33:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":147212,"visible":true,"origin":"","legend":"\u003cp\u003eBHB treatment reduces replication stress and genomic instability. (\u003cstrong\u003eA-J\u003c/strong\u003e) BHB alleviates replication stress. (\u003cstrong\u003eA\u003c/strong\u003e) Representative immunostaining images of γH2AX in cyclin A-positive U2OS cells treated with 5 or 10 mM BHB. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of cyclin A-positive cells with \u0026gt;5 γH2AX foci from (\u003cstrong\u003eA\u003c/strong\u003e). (\u003cstrong\u003eC\u003c/strong\u003e) Representative images of BrdU incorporation in cyclin A-positive cells under non-denaturing conditions. U2OS cells were treated with 5 or 10 mM BHB, labeled with 20 µM BrdU for 36 hours. (\u003cstrong\u003eD\u003c/strong\u003e) Quantification of cyclin A-positive U2OS cells with \u0026gt;5 BrdU foci from (\u003cstrong\u003eC\u003c/strong\u003e). (\u003cstrong\u003eE\u003c/strong\u003e) γH2AX intensity quantification from immunofluorescence experiments. (\u003cstrong\u003eF\u003c/strong\u003e) RPA2 intensity quantification from CSK buffer pre-extracted nuclei. (\u003cstrong\u003eG\u003c/strong\u003e) Western blot analysis of CHK1, and phosphorylated-S317-CHK1 in whole-cell lysates from U2OS cells treated with 10 mM BHB for 8 hours. (\u003cstrong\u003eH\u003c/strong\u003e) Western blot of chromatin-bound RPA2 and γH2AX in U2OS cells treated as (\u003cstrong\u003eG\u003c/strong\u003e). (\u003cstrong\u003eI-L\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/del\u003e) BHB mitigates genomic instability. \u003cdel\u003e(\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e) Representative immunostaining images of 53BP1 in cyclin A-positive U2OS cells treated with 5 or 10 mM BHB for 24 hours. (\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eJ\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e) Quantification of cyclin A-positive U2OS cells with \u0026gt;5 53BP1 foci from (\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e). \u003c/del\u003e(\u003cstrong\u003eI\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/del\u003e) Representative images of micronuclei, mitotic chromosome bridges, and chromosomal breaks in U2OS cells. Red arrows indicate micronuclei, bridges, or chromosomal breaks. (\u003cstrong\u003eJ\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003c/del\u003e) Quantification of micronuclei in U2OS cells. Cells were treated with 5 or 10 mM BHB for 24 hours, cultured in medium containing 2 mg/ml cytochalasin B for 16 hours to arrest cytokinesis. (\u003cstrong\u003eK\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003c/del\u003e) Quantification of mitotic chromosome bridges in U2OS cells. Cells were synchronized with 9 mM RO-3306 for 16 hours and released into fresh medium for 1.5 hours. (\u003cstrong\u003eL\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/del\u003e) Quantification of chromosomal breaks per chromosome spread. U2OS cells were treated with 0.2 µM aphidicolin (APH) for 24 hours and 200 ng/ml nocodazole for the last 5 hours. Mitotic cells were collected by shake-off, and chromosomes were spread and stained with DAPI. All data are represented mean ± S.D. from three independent experiments (*p \u0026lt; 0.01; **p \u0026lt; 0.05; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/9572e29e9c38e89b1c06ae1f.jpg"},{"id":87499082,"identity":"f4d4133f-c43b-4a7d-8c01-6d4edfee5cdf","added_by":"auto","created_at":"2025-07-24 13:33:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59648,"visible":true,"origin":"","legend":"\u003cp\u003eNAT10 functions as a β-hydroxybutyryl-transferase. (\u003cstrong\u003eA\u003c/strong\u003e) CREBBP (CBP), EP300 (p300), HAT1, and NAT10 of histone acetyl-transferases, are β-hydroxybutyrylated (Kbhb). U2OS cells were treated with 10 mM BHB, and Kbhb proteins were detected via LC-MS/MS. Notably, NAT10 was Kbhb at lysine 989 (K989). (\u003cstrong\u003eB\u003c/strong\u003e) Kbhb modification of HAT1, NAT10, and CBP/p300 is observed. Flag-tagged HATs were overexpressed in 293T cells treated with 10 mM BHB for 24 hours. Proteins were enriched and purified by immunoprecipitation with M2 beads under stringent conditions (500 mM NaCl), and detected using a\u003cdel\u003en\u003c/del\u003e pan anti\u003cdel\u003e-pan\u003c/del\u003e-β-hydroxybutyrylation antibody (BHB-K). (\u003cstrong\u003eC\u003c/strong\u003e) Endogenous NAT10 is Kbhb. U2OS cells treated with 10 mM BHB for 24 hours were subjected to anti-BHB-K immunoprecipitation. NAT10 was detected by immunoblotting with a NAT10 antibody. (\u003cstrong\u003eD\u003c/strong\u003e) NAT10 is \u003cem\u003ein vitro\u003c/em\u003e Kbhb and inhibited by remodelin. Flag-NAT10 wild type, inactive mutant (Flag-NAT10/G641E), Kbhb site mutant (Flag-NAT10/K989R) and 2-isohydroxybutyrylation-lysine (Khib) site mutant (Flag-NAT10/K823R) were enriched and purified from 293T cells transfected with respective plasmids. The \u003cem\u003ein vitro\u003c/em\u003e β-hydroxybutyrylation assay was performed with the purified NAT10 proteins. (\u003cstrong\u003eE\u003c/strong\u003e) NAT10 is Kbhb in U2OS cells. Flag-NAT10 was overexpressed in U2OS cells treated with 10 mM BHB and 10 µM remodelin for 24 hours. Flag tagged NAT10 was enriched and purified, and Kbhb modification was detected using an \u003cdel\u003eanti-pan β-hydroxybutyrylation-lysine antibody (\u003c/del\u003eBHB-K\u003cdel\u003e)\u003c/del\u003e. (\u003cstrong\u003eF\u003c/strong\u003e) NAT10 Kbhb depends on specific lysine residues. Flag-NAT10 and its mutants were overexpressed in U2OS cells treated with 10 mM BHB and10 µM remodelin for 24 hours. Modified proteins were enriched and analyzed as described in (\u003cstrong\u003eE\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/f6da22db9ff494e559485f44.jpg"},{"id":87500085,"identity":"4e16b115-845b-4461-a87b-bf1546429811","added_by":"auto","created_at":"2025-07-24 13:41:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":161036,"visible":true,"origin":"","legend":"\u003cp\u003eNAT10 β-hydroxybutyrylates histones at multiple sites. (\u003cstrong\u003eA\u003c/strong\u003e) Total levels of Kbhb histones reduces following NAT10 depletion in U2OS cells. Cells were transfected with NAT10 siRNA and treated with 10 mM BHB. Histones were extracted and analyzed by immunoblotting with a pan anti-BHB-K antibody(BHB-K). (\u003cstrong\u003eB\u003c/strong\u003e) Site-specific Kbhb levels of histones decreases in NAT10-depleted U2OS cells. Histones were extracted from U2OS cells treated with 10 mM BHB for 8 hours with NAT10 knock down by different siRNA oligos, and Kbhb histones were immunoblotted with site-special BHB-K antibodies. (\u003cstrong\u003eC\u003c/strong\u003e) NAT10-mediated \u003cem\u003ein vitro\u003c/em\u003ehistones Kbhb at multiple lysine sites, which are inhibited by remodelin. Histones were acid-extracted from U2OS cells treated with BHB and subjected to\u003cem\u003ein vitro\u003c/em\u003e β-hydroxybutyrylation assays. “p” indicates acids extract of BHB treated U2OS cells, “Nucleo” indicates mononucleosome, “BHB-CoA” indicates β-hydroxybutyrate-CoA (2.5 mM), “Remo” indicates remodelin (10 μM). (\u003cstrong\u003eD\u003c/strong\u003e) Site-specific Kbhb levels of histones reduces in NAT10\u003cdel\u003e \u003c/del\u003e\u0026nbsp;knockout HeLa cells. CRISPR-Cas9-mediated NAT10 knockout by different sgRNA were performed, and histones extracted from the mixture ofcells treated with 10 mM BHB were analyzed as in (\u003cstrong\u003eB\u003c/strong\u003e). (\u003cstrong\u003eE\u003c/strong\u003e) Wild-type NAT10 rescues histone Kbhb levels. HeLa cells with NAT10 knockout were reconstituted with wild-type or mutant NAT10. Histones were extracted from cells treated with 10 mM BHB and analyzed as in (\u003cstrong\u003eB\u003c/strong\u003e). Note: the Kbhb levels showed the following pattern: significantly higher in lane 6 compared to lane 4, comparable between lanes 8 and 4, and elevated in lane 10 relative to lane 4. (\u003cstrong\u003eF-G\u003c/strong\u003e) Total histone acetylation and Kbhb levels are altered by BHB or remodelin treatments. (\u003cstrong\u003eF\u003c/strong\u003e) Time-course analysis of histone acetylation and Kbhb levels in U2OS cells treated with 10 mM BHB. (\u003cstrong\u003eG\u003c/strong\u003e) Decreases in histone acetylation and Kbhb levels after NAT10 or CBP/p300/HAT1 triple depletion. Histones were analyzed by immunoblotting with pan-acetyl-K and pan-BHB-K antibodies. (\u003cstrong\u003eH\u003c/strong\u003e) Quantitative mass spectrometry analysis of Ac-K of histones. NAT10 kock down or CBP/p300 /HAT1 triple knock down were performed in U2OS cells treated with 10 mM BHB. Histones were analyzed via LC-MS/MS. Data are represented mean ± S.D. from three independent experiments (****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/43cf3140ded102bd4f43d497.jpg"},{"id":87499087,"identity":"41eca698-4e6e-4f91-818f-c1cfc9c9120d","added_by":"auto","created_at":"2025-07-24 13:33:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":159709,"visible":true,"origin":"","legend":"\u003cp\u003eβ-hydroxybutyrylation of NAT10 and histones reduces chromatin-bound NAT10. (\u003cstrong\u003eA-E\u003c/strong\u003e) NAT10 directly interacts with nucleosomes. (\u003cstrong\u003eA\u003c/strong\u003e) GST-NAT10 proteins and mono-nucleosomes were subjected to GST pull down assay. (\u003cstrong\u003eB\u003c/strong\u003e) GST-NAT10 proteins and mono-nucleosomes were subjected to GST pull down assay in different ionic concentration. H2B served as a marker for mono-nucleosomes, detected by H2B immunoblotting. (\u003cstrong\u003eC\u003c/strong\u003e) GST-NAT10 fragments and mono-nucleosomes were subjected to GST pull down assay. s: short exposure, l: long exposure. (\u003cstrong\u003eD\u003c/strong\u003e) GST-NAT10 or Kbhb GST-NAT10 proteins (generated via \u003cem\u003ein vitro\u003c/em\u003e Kbhb modification) and mono-nucleosomes were subjected to GST pull down assay. (\u003cstrong\u003eE\u003c/strong\u003e) GST-NAT10 and Kbhb-modified mono-nucleosomes purified from BHB-treated U2OS cells, were subjected to GST pull down assay. (\u003cstrong\u003eF\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003e-H\u003c/strong\u003e\u003c/del\u003e) Chromatin-bound NAT10 levels decrease with BHB treatment. \u003cdel\u003e(\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e) \u003c/del\u003eU2OS cells treated with 10 mM BHB for 16 hours were fractionated into cytoplasm (cyto), nuclear-soluble (nucl), chromatin-bound (chr) fractions and whole-cell lysate (wcl). Lysates were analyzed by immunoblotting. \u003cdel\u003e(\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eG-H\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e) Chromatin-bound NAT10 levels were assessed under different lysis conditions: RIPA buffer (\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e) and CSK buffer with RNase A (\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e). \u003c/del\u003e(\u003cstrong\u003eG\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/del\u003e) Intensity of chromatin-bound NAT10 reduces with BHB treatment. U2OS cells were treated with 10 mM BHB for different hours and pre-extracted with CSK buffer, and NAT10 intensity was assessed by immunostaining. (\u003cstrong\u003eH\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eJ\u003c/strong\u003e\u003c/del\u003e) Quantitative analysis of chromatin-bound NAT10 intensity from (\u003cstrong\u003eG\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/del\u003e). (\u003cstrong\u003eI\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/del\u003e\u003cstrong\u003e-M\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003c/del\u003e) Genome-wide changes in NAT10 localization upon BHB treatment with ChIP-seq analysis. (\u003cstrong\u003eI\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/del\u003e) NAT10 predominantly localizes to intergenic regions. (\u003cstrong\u003eJ\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003c/del\u003e) Venn diagram of NAT10 ChIP-seq peaks showing decreased and relocalized peaks after 10 mM BHB treatment. (\u003cstrong\u003eK\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003c/del\u003e) Genome browser views of represent region. (\u003cstrong\u003eL\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/del\u003e) heatmaps demonstrate decreased and relocalized NAT10 ChIP-seq signals with BHB treatment. (\u003cstrong\u003eM\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003c/del\u003e) Quantitative analysis confirms reduced NAT10 binding at these regions. \u003cdel\u003e(\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e) Subcellular fraction assay reveals chromatin-bound NAT10 levels decreased following NAT10 knockdown and BHB treatment. U2OS cells were treated with 10 mM BHB and lysed with CSK buffer to separate supernatant (supern) and chromatin fraction (pellet). (\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eQ\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e) Represent images of NAT10 intensity following NAT10 knock down and BHB treatment. Cells with NAT10 knock down, were treated with 10 mM BHB, pre-extracted with CSK buffer, and immunostained with NAT10 antibodies. (\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e) Quantitative analysis of NAT10 intensity from (\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eQ\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e). \u003c/del\u003e(\u003cstrong\u003eN\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/del\u003e\u003cstrong\u003e-P\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/del\u003e) Subcellular fraction assay shows chromatin-bound NAT10 levels decreased. \u003cdel\u003e(\u003c/del\u003e\u003cdel\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/del\u003e\u003cdel\u003e) U2OS were transfected with NAT10 plasmid to overexpress NAT10, treated with 10 mM BHB, and lysed with CSK buffer to seperate supernatant (supern) and chromatin fraction (pellet). \u003c/del\u003e(\u003cstrong\u003eN\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/del\u003e) U2OS cells were treated with 10 mM BHB or 10 uM remodelin, and lysed with CSK buffer. (\u003cstrong\u003eO\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eU\u003c/strong\u003e\u003c/del\u003e) Represent images of chromatin-bound NAT10 intensity. U2OS cells were treated with 10 mM BHB or 10 uM remodelin, pre-extracted with CSK buffer and immunostained with NAT10 antibodies. (\u003cstrong\u003eP\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003c/del\u003e) Quantitative analysis of NAT10 intensity from (\u003cstrong\u003eO\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eU\u003c/strong\u003e\u003c/del\u003e). (\u003cstrong\u003eQ\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eW\u003c/strong\u003e\u003c/del\u003e\u003cstrong\u003e-T\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eX\u003c/strong\u003e\u003c/del\u003e) Chromatin accessibility changes upon BHB treatment with ATAC-seq analysis. (\u003cstrong\u003eQ\u003c/strong\u003e)Volcano plot of chromatin accessibility changes with ATAC-seq analysis. Differential analysis between control siRNA (A) and control siRNA + BHB (B) groups using DESeq2 (Wald test, FDR-adjusted p \u0026lt; 0.0001, |log2FoldChange| \u0026gt;1). Red points indicate 1,043 significantly upregulated peaks (B \u0026gt; A), blue points denote 1,282 downregulated peaks (A \u0026gt; B). Dashed lines mark significance thresholds (FDR \u0026lt;0.05, FoldChange \u0026gt;2). (\u003cstrong\u003eR\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eX\u003c/strong\u003e\u003c/del\u003e) Genome browser views of ATAC-seq signals at Chr2:121,198,971-121,248,090 (hg38). Dashed boxes highlight regions with differential accessibility between groups A (control siRNA), groups B (control siRNA + BHB), groups C (NAT10 siRNA), and groups D (NAT10 siRNA + BHB). (\u003cstrong\u003eS\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eY\u003c/strong\u003e\u003c/del\u003e\u003cstrong\u003e-T\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eZ\u003c/strong\u003e\u003c/del\u003e) Coordinated chromatin accessibility and replication-associated protein binding dynamics. (\u003cstrong\u003eS\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eY\u003c/strong\u003e\u003c/del\u003e) Comparative landscape of chromatin accessibility and protein occupancy. Integrated visualization of: ATAC-seq differential regions (control siRNA vs. siRNA+BHB); ChIP-seq binding changes. Track annotations: Top: ATAC-seq signals (orange: accessibility loss in BHB; purple: gain in BHB); Bottom: ChIP-seq signals (blue: binding reduction; red: binding enhancement). Dashed box highlights loci with concurrent chromatin accessibility shifts (|log2FC| \u0026gt;1) and protein occupancy alterations. (\u003cstrong\u003eT\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eZ\u003c/strong\u003e\u003c/del\u003e) Functional interplay between chromatin structure and protein recruitment. Venn analysis reveals 288 overlapping genomic loci between:2,325 chromatin accessibility changes; 5,466 protein binding alterations. This significant overlap indicates that 12.4% of BHB-induced chromatin structural changes spatially coincide with NAT10 removement. Functional annotation highlights association with DNA metabolic processes. All data are represented mean ± S.D. from three independent experiments (*p \u0026lt; 0.01; **p \u0026lt; 0.05; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Slide5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/e955571c29c430a5f3360441.jpg"},{"id":87499075,"identity":"353f8c68-556f-4524-b7cb-43fcb980b225","added_by":"auto","created_at":"2025-07-24 13:33:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":185238,"visible":true,"origin":"","legend":"\u003cp\u003eNAT10 knockdown or inhibition by remodelin negates the effects of BHB treatment on replication fork dynamics and S-phase progression. (\u003cstrong\u003eA-B\u003c/strong\u003e) Cell viability after NAT10 knockdown or remodelin treatment. (\u003cstrong\u003eA\u003c/strong\u003e) U2OS cells transfected with NAT10 siRNA or (\u003cstrong\u003eB\u003c/strong\u003e) treated with 10 µM remodelin were exposed to 10 mM BHB. Cell viability was assessed via the CCK8 assay. (\u003cstrong\u003eC-D\u003c/strong\u003e) DNA synthesis assessed. U2OS cells were (\u003cstrong\u003eC\u003c/strong\u003e) transfected with NAT10 siRNA or (\u003cstrong\u003eD\u003c/strong\u003e) treated with remodelin and exposed to 10 mM BHB for different durations. EdU-positive cells were quantified by immunostaining. (\u003cstrong\u003eE-F\u003c/strong\u003e) DNA replication completion following NAT10 knockdown or remodelin treatment. U2OS cells were synchronized at the G1/S boundary using thymidine, released into S-phase, and treated with (\u003cstrong\u003eE\u003c/strong\u003e) NAT10 siRNA or (\u003cstrong\u003eF\u003c/strong\u003e) remodelin in the presence of 10 mM BHB. DNA content was analyzed by FACS following PI staining. (\u003cstrong\u003eG-H\u003c/strong\u003e) NAT10 knockdown (\u003cstrong\u003eG\u003c/strong\u003e) or remodelin treatment (\u003cstrong\u003eH\u003c/strong\u003e) mitigates the effect of BHB treatment on replication fork speed and symmetry. Mean fork speed and IdU/CldU ratios were derived from three independent experiments. (\u003cstrong\u003eI-K\u003c/strong\u003e) Representative images and quantitative analyses of DNA fiber analysis from remodelin-treated cells. (\u003cstrong\u003eI\u003c/strong\u003e) Representative DNA fiber images; (\u003cstrong\u003eJ\u003c/strong\u003e) replication fork speed; and (\u003cstrong\u003eK\u003c/strong\u003e) fork symmetry. (\u003cstrong\u003eL-M\u003c/strong\u003e) S-phase progression following NAT10 knockdown. (\u003cstrong\u003eL\u003c/strong\u003e) BrdU and EdU pulse-labeling experimental design and representative images. (\u003cstrong\u003eM\u003c/strong\u003e) Quantitative analysis of S-phase progression based on three independent experiments with n \u0026gt; 250 nuclei. (\u003cstrong\u003eN-O\u003c/strong\u003e) EdU FACS analysis in NAT10 knockdown or remodelin-treated U2OS cells. U2OS cells were treated with NAT10 siRNA, or remodelin (10 uM), and exposed to 10 mM BHB for 8 hours, and 20 µM EdU was added for the last 20 minutes of treatment. (\u003cstrong\u003eN\u003c/strong\u003e) Percentage of S-phase cells. (\u003cstrong\u003eO\u003c/strong\u003e) Fold changes in EdU incorporation in early and late S-phases post-release. All data are represented mean ± S.D. from three independent experiments (*p \u0026lt; 0.01; **p \u0026lt; 0.05; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Slide6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/d430749636f209ca435bbd6b.jpg"},{"id":87499071,"identity":"2e179b6e-09a5-4d91-b32f-ae41f704670b","added_by":"auto","created_at":"2025-07-24 13:33:43","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":133545,"visible":true,"origin":"","legend":"\u003cp\u003eNAT10 knockdown or inhibition by remodelin negates the effects of BHB treatment on replication stress and genomic instability. \u003cstrong\u003e(A-E)\u003c/strong\u003eReplication stress is alleviated by NAT10 knockdown or remodelin treatment. (\u003cstrong\u003eA\u003c/strong\u003e) Quantification of cyclin A-positive cells with \u0026gt;5 γH2AX foci following 10 mM BHB treatment. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of cyclin A-positive cells with \u0026gt;5 BrdU foci under non-denaturing conditions. (\u003cstrong\u003eC-D\u003c/strong\u003e) Western blot analysis of CHK1 and phosphorylated S317-CHK1 (\u003cstrong\u003eC\u003c/strong\u003e), and chromatin-bound RPA2, and γH2AX (\u003cstrong\u003eD\u003c/strong\u003e) in U2OS cells with NAT10 knockdown. (\u003cstrong\u003eE-F\u003c/strong\u003e) Quantification of RPA2 intensity (\u003cstrong\u003eE\u003c/strong\u003e) and γH2AX (\u003cstrong\u003eF\u003c/strong\u003e) from immunofluorescence experiments. (\u003cstrong\u003eG-K\u003c/strong\u003e) NAT10 knockdown or remodelin treatment mitigates genomic instability. (\u003cstrong\u003eG\u003c/strong\u003e) Quantification of micronuclei. (\u003cstrong\u003eH\u003c/strong\u003e) Quantification of mitotic chromosome bridges. (\u003cstrong\u003eI\u003c/strong\u003e) Quantification of chromosomal breaks per spread. (\u003cstrong\u003eJ\u003c/strong\u003e) Representative images of EdU incorporation on metaphase chromosomes. (\u003cstrong\u003eK\u003c/strong\u003e) Quantitative analysis of EdU incorporation on metaphase chromosomes. All data are represented mean ± S.D. from three independent experiments (*p \u0026lt; 0.01; **p \u0026lt; 0.05; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Slide7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/2676aee025abd88e9348fad0.jpg"},{"id":87499089,"identity":"f1d2489d-3303-4157-9eee-5ce65a332401","added_by":"auto","created_at":"2025-07-24 13:33:43","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":122759,"visible":true,"origin":"","legend":"\u003cp\u003eNAT10-mediated β-hydroxybutyrylation accelerates replication, reduces replication stress, and mitigates genomic instability in WI-38 cells. (\u003cstrong\u003eA\u003c/strong\u003e) Representative DNA fiber images in NAT10-depleted cells treated with 10 mM BHB. Cells were pulse-labeled with 25 µM CldU for 20 minutes, followed by 250 µM IdU for another 20 minutes. DNA fibers were spread and visualized. Scale bar: 10 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of replication fork speed from (\u003cstrong\u003eA\u003c/strong\u003e). (\u003cstrong\u003eC\u003c/strong\u003e) DNA fiber analysis shows replication fork symmetry from (\u003cstrong\u003eA\u003c/strong\u003e). IdU/CldU ratios were calculated from values in (\u003cstrong\u003eB\u003c/strong\u003e). (\u003cstrong\u003eD\u003c/strong\u003e) Representative images of BrdU incorporation in cyclin A-positive cells under non-denaturing conditions. (\u003cstrong\u003eE\u003c/strong\u003e) Quantification of cyclin A-positive cells with \u0026gt;5 BrdU foci. (\u003cstrong\u003eF\u003c/strong\u003e) Quantification of micronuclei. Cells, with NAT10 knockdown or 10 µM remodelin, were exposed to 10 mM BHB for 24 hours and cultured in medium containing 2 mg/mL cytochalasin B for 16 hours. (\u003cstrong\u003eG\u003c/strong\u003e) Quantification of mitotic chromosome bridges. Cells, with NAT10 knockdown or 10 µM remodelin, were synchronized with 9 mM RO-3306 for 16 hours, released into medium for 1.5 hours. (\u003cstrong\u003eH\u003c/strong\u003e) Quantification of chromosomal breaks per chromosome spread. Cells, with NAT10 knockdown or 10 µM remodelin, were exposed to 0.2 µM aphidicolin (APH) for 24 hours and 200 ng/mL nocodazole for the last 5 hours. (\u003cstrong\u003eI\u003c/strong\u003e) Quantification of EdU incorporation on metaphase chromosomes. Cells, with NAT10 knockdown or 10 µM remodelin, were labeled with 10 µM EdU for 45 minutes, exposed to 0.4 µM APH for 24 hours, and treated with 200 ng/mL nocodazole for the last 5 hours. All data are represented mean ± S.D. from three independent experiments (*p \u0026lt; 0.01; **p \u0026lt; 0.05; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Slide8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/81e8adb614d7400c9f447669.jpg"},{"id":87499085,"identity":"d9092e2d-a762-4d40-bfc5-27c040b8c239","added_by":"auto","created_at":"2025-07-24 13:33:43","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":133520,"visible":true,"origin":"","legend":"\u003cp\u003eNAT10-mediated β-hydroxybutyrylation accelerates replication, reduces replication stress, and mitigates genomic instability in 2BS cells. (\u003cstrong\u003eA\u003c/strong\u003e) Representative DNA fiber images analysis in control or NAT10-depleted cells treated with 10 mM BHB. Cells were pulse-labeled with 25 µM CldU for 20 minutes, followed by 250 µM IdU for another 20 minutes. DNA fibers were spread and visualized. Scale bar: 10 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of replication fork speed from (\u003cstrong\u003eA\u003c/strong\u003e). (\u003cstrong\u003eC\u003c/strong\u003e) DNA fiber analysis shows replication fork symmetry treated from(\u003cstrong\u003eA\u003c/strong\u003e). IdU/CldU ratios were calculated from values in (\u003cstrong\u003eB\u003c/strong\u003e). (\u003cstrong\u003eD\u003c/strong\u003e) Representative images of BrdU incorporation in cyclin A-positive cells under non-denaturing conditions. (\u003cstrong\u003eE\u003c/strong\u003e) Quantification of cyclin A-positive cells with \u0026gt;5 BrdU foci. (\u003cstrong\u003eF\u003c/strong\u003e) Quantification of micronuclei. Cells, with NAT10 knockdown or 10 µM remodelin, were exposed to 10 mM BHB for 24 hours and cultured in medium containing 2 mg/mL cytochalasin B for 16 hours. (\u003cstrong\u003eG\u003c/strong\u003e) Quantification of mitotic chromosome bridges. Cells, with NAT10 knockdown or 10 µM remodelin, were synchronized with 9 mM RO-3306 for 16 hours, released into medium for 1.5 hours. (\u003cstrong\u003eH\u003c/strong\u003e) Quantification of chromosomal breaks per chromosome spread. Cells, with NAT10 knockdown or 10 µM remodelin, were exposed to 0.2 µM aphidicolin (APH) for 24 hours and 200 ng/mL nocodazole for the last 5 hours. (\u003cstrong\u003eI\u003c/strong\u003e) Quantification of EdU incorporation on metaphase chromosomes. Cells, with NAT10 knockdown or 10 µM remodelin, were labeled with 10 µM EdU for 45 minutes, exposed to 0.4 µM APH for 24 hours, and treated with 200 ng/mL nocodazole for the last 5 hours. All data are represented mean ± S.D. from three independent experiments (*p \u0026lt; 0.01; **p \u0026lt; 0.05; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Slide9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/4b76c9711718c6d0b3295e96.jpg"},{"id":94473122,"identity":"b3140a83-6528-4a4f-9e32-59921f9dfaf2","added_by":"auto","created_at":"2025-10-27 15:42:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2718599,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/afff35d0-0adc-4970-bdce-a5f07016e633.pdf"},{"id":87499078,"identity":"80768e37-7e13-40b2-af44-31e798117a04","added_by":"auto","created_at":"2025-07-24 13:33:43","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":952777,"visible":true,"origin":"","legend":"Supplementary Table 1_OriginFiringFactors-MS","description":"","filename":"SupplementaryTable1OriginFiringFactorsMS.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/c2e18b55ab5cf4eddb884469.xlsx"},{"id":87499091,"identity":"8c5eebcd-15d6-4c4c-b90f-bc886fdfba01","added_by":"auto","created_at":"2025-07-24 13:33:43","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":228234,"visible":true,"origin":"","legend":"Supplementary Tabl2 2_AcetylatedHistones-MS","description":"","filename":"SupplementaryTable2AcetylatedHistonesMS.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/218a6cdba83d66e4786bc13c.xlsx"},{"id":87500089,"identity":"3bbf4a4c-44b6-4db1-9a52-d0c5fb4dca83","added_by":"auto","created_at":"2025-07-24 13:41:44","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14208290,"visible":true,"origin":"","legend":"Supplementary","description":"","filename":"SupplementaryV96.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7110451/v1/b5d2cb0a9a0aef7919a724dc.pptx"}],"financialInterests":"There is no duality of interest","formattedTitle":"NAT10-mediated β-hydroxybutyrylation Affects DNA Replication","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHistone posttranslational modifications (PTM) are critical regulators of DNA replication in eukaryotic cells, influencing chromatin structure and replication timing\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Among these, histone acetylation and methylation are particularly well-studied, with high levels of acetylation accelerating replication in distinct genomic regions\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Open chromatin marks, such as H3K4me1/2/3, H3K9ac, H3K18ac and H3K27ac are consistently enriched in early-replicating regions, highlighting their pivotal role in replication process\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eLysine β-hydroxybutyrylation (Kbhb), a recently identified histone PTM, may play a role in DNA replication. Kbhb is derived from β-hydroxybutyrate (BHB), a key component of ketone bodies, which is converted into β-hydroxybutyryl-CoA to provide β-hydroxybutyryl group to conjugate to lysine. Kbhb is a conserved PTM observed across species from yeast to human\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The identified histone Kbhb sites in mice and human include H3K4, H3K9, H3K18 and H3K27 lysine residues, whose acetylation marks open chromatin structure and early replication genomic regions\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. H3K9bhb is associated with upregulation of genes involved in starvation-responsive pathways\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. H3K56bhb colocalized at the super-enhancer regions with coactivators such as BRD4\u003csup\u003e12\u003c/sup\u003e. These clues imply that histone Kbhb may affect DNA replication. Until now, there is lacking experimental evidence linking histone Kbhb to DNA replication.\u003c/p\u003e\u003cp\u003eN-acetyltransferase 10 (NAT10) is a candidate regulator of Kbhb and DNA replication. NAT10, as a histone acetyltransferases (HATs), catalyzes RNA acetylation (N4-acetylation of Cytidine of RNA, ac4C) and lysine acetylation (acetylation of histones, UBF1, p53, etc.)\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. NAT10 has 2-hydroxyisobutyrylation (Khib), a modification with a chemical structure closely resembling to Kbhb\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Other HATs, such as CBP/p300, exhibit lysine β-hydroxybutyryl-transferase activity\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Those clues raise the possibility that NAT10 also catalyze Kbhb besides Kac and ac4C. Furthermore, NAT10 is enriched in mitotic chromosome scaffold fraction and play critical roles in genome integrity, bipolar assembly, and chromatin segregation during mitosis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. NAT10 depletion or inhibition activity promote DNA replication\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. These clues suggest NAT10 might regulate DNA replication. However, direct evidence linking NAT10 to Kbhb modification and its functional implication in DNA replication has remained elusive.\u003c/p\u003e\u003cp\u003eHere, we demonstrate that NAT10 as a β-hydroxybutyryl-transferase catalyzes Kbhb at multiple lysine residues of histones, thereby facilitating DNA replication, lower replication stress and better maintenance of genomic stability. Mechanistically, we show that NAT10-mediated Kbhb enhances replication dynamics by accelerating replication fork speed and shortening the S phase duration. NAT10-mediated Kbhb results in lower replication stress, as evidenced by significant reductions in single-stranded DNA (ssDNA) accumulation, γH2AX foci formation, RPA2 foci localization, and phospho-S317 CHK1 levels. NAT10-mediated Kbhb possibly leads to preserved genomic instability, as indicted by lower frequency of micronuclei, chromosome bridge and DNA breakage occurrence. Notably, we find that NAT10 depletion or NAT10-mediated Kbhb of itself dynamically reduces chromatin-bound NAT10 and chromatin accessibility, highlighting its regulatory role in chromatin structure. Our findings provide direct evidence linking NAT10 to histone Kbhb and uncover its critical role in promoting DNA replication, reducing replication stress, and preserving genomic stability. These insights establish a previously unrecognized regulatory pathway connecting metabolic signaling, histone modification, and DNA replication.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eBHB treatment accelerates DNA replication\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the impacts of sodium β-hydroxybutyrate (BHB) on cellular processes, we first assessed its impact on U2OS cell viability. CCK8 assays revealed a dose-dependent enhancement in cell viability \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, suggesting that BHB positively influences cell growth. Subsequent flow cytometric analysis of synchronized cells released from G1/S boundary arrest demonstrated accelerated S-phase progression under BHB treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo explore the effects of BHB on DNA replication, we conducted DNA fiber analysis to evaluate replication fork progression. Single-molecule DNA fiber assays revealed a significant increase in replication fork velocity with preserved fork symmetry in BHB-treated cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Dual-pulse BrdU/EdU labeling corroborated these findings, showing redistribution of S-phase populations toward early-middle and middle-late replication stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Temporal EdU FACS quantification analysis revealed that 8-hour BHB treatment transiently elevated both the proportion of S-phase cell and EdU incorporation intensity. These effects progressively diminished at 16\u0026ndash;24 hours but persisted when cells were released into fresh media post-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM, \u003cb\u003eSupplementary Fig.\u0026nbsp;1A-1B\u003c/b\u003e). These findings indicate that BHB as a temporal accelerator of S-phase progression, operating within a defined, time-restricted window of EdU labeling.\u003c/p\u003e\u003cp\u003eTo determine whether BHB impacts replication origin firing\u0026mdash;a critical regulatory step in DNA replication dynamics\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, we analyzed chromatin-bound replication initiation complexes by intensity-based absolute quantification (iBAQ) mass spectrometry. Quantitative proteomics revealed no significant changes in replication initiation factor abundance following BHB treatment \u003cb\u003e(Supplementary Fig.\u0026nbsp;1C-1E)\u003c/b\u003e, demonstrating that BHB accelerates DNA replication without significantly impacting origin firing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBHB treatment reduces replication stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAccelerated DNA replication fork progression is known to induces replication stress and genomic instability\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Therefore, we further evaluated whether the BHB-induced replication acceleration results in replication stress. Initial evaluation of γH2AX foci formation in S-phase cells demonstrated a significant decrease following BHB treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Similarly, non-denaturing BrdU incorporation assays showed a reduction in ssDNA-positive S-phase populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Concordant reductions in γH2AX and RPA2 signal intensities were observed by immunostaining analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). Western blot analysis of S317-CHK1 phosphorylation, γH2AX, and RPA2 levels confirmed these observations, showing marked reductions following BHB treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Collectively, these data demonstrate that BHB enhances DNA replication efficiency \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e while simultaneously alleviating replication stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBHB treatment reduces genomic instability\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that rapid replication fork progression can induce genomic instability\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, manifesting as micronuclei formation, sister-chromatid bridges, and chromatid breaks or gaps during mitosis\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. We assessed whether BHB-induced acceleration exacerbates these defects. We performed immunostaining analysis to measure the frequency of the DAPI-stained micronuclei and anaphase-bridge in BHB-treated U2OS cells. BHB treatment modestly reduced the incidence of micronuclei and anaphase-bridges (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-K). Furthermore, under genomic instability induced by low-dose (0.2 \u0026micro;M) aphidicolin (APH)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, BHB-treated cells exhibited fewer chromatid breaks and gaps compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL).\u003c/p\u003e\u003cp\u003eThese findings demonstrate that BHB promotes DNA replication by accelerating replication fork progression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e while concurrently suppressing both replication stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) and genomic instability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL), thereby preserving genomic integrity despite accelerated replication dynamics.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNAT10 is a β-hydroxybutyryl-transferase\u003c/b\u003e\u003c/p\u003e\u003cp\u003eβ-Hydroxybutyrate (BHB), the main component of ketone body, predominantly synthesized during hepatic fatty acid oxidation, circulates systemically to serve as an energy substrate through conversion to acetyl-CoA and eventually to ATP. Beyond its metabolic role, BHB acts as a signaling metabolite via β-hydroxybutyryl-CoA-mediated β-hydroxybutyryl group to conjugate to lysine residue (Kbhb), a protein posttranslational modification\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGiven that BHB treatment accelerates DNA replication and reduces replication stress and genomic instability, and that similar dosage of BHB induce widespread histone Kbhb in human cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, we hypothesized that BHB affects replication processes through Kbhb modification. We performed LC-MS/MS analysis of protein extracts from BHB-treated U2OS cells, identified NAT10 as a Kbhb-modified protein at lysine 989 (K989), alongside known acetyltransferases CBP, p300, and HAT1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven that CBP/p300-mediated Kbhb of histones and p53 is established\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, CBP/p300 are known to exhibit diverse of acyl-transferase activities\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and NAT10 previously linked to lysine 2-hydroxyisobutyrylation (Khib) modification\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, we predictes that NAT10 is a potential β-hydroxybutyryl-transferase. To validate this, we overexpressed ten Flag-tagged HATs in 293T cells under BHB treatment. The proteins were purified and enriched under very stringent condition by immunoprecipitation (IP) with M2 beads, and detected using a pan anti-β-hydroxybutyrylation antibody (BHB-K). The results confirmed that CBP, p300, HAT1, and NAT10 are Kbhb proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We also purified and enriched the Kbhb proteins by IP with BHB-K antibody from BHB treated cells lysate, and detected endogenous NAT10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eWe next interrogated NAT10\u0026rsquo;s catalytic mechanism. Studies implicate G641 as essential for NAT10\u0026rsquo;s acetyltransferase activity, which is inhibited by remodelin\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. To verify whether G641 is also critical for β-hydroxybutyryl-transferase activity of NAT10, whether K989 is the key Kbhb site and whether the Khib site K823 affects its Kbhb activity, we performed \u003cem\u003ein vitro\u003c/em\u003e β-hydroxybutyrylation assays with the purified NAT10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The results demonstrated that wild-type NAT10 and NAT10/K823R mutants retained activity, whereas NAT10/K989R and NAT10/G641E mutants abolished Kbhb modification \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Remodelin treatment similarly suppressed β-hydroxybutyryl-transferase activity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTo corroborate these findings, we performed \u003cem\u003ein vivo\u003c/em\u003e β-hydroxybutyrylation assays by expressing Flag-tagged NAT10 wild-type and mutants in U2OS cells. Cells were treated with BHB and remodelin, and proteins were purified and enriched under very stringent condition by IP with M2 beads, and detected using BHB-K (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The \u003cem\u003ein vivo\u003c/em\u003e results were consistent with the \u003cem\u003ein vitro\u003c/em\u003e findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eTogether, these results indicated that NAT10 is a β-hydroxybutyryl-transferase.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNAT10 β-hydroxybutyrylates histones at multiple lysine sites\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNAT10, predominantly localized in the nucleolar\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, was investigated for its potential role in histone β-hydroxybutyrylation (Kbhb). We depleted NAT10 in U2OS cells treated with BHB using a pool of siRNA oligonucleotides. Immunoblotting analysis of extracted histones using an BHB-K antibody revealed a significant reduction in total levels of Kbhb histones in NAT10-depleted cells compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To further verify the role of NAT10 in histone Kbhb, we conducted additional NAT10 knockdown experiments with distinct siRNA oligonucleotides in U2OS cells. Immunoblotting with site-specific BHB-K antibodies demonstrated reduction in Kbhb levels at multiple lysine sites, except H3K14bhb and H4K8bhb no changed, in NAT10-depleted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe performed \u003cem\u003ein vitro\u003c/em\u003e β-hydroxybutyrylation assay. The purified recombinant human NAT10 expressed in 293T cells was incubated with mononucleosome in the presence of BHB-CoA in an \u003cem\u003ein vitro\u003c/em\u003e Kbhb assay. With wildtype NAT10, histone Kbhb was detected at multiple lysine residues, whereas addition of remodelin in the assay completely inhibited histone Kbhb. Furthermore, with NAT10/K989R mutant, the levels of Kbhb histone were reduced. Finally, with NAT10/G641E inactive mutant, Kbhb histones was completely abolished (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The \u003cem\u003ein vitro\u003c/em\u003e assay confirmed the previous findings on NAT10 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo validate the β-hydroxybutyryl-transferase activity of endogenous NAT10, we employed a CRISPR-Cas9 system to knock out NAT10 in HeLa cells transiently, as previously described\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Histones extracted from the pool of NAT10 knockout cells showed significantly lower Kbhb levels at multiple lysine residues, except H3K14bhb and H4K8bhb no changed, compared to histones from parental HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). To further confirm this activity, rescue experiments were conducted by re-expressing wild-type NAT10, NAT10/G641E, or NAT10/K989R in NAT10 knockout cells. Only wild-type NAT10 restored histone Kbhb levels in the presence of BHB, while neither the G641E nor K989R mutants were able to recover histone Kbhb (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eWe analyzed the global patterns of acetylated and β-hydroxybutyrylated proteins in whole-cell lysates using pan-acetylation and pan-Kbhb antibodies, respectively. With increasing durations of BHB treatment, total Kbhb levels in whole-cell lysates were significantly elevated, while global protein acetylation levels remained largely unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). NAT10 depletion selectively reduced histone Kbhb, whereas CBP/p300/HAT1 triple knockdown showed minimal effect. Neither perturbation influenced acetylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Quantitative histone mass spectrometry analysis further established NAT10\u0026rsquo;s essential role in regulating Kbhb. Notably, NAT10 does not modulate H3K24ac, H4K6ac, H4K13ac, or H4K17ac upon BHB treatment. Instead, CBP/p300/HAT1 are the key mediators of H4K6ac, H4K13ac, and H4K17ac in response to BHB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, \u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eNAT10-mediated β-hydroxybutyrylation dynamically reduces chromatin-bound NAT10 and alteres chromatin accessibility\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that NAT10 mediates histone Kbhb at multiple lysine sites, we investigated the mechanism deeply of this modification on NAT10 and chromatin interaction and chromatin accessibility.\u003c/p\u003e\u003cp\u003eTo determine whether Kbhb affects NAT10 binding to chromatin, we performed GST pull-down assays. The results demonstrated that NAT10 directly interacts with nucleosomes, and both Kbhb NAT10 and Kbhb histones exhibited significantly reduces interactions with nucleosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Subcellular fractionation showed BHB treatment selectively reduced chromatin-associated NAT10 levels while leaving whole-cell lysates and soluble nuclear pools unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Immunostaining of nuclei pre-extracted to eliminate RNA-bound and free NAT10 confirmed diminished chromatin occupancy of NAT10 post-BHB treatment, despite preserved nucleolar localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). ChIP-seq analysis demonstrated NAT10 enrichment in intergenic regions under basal conditions, with a two-thirds reduction in binding peaks following BHB treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). These finding consistent with the reduction of chromatin-bound NAT10 observed in immunoblotting and immunostaining experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo explore whether the BHB-induced reduction of chromatin-bound NAT10 is caused by NAT10-mediated β-hydroxybutyrylation, we used remodelin to inhibit NAT10 activity. Subcellular fractionation and immunostaining of chromatin-bound NAT10 in nuclei pre-extracted with CSK buffer showed that BHB treatment and NAT10 inhibition individually caused a modest reduction in chromatin-bound NAT10. However, the reduction observed in NAT10-inhibited cells treated with BHB was not significantly greater than in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eP). Importantly, the BHB-induced reduction in chromatin-bound NAT10 was reversed by pre-treatment with remodelin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eP). These results indicate that both NAT10 depletion and NAT10-mediated β-hydroxybutyrylation negatively regulate NAT10 binding to chromatin \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTo further explore the effects of BHB treatment on chromatin architecture, we conducted ATAC-seq to assess chromatin accessibility. Differential chromatin accessibility regions were identified by comparing the following groups: group A, cells treated with control siRNA; group B, cells treated with control siRNA and BHB; group C, cells treated with NAT10 siRNA; and group D, cells treated with NAT10 siRNA and BHB (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and fold change\u0026thinsp;\u0026gt;\u0026thinsp;2). Among these regions, 1043 were up-regulated in group B compared to group A, of which 398 (38.2%) exhibited a normalized average count of 0 in group B. Additionally, 1282 regions were down-regulated in group B compared to group A, with 445 (44.9%) showing a normalized average count of 0 in group A (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, fold change\u0026thinsp;\u0026gt;\u0026thinsp;2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eQ-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eT, \u003cb\u003eSupplementary Fig.\u0026nbsp;4A-4H\u003c/b\u003e). Importantly, the differential chromatin accessibility regions observed between groups B and A (1043 up-regulated and 1282 down-regulated) were not detected between groups D and C, indicating that the BHB-induced changes in chromatin accessibility are dependent on NAT10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eW-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eX, \u003cb\u003eSupplementary Fig.\u0026nbsp;4A-4H\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo investigate the association between chromatin accessibility dynamics and protein-DNA interactions, we performed integrated analysis of ATAC-seq and ChIP-seq datasets. Technical divergence in sequencing read lengths between these methodologies (ATAC-seq vs ChIP-seq) necessitated length-normalized comparison, restricting direct analysis to regions with equivalent peak widths. Cross-platform evaluation identified 288 overlapping genomic loci from 2,325 chromatin accessibility changes and 5,466 protein binding alterations, representing a 12.4% spatial concordance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eS-T; \u003cb\u003eSupplementary Fig.\u0026nbsp;4A-4H\u003c/b\u003e). These findings suggest coordinated chromatin restructuring and replication complex recruitment during BHB exposure. Further analysis demonstrated that NAT10-mediated Kbhb modulates chromatin occupancy patterns, with reduced NAT10 binding correlating spatially with differential ATAC-seq accessibility regions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNAT10-mediated β-hydroxybutyryltion accelerates DNA replication\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrevious results demonstrated that BHB treatment accelerates DNA replication, induces NAT10-mediated Kbhb at multiple lysine sites on histones, concurrently reducing chromatin-bound NAT10 and altering chromatin accessibility. We further investigated whether the effects of BHB-induced β-hydroxybutyrylation on DNA replication require NAT10 activity.\u003c/p\u003e\u003cp\u003eBHB increased cell viability in control U2OS cells, consistent with replication enhancement. However, NAT10 depletion or remodelin treatment abolished this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Immunostaining revealed that BHB-induced increases the proportion of EdU-positive cells, indicative of enhanced DNA synthesis. This increase was absent in NAT10-knockdown or remodelin-treated cells, further implicating NAT10 in mediating this response (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e). Flow cytometry-based DNA content analysis showed BHB-treated controls completed replication earlier than untreated cells, whereas NAT10-depleted or remodelin-treated cells exhibited no such acceleration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Consistently, DNA fiber assays confirmed BHB increased replication fork speed and symmetrical fork progression in controls, effects attenuated by NAT10 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Dual-pulse BrdU/EdU labeling demonstrated BHB promoted early-to-late S-phase transition in controls, but NAT10 depletion or remodelin treatment failed to recapitulate this progression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL-M\u003cb\u003e)\u003c/b\u003e. Similarly, BHB enhanced both S-phase cell proportion and EdU incorporation intensity in controls, with no changes observed in NAT10-deficient cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN-O, \u003cb\u003eSupplementary Fig.\u0026nbsp;6)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings establish NAT10-mediated β-hydroxybutyrylation plays a pivotal role in BHB-driven replication acceleration. Increased fork velocity, S-phase progression, and replication timing advance are strictly dependent on NAT10 activity, mechanistically linking β-hydroxybutyrylation to replication dynamics.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNAT10-mediated β-hydroxybutyrylation reduces replication stress and genomic instability\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate whether NAT10 mediates BHB-induced suppression of replication stress and genomic instability, we analyzed these phenotypes in NAT10-depleted or remodelin-treated U2OS cells under BHB treatment.\u003c/p\u003e\u003cp\u003eBHB treatment reduced the proportion of γH2AX foci-positive S-phase cells\u0026mdash;a replication stress marker\u0026mdash;in control cells, whereas NAT10-depleted cells showed no such reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Similarly, BrdU-based quantification of ssDNA-positive S-phase cells revealed decreased replication stress in BHB-treated controls but not in NAT10-depleted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Western blot analysis further revealed that replication stress markers, including phosphorylated S317-CHK1, γH2AX, and RPA2, were reduced in control cells following BHB treatment. These effects were abolished in NAT10-depleted cells regardless of BHB treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Notably, NAT10 depletion alone increased replication stress, as evidenced by unchanged or mildly elevated levels of these markers compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Consistent with these, immunostaining showed diminished RPA2 and γH2AX signals in BHB-treated control cells but not in NAT10-depleted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, \u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e). Strikingly, NAT10 depletion alone further increased RPA2 and γH2AX signals, while remodelin plus BHB treatment had no effect compared to controls, which reinforce its role in promoting replication stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Together, these results demonstrate that NAT10-mediated β-hydroxybutyrylation reduces replication stress, but NAT10 depletion alone enhances it.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate genomic stability, we examined chromosomal abnormalities. BHB treatment reduced the incidence of micronuclei and anaphase bridges in control cells, whereas NAT10 depletion or remodelin treatment abrogated these protective effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). BHB treatment also decreased chromosomal aberrations induced by low-dose aphidicolin (APH). In control cells, the frequency of chromatid breaks and gaps was significantly reduced following BHB treatment. This protection was lost in NAT10-depleted or remodelin-treatment-treated cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). EdU incorporationon metaphase chromosome spreads, reduced by BHB in control cells, remained unchanged in NAT10-depleted or remodelin-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK).\u003c/p\u003e\u003cp\u003eThese findings demonstrate that NAT10-mediated β-hydroxybutyrylation is essential for BHB\u0026rsquo;s suppression of replication stress and genomic instability. Genetic or pharmacological inhibition of NAT10 abolishes these protective effects of BHB treatment, highlighting the essential function of NAT10 in mediating these responses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNAT10-mediated β-hydroxybutylation accelerates replication, reduces replication stress and genomic instability in WI-38 cells and in 2BS cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe further explored the role of NAT10-mediated β-hydroxybutyrylation in the primary human cell lines WI-38 and 2BS, in addition to the previously studied U2OS cells, a human osteosarcoma cell line. In WI-38 cells, BHB treatment significantly increased replication fork speed and maintained replication fork symmetry, as demonstrated by DNA fiber assays. BrdU incorporation analysis under non-denaturing conditions revealed a reduction in ssDNA levels, indicating decreased replication stress. Similarly, NAT10 knockdown or inhibition by remodelin abolished the protective effects of BHB, as evidenced by increased micronuclei formation, anaphase bridges, and chromosomal aberrations, including broken chromosomes and EdU incorporation events on metaphase chromosome spreads (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI, \u003cb\u003eSupplementary Fig.\u0026nbsp;8\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn 2BS cell, consistent results were observed. BHB treatment accelerated replication fork progression and preserved fork symmetry, while ssDNA levels were reduced, indicating alleviation of replication stress. NAT10 depletion or inhibition negated these effects, leading to increased genomic instability marker, such as micronuclei, mitotic chromosome bridges, and chromatid breaks. Additionally, EdU incorporation analysis revealed that the BHB-induced reduction in replication-associated abnormalities was dependent on NAT10 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI, \u003cb\u003eSupplementary Fig.\u0026nbsp;9\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCollectively, these findings demonstrate that NAT10-mediated β-hydroxybutyrylation plays a critical role in reducing replication stress and genomic instability. Knockdown of NAT10 or its inhibition by remodelin negates the protective effects of BHB treatment, highlighting the essential function of NAT10 in mediating these responses.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe faithful execution of DNA replication is fundamental to cellular homeostasis, and disruptions in replication dynamics can lead to replication stress and genomic instability, hallmarks of cancer, aging, and various metabolic disorders\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In this study, we observe that NAT10-mediated β-hydroxybutyrylation appears to function as an epigenetic mechanism that supports DNA replication, while potentially reducing replication stress and genomic instability. These effects seem to occur not only in cancer cells (U2OS) but also in primary human fibroblasts (WI-38 and 2BS), suggesting NAT10 may play a role in replication dynamics across different cell types. Our data indicate that NAT10 may modify histones through β-hydroxybutyrylation, possibly influencing chromatin accessibility to facilitate DNA synthesis and help manage replication stress. When NAT10 is depleted or inhibited, these effects appear to be diminished, which may point to its involvement in replication homeostasis. These findings could contribute to our understanding of how metabolic signaling, chromatin structure, and genome stability interact, with NAT10 potentially being one factor in replication regulation and cellular stress responses.\u003c/p\u003e\u003cp\u003eNAT10-mediated β-hydroxybutyrylation appears to be a potential regulator of replication efficiency and genome stability. Our findings suggest that NAT10-mediated β-hydroxybutyrylation may represent a previously unexplored chromatin modification that could influence replication efficiency. DNA fiber assays indicated that BHB-induced β-hydroxybutyrylation might be associated with faster replication fork progression while preserving fork symmetry, which is often linked to regulated replication dynamics. This effect appears to rely on NAT10 activity, as both genetic depletion and chemical inhibition (e.g., remodelin treatment) reduced the observed replication acceleration. In contrast to histone acetylation, which is primarily known to open chromatin for transcription, β-hydroxybutyrylation might modulate chromatin accessibility in a way that supports replication origin licensing and firing. These differences could imply that β-hydroxybutyrylation contributes to a distinct form of epigenetic regulation, possibly specialized for replication-associated chromatin remodeling.\u003c/p\u003e\u003cp\u003eFurthermore, our results suggest that NAT10-mediated β-hydroxybutyrylation may help reduce replication stress by stabilizing replication forks and decreasing ssDNA accumulation. In BHB-treated cells, we observed lower levels of replication stress markers such as γH2AX, phosphorylated CHK1, and RPA2, though these effects were not seen when NAT10 was knocked down or inhibited. The observed decrease in ssDNA, along with preserved replication fork symmetry, could indicate that β-hydroxybutyrylation influences chromatin structure in ways that might limit excessive fork stalling or collapse. Since replication stress contributes to genomic instability, these findings raise the possibility that NAT10 could play a role in maintaining genomic stability during replication.\u003c/p\u003e\u003cp\u003eNAT10 as a potential chromatin organizer: Implications for genome stability and human disease. Chromatin structure is dynamically regulated during replication, ensuring that DNA synthesis proceeds efficiently while maintaining accessibility for replication and repair factors. Our results imply that NAT10 may contribute to this process by modifying histones, which could affect chromatin-bound protein interactions and replication-associated chromatin remodeling. ChIP-seq and ATAC-seq data indicate that NAT10-mediated β-hydroxybutyrylation might influence chromatin accessibility in response to BHB treatment, potentially creating conditions favorable for replication progression. It's worth noting that NAT10 depletion appeared to reduce chromatin-bound NAT10 levels, possibly pointing to an autoregulatory mechanism where β-hydroxybutyrylation helps regulate NAT10-chromatin interactions to maintain balanced replication activity.\u003c/p\u003e\u003cp\u003eThese findings could have implications for human diseases involving chromatin dysregulation and replication stress. In aging cells, replication stress tends to accumulate, possibly due to less efficient origin activation, fork stalling, and compromised repair pathways. Since WI-38 and 2BS cells are commonly used as models of cellular senescence, our observation that NAT10 appears to reduce replication stress and genomic instability in these cells raises the possibility that β-hydroxybutyrylation might play a role in mitigating age-related replication defects. If further validated, modulating NAT10 function could be explored as a potential strategy to address replication stress in aging and age-related conditions.\u003c/p\u003e\u003cp\u003eThese findings appear to align with prior studies suggesting NAT10's involvement in chromatin structure during mitosis, particularly in Hutchinson-Gilford Progeria Syndrome (HGPS). NAT10 may contribute to maintaining genome integrity during mitosis, possibly helping preserve genetic fidelity, though this effect has been primarily linked to NAT10's acetylation of CCDC84 and Eg5, which are involved in centrosome duplication\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Additionally, some studies have reported that depleting NAT10 reduces DNA damage (γH2AX) accumulation in Lmna\u003csup\u003eG609G\u003c/sup\u003e HGPS mouse models and HGPS patient-derived cells, which often exhibit various genetic abnormalities and nuclear deformities\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn the context of cancer, replication stress is a double-edged sword. While excessive replication stress drives genomic instability, which fuels tumorigenesis, controlled modulation of replication stress can be exploited for cancer therapy. Our observations raise the possibility that NAT10 overexpression could potentially support cancer cell proliferation by alleviating replication stress. On the other hand, inhibiting NAT10 might preferentially elevate replication stress in malignant cells, possibly making them more vulnerable to agents like ATR or CHK1 inhibitors. These findings could open avenues for exploring therapeutic approaches that leverage NAT10 activity to differentially target cancer cells while maintaining normal cellular replication.\u003c/p\u003e\u003cp\u003eBeyond aging and cancer, metabolic disorders such as diabetes and obesity have been linked to chromatin modifications that alter gene expression and replication dynamics. Our findings position NAT10 as a potential metabolic sensor that integrates ketone body availability with chromatin regulation. The interplay between metabolic states and chromatin accessibility remains poorly understood, and our study provides a framework for exploring how metabolic signals influence epigenetic modifications to maintain genome stability.\u003c/p\u003e\u003cp\u003eLethality of NAT10 depletion: Limitations and future directions. A key limitation in targeting NAT10 is its essential role in cellular viability. Our observations indicate that NAT10 depletion appears to lead to significant replication stress, elevated genomic instability, and possible cell cycle arrest, highlighting its potentially unique role in replication regulation. The essential nature of NAT10 for cell viability suggests it may play a critical role in maintaining replication fork stability and origin activation. However, it remains unclear whether these effects are directly mediated by its β-hydroxybutyrylation activity or involve other epigenetic mechanisms.\u003c/p\u003e\u003cp\u003eOne possible explanation for the essential nature of NAT10 for cell viability is its role in stabilizing replication forks and coordinating replication-transcription conflicts. The loss of NAT10 may lead to uncoordinated origin firing, increased replication-transcription collisions, or failure to resolve stalled forks, ultimately triggering replication catastrophe. Additionally, NAT10 has been implicated in RNA modification and ribosomal biogenesis, raising the possibility that its essential function extends beyond chromatin remodeling. Future studies should focus on dissecting whether NAT10 replication-associated functions are distinct from its roles in RNA metabolism or whether these pathways are interdependent.\u003c/p\u003e\u003cp\u003eAnother key question is whether NAT10 functions independently or as part of a larger chromatin remodeling complex. Since its dynamic association with chromatin, NAT10 may interact with histone acetyltransferases, chromatin remodelers, or replication-associated factors to coordinate replication and repair. Identifying these interacting partners will provide deeper mechanistic insights into how NAT10 regulates chromatin structure and genome stability.\u003c/p\u003e\u003cp\u003eFrom a therapeutic perspective, the essential nature of NAT10 for cell viability presents both challenges and opportunities. While complete loss of NAT10 is detrimental, partial inhibition or selective modulation of its β-hydroxybutyrylation activity may offer a way to fine-tune replication stress responses without inducing cytotoxicity. Developing small-molecule inhibitors that target specific enzymatic activities of NAT10, while preserving its essential cellular functions, will be crucial for future translational applications.\u003c/p\u003e\u003cp\u003eIn summary, this study suggests that NAT10-mediated β-hydroxybutyrylation may represent an important mechanism connecting metabolic signaling with chromatin remodeling, replication efficiency, and genomic stability. Our data indicate that NAT10 appears to contribute to maintaining a chromatin environment that ensures efficient and faithful DNA synthesis. These observations offer potential new perspectives on how chromatin modifications might interact with replication control, which could have relevance for understanding aging, cancer, and metabolic disorders. Further investigation of NAT10's regulatory network may help evaluate its possible utility as a therapeutic target in replication stress-associated conditions.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003ePlasmid Construction and Cloning\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cDNA encoding wild-type NAT10 was amplified via PCR and inserted into the pcDNA3.1(+) vector, including an N-terminal FLAG or HA tags. Site-directed mutagenesis was employed to generate NAT10 mutants (G641E, K989R and K823R) using the Fast Mutagenesis System. Full-length NAT10 and its truncated variants were cloned into the pGEX-4T-3 vector to generate GST-fusion proteins.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Culture, siRNA and Plasmid Transfections\u003c/b\u003e\u003c/p\u003e\u003cp\u003eU2OS, HeLa, and 293T cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS, Gibco, A5670401), essential amino acids, and 1% streptomycin/penicillin (100 U/ml) at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. WI-38 and 2BS cells were maintained in RPMI 1640 medium with 15% FBS, essential amino acids, and 1% streptomycin/penicillin.\u003c/p\u003e\u003cp\u003eTo knock down NAT10 expression, U2OS cells (30\u0026ndash;40% confluence) were transfected with 120 pmol siRNA oligonucleotides per well in 6-well plates using Entranster\u0026trade;-in vivo Transfection Reagent (Engreen Biosystem, 18668-11-1). The medium was refreshed after 24 hours, and a second transfection was performed. Cells were treated 48 hours later with chemical agents, including (\u0026plusmn;)-sodium 3-hydroxybutyrate (BHB, Sigma-Aldrich, 54965), aphidicolin (APH, Santa Cruz, sc-201535), RO-3306 (Sigma-Aldrich, SML0569), or nocodazole (Selleck, S2775).\u003c/p\u003e\u003cp\u003eThe siRNA sequences targeting NAT10 were:\u003c/p\u003e\u003cp\u003eHomo siNAT10 #1: 5\u0026rsquo;-GAGCAUGGACCUCUCUGAAUACAUA-3\u0026rsquo;\u003c/p\u003e\u003cp\u003eHomo siNAT10 #2: 5\u0026rsquo;-CAAACAUUCGCUACUGCUACUACAA-3\u0026rsquo;\u003c/p\u003e\u003cp\u003eHomo siNAT10 #3: 5\u0026rsquo;-CAGGCUGAACUAGUUGUGAUUGAUG-3\u0026rsquo;\u003c/p\u003e\u003cp\u003ePlasmid transfection in 293T and U2OS cells were performed using the NEOFECT\u0026trade; DNA Transfection Reagent (Neofect Biotech) following the manufacturer\u0026rsquo;s protocol. siRNA transfections in WI-38 and 2BS cells utilized RFectPM (Baidai Bio, 11014).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntibodies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe primary antibodies utilized included: anti-NAT10 (Santa Cruz, B-4, sc-271770; C-10, sc-271141), anti-53BP1 (Cell Signaling Technology, 4937S), anti-Phospho H2A.X S139 (Cell Signaling Technology, 9718), anti-Cyclin A (B-8) (Santa Cruz, sc-271682), anti-β-actin (Sigma-Aldrich, A2228), anti-Flag M2 (Sigma-Aldrich, F1804), anti-H2B (V119) (Cell Signaling Technology, 8135S), anti-HA-Tag (6E2) (Cell Signaling Technology, 2367) and anti-COX-IV (Cell Signaling Technology, 4844), anti-phosphorylation S317-CHK1(Proteintech, 28807-1-AP), anti-RPA2(Cell Signaling Technology, 35869S), anti-BrdU (Serotec, OBT0030) and anti-BrdU (Becton Dickinson, 347580).\u003c/p\u003e\u003cp\u003eThe β-hydroxybutyrylation-lysine site specific antibodies are from Jingjie PTM BioLab, Co. Ltd, Hangzhou, China: anti-pan β-hydroxybutyrylation-lysine (BHB-K) antibody (PTM-1201), anti-H2AK5BHB (PTM-1220), anti-H2AK118BHB (PTM-1224), anti-H2BK5BHB (PTM-1230), anti-H2BK11BHB (PTM-1231), anti- H2BK16 BHB (PTM-1234), anti- H2BK20BHB (PTM-1235), anti-H2BK23BHB (PTM-1236), anti- H2BK34 BHB (PTM-1238), anti-H3K9BHB (PTM-1250), anti-H3K14BHB (PTM-1251), anti-H3K23BHB (PTM-1300), anti-H4K5BHB (PTM-1205), anti-H4K8BHB (PTM-1253), anti-H4K9BHB (PTM-1210), anti-H4K12BHB (PTM-1206).\u003c/p\u003e\u003cp\u003eSecondary antibodies used were Goat anti-Rabbit IgG Secondary Antibody HRP conjugated (Jackson ImmunoResearch, 111-035-003) and Goat anti-Mouse IgG Secondary Antibody HRP conjugated (Jackson ImmunoResearch, 115-035-003).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Proliferation Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell proliferation was evaluated using the Cell Counting Kit-8 (Selleck, B34302). U2OS cells were plated in 96-well plates at appropriate densities. After 24 hours of attachment, 10 \u0026micro;l of CCK-8 reagent was added per well, and absorbance at 450 nm was measured after a 2-hour incubation using a microplate reader. Cell viability was monitored at 0, 12, 24, 36, 48, and 72 hours post-seeding.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence Assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells plated on coverslips with siRNA transfected or treated with drugs were fixed in 4% PFA/PBS for 15 minutes and neutralized with Glycine. After washing twice with PBS, the cells were permeabilized with 0.3% Triton-X/PBS for 10 minutes. After blocked with 5% BSA/PBS for 30 minutes, the cells were incubated with the indicated primary antibody for 60 minutes at room temperature, following by incubation with the Alexa Fluor\u0026reg; 488 AffiniPure Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) antibody (Jackson ImmunoResearch, 111-545-003) or Alexa Fluor\u0026reg; 594 AffiniPure Goat Anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) antibody (Jackson ImmunoResearch, 111-585-003) for 45 minutes. After staining nuclei with 2 \u0026micro;g/ml diamidinophenylindole (DAPI) (Roche, 28718-90-3) for 5 minutes, the coverslips were mounted with Vectashield medium. Images were obtained on a ZEISS LSM880 confocal system with a 63x/1.40 (oil) or 100x/1.40 (oil) objective lens or a high content screening system (Operetta\u0026trade;, Perkin Elmer) with a 60\u0026times;, 0.95-NA objective at the perfect focus. Confocal and super-resolution images were analyzed with ZEN (Blue edition) software from Carl Zeiss Microscopy, and fluorescence intensity was calculated with Harmony 3.5 software.\u003c/p\u003e\u003cp\u003eNote that for immunostaining of pre-extracted cells, cells grown on coverslips were incubated in CSK buffer containing 0.3 mg/ml RNase A for 15 minutes at room temperature before fixed as descripted above.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEdU Labeling and Detection in Mitotic Cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were incubated with 9 mM RO-3306 and 0.4 \u0026micro;M APH (Santa Cruz, sc-201535) for 16 hours to synchronize in late G2 phase, subsequently washed three times with PBS for 5 minutes at room temperature, and then released into pre-warmed fresh medium containing 20 mM 5-ethynyl-2\u0026rsquo;-deoxyuridine (EdU, Sigma-Aldrich, 61135-33-9) and 0.1 mg/ml Colcemid (Thermo Fisher Scientific, 15210040) for 60 minutes. Cells were harvested by centrifuged at 2,000 g for 5 minutes at 4\u0026deg;C, and resuspended and incubated in 8 ml pre-warmed 75 mM KCl for 15 minutes at 37\u0026deg;C. Swollen mitotic cells were subsequently fixed in Carnoy\u0026rsquo;s buffer (75% methanol, 25% glacial acetic acid) three times for 15 minutes, spread on pre-hydrated slides (Thermo Fisher Scientific) and dried overnight at room temperature followed by EdU detection with Click-IT Plus EdU Alexa fluor 488 Imaging Kit (Thermo Fisher Scientific, C10337) according to the manual. After staining chromosomes with 2 \u0026micro;g/ml DAPI (Roche, 28718-90-3) for 5 minutes and mounting slides with vectashield mounting medium, images were captured on a ZEISS LSM880 confocal system with a 100\u0026times;/1.40 (oil) objective. At least 150 metaphase chromosomes were analyzed in three independent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFlow Cytometer\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor DNA content analysis, cells were synchronized at the G1/S boundary with 2 mM thymidine for 18 hours, followed by release every two hours. Cells were harvested with trypsin and fixed overnight in pre-chilled 100% methanol at -20\u0026deg;C. Subsequently, cells were stained with 40 \u0026micro;g/ml propidium iodide (PI, Sigma-Aldrich, 25535-16-4) in PBS, containing 100 \u0026micro;g/ml RNase A, for 15 minutes at room temperature in the dark. Flow cytometric analysis was performed immediately using a FACSCalibur system (Becton Dickinson). DNA content was analyzed using CellQuest or FlowJo VX software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEdU FACS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAsynchronously growing U2OS cells were labeled with 20 uM EdU for 20 minutes. After labeling, cells were harvested. Cells were washed once with PBS and fixed in 70% ethanol over night at 4\u0026deg;C. Cells were incubated in 2 N HCl at room temperature for 20 mins, washed in PBS. EdU incorporation was detected using the YF\u003csup\u003e@\u003c/sup\u003e 648A Click-iT EdU Flow Cytometry Assay Kit (UE, C6046S) following the manufacturer\u0026rsquo;s protocol. Nuclei were stained with 40 \u0026micro;g/ml propidium iodide (PI, Sigma-Aldrich, 25535-16-4) in PBS, containing 100 \u0026micro;g/ml RNase A, for 15 minutes at room temperature in the dark. Flow cytometric analysis was conducted using a FACSCalibur system, and data were processed with FlowJo VX software to assess cell cycle progression and EdU-positive populations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA fiber analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDNA fiber analysis was conducted as described previously \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In summary, cells were either directly treated with 5 mM or 10 mM BHB (Sigma-Aldrich, 54965) for 24 hours, or transfected with siRNA or treated with 10 \u0026micro;M remodelin (Selleck, S7641) with or without 10 mM BHB. Following treatment, cells were pulse-labeled with 25 \u0026micro;M chlorodeoxyuridine (CldU, Sigma-Aldrich, 50-90-8) for 20 minutes, washed, and labeled with 250 \u0026micro;M iododeoxyuridine (IdU, Sigma-Aldrich, 54-42-2) for another 20 minutes.\u003c/p\u003e\u003cp\u003eCldU and IdU were detected using a rat anti-BrdU (Serotec, OBT0030) and mouse anti-BrdU (Becton Dickinson, 347580) primary antibodies, respectively, followed by DyLight 550 anti-rat (Thermo Fisher, SA5-10019) and Alexa Fluor 488 anti-mouse (Thermo Fisher, A-10680) secondary antibodies.DNA fibers were imaged with a ZEISS LSM880 confocal microscope equipped with a 100\u0026times;/1.40 oil objective. Images were acquired using autofocus and tile-array methods, and double-labeled replication forks were analyzed manually with ZEN software. For each slide, 50\u0026ndash;100 forks were evaluated, with data aggregated from three independent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003essDNA Visualization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSingle-stranded DNA (ssDNA) in S-phase cells was visualized by detecting BrdU foci without DNA denaturation. Cells labeled with 10 \u0026micro;M BrdU for 48 hours were fixed in 4% formaldehyde for 15 minutes, permeabilized with 0.3% Triton X-100 for 10 minutes, and blocked with 5% BSA for 30 minutes. Cells were incubated with primary anti-BrdU (1:400) and anti-cyclin A (1:400) antibodies (Santa Cruz, sc-271682) for 60 minutes, followed by Alexa Fluor\u0026reg; 488 (1:200, Jackson ImmunoResearch, 111-545-003) or Alexa Fluor\u0026reg; 594 (1:200, Jackson ImmunoResearch, 111-585-003) secondary antibody for 45 minutes. Nuclei were stained with 2 \u0026micro;g/ml DAPI for 5 minutes, and images were acquired using a high-content screening system (Perkin Elmer) with a 60\u0026times;, 0.95-NA objective. Images were obtained on a ZEISS LSM880 confocal system with a 63x/1.40 (oil) or 100x/1.40 (oil) objective lens or a high content screening system (Operetta\u0026trade;, Perkin Elmer) with a 60\u0026times;, 0.95-NA objective at the perfect focus. Confocal and super-resolution images were analyzed with ZEN (Blue edition) software from Carl Zeiss Microscopy, and fluorescence intensity was calculated with Harmony 3.5 software.\u003c/p\u003e\u003cp\u003eSingle-stranded DNA (ssDNA) in S-phase cells was also visualized by detecting RPA2 foci in pre-extracted nuclei. Cells grown on coverslips were incubated in CSK buffer containing 0.3 mg/ml RNase A for 15 minutes at room temperature before fixed. Cells were immunostained by RPA2 antibody (1:400), and imagines were obtained as described above.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMass Spectrometry Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProtein extraction, digestion, and mass spectrometry analysis were outsourced to (Beijing Proteome Research Center). Proteins were subjected to intensity-based absolute quantification (iBAQ) mass spectrometry to analyze.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOrigin Firing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOrigin firing analysis was conducted as described previously\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Briefly, chromatin-binding proteins were enriched, and were analysis by label-free quantitative proteomic analysis. U2OS cells were lysed with cytoplasmic lysis buffer (10 mM HEPES pH 7.9, 340 mM sucrose, 3 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 2 mM MgOAc, 0.1 mM EDTA, 1 mM DTT, 0.5% Triton X-100 and protease inhibitors) for 20 min on ice. Intact nuclei were pelleted by centrifugation at 3,500 \u0026times; g for 15 min. Nuclei were washed with cytoplasmic lysis buffer without Triton X-100. Nuclei were lysed with nuclear lysis buffer (20 mM HEPES pH 7.9, 150 mM K\u003csub\u003e2\u003c/sub\u003eOAc, 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 3 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 10% glycerol, and protease inhibitors) for 30 min on ice. The nucleoplasmic fraction was cleared by centrifugation at 15,000 \u0026times; g for 30 min. The chromatin-enriched pellet was resuspended in 50 \u0026micro;l denaturation buffer (8 M Urea; 100 mM Tris pH 8, 5 mM DTT) and incubated at 22\u0026deg;C for 30 min. The sample was cleared by centrifugation at 20,000 \u0026times; g for 30 min, and the supernatant containing the solubilized denature chromatin proteins was collected. The chromatin-bound proteins were subjected to intensity-based absolute quantification (iBAQ) mass spectrometry to analyze replication initiation factors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReplication Program\u003c/b\u003e\u003c/p\u003e\u003cp\u003eU2OS cells in logarithmic phase were pulse-labeled with 10 \u0026micro;M BrdU (Sigma-Aldrich, 59-14-3) for 45 minutes, washed twice with warm medium and chased in fresh medium for 4 hours. Cells were then labeled with 10 \u0026micro;M EdU for another 45 minutes. Fixed cells (4% formaldehyde,15 minutes) were permeabilized with 0.3% Triton X-100 for 10 minutes, and DNA was denatured with 2N HCl for 30 minutes at room temperature, followed by neutralization with 0.1M sodium borate buffer (pH 8.5). EdU was detected using the Cell-Light EdU Apollo567 In Vitro Kit (Ribobio, C10310), and BrdU was visualized with an anti-BrdU antibody (1:400) followed by Alexa Fluor\u0026reg; 488 secondary antibody (Jackson ImmunoResearch, 111-545-003), after blocking with 5% BSA for 1 hour. Nuclei were counterstained with 2 \u0026micro;g/ml DAPI, and images were captured using a high-content screening system (Perkin Elmer) with a 60\u0026times;, 0.95-NA objective, and BrdU or EdU intensity per nucleus was quantified using Harmony 3.5 software. Replication patterns and S phase progression were analyzed as previously described\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The data obtained from \u0026gt;\u0026thinsp;250 nuclei across three independent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnaphase Bridge Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were synchronized with 9 mM RO-3306 for 16 hours, washed with PBS, and released into DMEM for 1.5 hours at 37\u0026deg;C. Cells were then cross-linked and permeabilized in PTEMF buffer (20 mM PIPES, pH 6.8, 10 mM EGTA, 1 mM MgCl₂, 0.2% Triton X-100, 4% formaldehyde) and stained with 2 \u0026micro;g/ml DAPI (Roche, 28718-90-3). Anaphase chromatin structures were imaged using a ZEISS LSM880 confocal microscope with a 63\u0026times;/1.40 oil objective. At least 150 anaphase cells were analyzed per experiment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicronuclei Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells treated with siRNA or drugs were cultured in medium containing 2 mg/ml cytochalasin B (Sigma-Aldrich, C6762) for 16 hours to arrest cytokinesis. Fixed cells were stained with 4% formaldehyde and 2 \u0026micro;g/ml DAPI (Roche, 28718-90-3) for nuclear visualization. Micronuclei were quantified using a ZEISS LSM880 confocal microscope. At least 150 binucleated cells per condition were scored, considering only distinct micronuclei adjacent to DAPI-stained nuclei.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation and Analysis of Chromosome Spreads\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were treated with 0.2 \u0026micro;M aphidicolin (Santa Cruz, sc-201535) for 24 hours, followed by 200 ng/ml nocodazole (Selleck, S2775) for the final 5 hours. Mitotic cells were collected by shake-off, centrifuged at 2,000 g for 5 minutes at 4\u0026deg;C. The pellet was resuspended in 1 ml PBS, followed by the addition of 8 ml pre-warmed 75 mM KCl, and incubated at 37\u0026deg;C for 15 minutes. After this incubation, 5 ml freshly prepared Carnoy\u0026rsquo;s buffer (75% methanol, 25% glacial acetic acid) was added for 15 minutes. Cells were fixed with Carnoy\u0026rsquo;s buffer (three times, 15 minutes each), spread onto slides and dried overnight at room temperature. Chromosomes were stained with 2 \u0026micro;g/ml DAPI and imaged using a ZEISS LSM880 confocal microscope with a 100\u0026times;/1.40 oil objective. At least 150 spreads were analyzed for chromatid breaks and gaps.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein Expression, Purification, and Pull-Down Assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo purify GST-NAT10 (full length or various fragments), bacterial expression constructs were transformed into \u003cem\u003eE. coli\u003c/em\u003e Rosetta (DE3) (Novagen, 70956). Selected clones were cultured in 500 ml LB medium at 37\u0026deg;C until the OD\u003csub\u003e600\u003c/sub\u003e reached\u0026thinsp;~\u0026thinsp;0.6, at which point protein expression was induced with 1 mM IPTG (AppliChem, A1008) at 20\u0026deg;C overnight. Harvested cell were resuspended in BC500 buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM KCl, 0.5% Triton X-100, 20% glycerol) supplemented with 1 mM PMSF and protease inhibitors. The suspension was lysed by sonication (70% amplitude, 10 seconds on/10 seconds off for 20 minutes) using a Hielscher-Ultrasound Technology Sonicator. Clarified lysates, obtained by centrifugation at 20,000 g for 30 minutes at 4\u0026deg;C, were incubated overnight at 4\u0026deg;C with pre-equilibrated glutathione Sepharose 4B beads (GE Healthcare, 17527902). Beads were washed once and resuspended in BC100 buffer (20 mM Tris-HCl, pH 7.9, 100 mM NaCl, 1.5 mM MgCl₂, 10 mM KCl, 0.1% Triton X-100, 20% glycerol) containing 50 U/ml Benzonase Nuclease (Sigma, 9025-65-4) on ice for 30 minutes, followed by four additional washes with BC100 buffer. The GST-fusion proteins were eluted with 20 mM glutathione (GSH) in BC100 buffer, dialyzed overnight against BC100 buffer, and stored for pull-down assays. Protein concentrations were determined using the Bradford assay.\u003c/p\u003e\u003cp\u003eFor FLAG-tagged proteins, 293T cells overexpressing the indicated constructs were lysed in BC500 buffer supplemented with 1 mM PMSF and protease inhibitors, followed by sonication. Lysates (2 mg total protein) were incubated with 40 \u0026micro;l anti-FLAG M2 agarose beads (Sigma-Aldrich). Beads were washed once with BC500 buffer and resuspended in BC100 buffer containing 50 U/ml Benzonase Nuclease (Sigma, 9025-65-4) on ice for 30 minutes, followed by four times washes with BC100 buffer. FLAG-tagged proteins were eluted using 0.5 \u0026micro;g/\u0026micro;l FLAG peptide in BC100 buffer.\u003c/p\u003e\u003cp\u003eGST pull-down assays were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Briefly, 5 \u0026micro;g GST-fusion protein was incubated with 2 \u0026micro;g eukaryotic purified protein or 100 \u0026micro;g purified mononucleosomes in 1 ml BC100 buffer or various binding buffers containing different NaCl concentrations (100 mM, 150 mM, 200 mM, 300 mM, 500 mM). The mixture was incubated overnight at 4\u0026deg;C with gentle shaking, followed by 4-hour incubation with glutathione Sepharose (GE Healthcare, 17527902). After five washes with the respective buffer, bound proteins were eluted with 1\u0026times; SDS loading buffer and analyzed by SDS-PAGE.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMononucleosome Extraction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMononucleosomes for use in \u003cem\u003ein vitro\u003c/em\u003e pull-down and β-hydroxybutyrylation assays were isolated using micrococcal nuclease\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Briefly, U2OS cells, treated with or without BHB, were lysed in BC300 buffer on ice for 30 minutes. Chromatin pellet, obtained by centrifugation at 1,000 g for 3 minutes, were washed and digested with 60 U/ml micrococcal nuclease (MNase) in digestive buffer (10 mM Tris-Cl pH 8.0, 10 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.05 mM DTT, and 0.01 mM PMSF) at 30\u0026deg;C for 30 minutes, and the reaction was stopped with 10 mM EDTA. The first supernatant (S1) was collected after centrifugation, and mononucleosomes were purified using gel filtration chromatography on the SMART\u0026trade; FPLC system (Amersham Biosciences). Purity was confirmed by agarose gel electrophoresis (140 bp band) and SDS-PAGE (histones only).\u003c/p\u003e\u003cp\u003e\u003cb\u003eβ-hydroxybutyrylation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate NAT10 as a β-hydroxybutyryltransferase, 293T cells transfected with Flag-NAT10-expressing plasmids were treated with 10 mM BHB for 24 hours. Cell was resuspended in BC500 buffer with 10 mM BHB, sonicated, and the lysate was subjected to immunoprecipitation and western blot analysis. Alternatively, β-hydroxybutyrylated proteins were enriched using pan-β-hydroxybutyrylated lysine antibodies in the BC100 buffer.\u003c/p\u003e\u003cp\u003eTo confirm NAT10-mediated histone β-hydroxybutyrylation \u003cem\u003ein vivo\u003c/em\u003e, NAT10 (wild type or G641E, K989R and K823R mutants) was overexpressed or knocked down in cells, followed by 10 mM BHB treatment for 6 hours. In parallel, cells transfected with NAT10 sgRNA and Cas9 plasmids were screened with puromycin (3 \u0026micro;g/ml) and hygromycin (1 \u0026micro;g/ml) for 72 hours prior to BHB treatment. Histones were purified using standard acid extraction protocols \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Briefly, intact nuclei were isolated from cells cracked with pre-cold hypotonic HB buffer, resuspended in 2N HCl and incubated overnight on a rotator. Nuclear debris was removed by centrifugation at 10,000 g for 10 minutes at 4\u0026deg;C, and the supernatant was neutralized with Tris (pH 8.0). Extracted histones were analyzed by SDS-PAGE and immunoblotting with specific antibodies.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e assay, purified NAT10 (wild type or G641E, K989R and K823R) was co-incubated with 25 \u0026micro;M DL-β-Hydroxybutyryl coenzyme A (BHB-CoA, Sigma, H0261) with or without 10 \u0026micro;M remodelin (Selleck, S7641) in 30 \u0026micro;l reaction buffer (50 mM HEPES pH 8.0, 10% glycerol, 1 mM DTT, 1 mM PMSF). Alternatively, 1 \u0026micro;g NAT10 was incubated with 2 \u0026micro;g mononucleosome in the same buffer. Reactions were performed at 30\u0026deg;C for 60 minutes, stopped with SDS buffer, and analyzed by Western blotting.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSubcellular Fractionation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eU2OS cells (1 \u0026times; 10⁷) were homogenized in HB buffer (10 mM Tris-Cl, pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 1 mM DTT, and 1\u0026times; EDTA-free protease inhibitor cocktail, Sigma-Aldrich, 11873580001) until ~\u0026thinsp;90% lysis, confirmed by trypan blue. Lysates were centrifuged at 1,000 g for 15 minutes at 4\u0026deg;C to separate the cytoplasmic fraction (Cyto), which was supplemented with 100 mM NaCl and 0.1% Triton X-100. The nuclear pellet was resuspended in BC300 buffer (20 mM Tris-HCl pH 7.9, 300 mM NaCl, 10 mM KCl, 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.1% Triton X-100, 20% glycerol, and 1\u0026times; protease inhibitor cocktail), vortexed for 30 minutes and centrifuged. The supernatant was collected as the nuclear soluble fraction (Nucl) and adjusted to 100 mM NaCl.\u003c/p\u003e\u003cp\u003eChromatin fractions were isolated by lysing cells in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100) supplemented with 0.2 mM PMSF and protease inhibitors. The lysates were centrifuged at 20,000 g for 15 minutes, and the supernatant was designated as the soluble fraction (Sup). The insoluble chromatin fraction was sonicated in RIPA buffer containing 1% SDS, centrifuged at 20,000 g for 15 minutes at 4\u0026deg;C, and the supernatant was collected as the chromatin soluble fraction (Chr).\u003c/p\u003e\u003cp\u003eAlternatively, cells were lysed in CSK buffer (10 mM HEPES pH 7.9, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5% Triton X-100) with 300 \u0026micro;g/ml RNase A. After centrifugation, the supernatant was collected as the soluble fraction (Sup), and the chromatin-containing pellet was sonicated in RIPA buffer with 0.1% SDS to obtain the pellet fraction (Pellet).\u003c/p\u003e\u003cp\u003e\u003cb\u003eChromatin immunoprecipitation and sequencing (ChIP-seq)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChromatin immunoprecipitation was conducted using the ChIP-IT Express Kit (Active Motif, 53008). Briefly, U2OS cells cultured on 15 cm plates were treated with 10 mM BHB or left untreated for 12 hours. Cells were fixed with 1% formaldehyde for 15 minutes at room temperature, and cross-linking was quenched with 125 mM glycine for 5 minutes. After centrifugation at 1,250 g for 3 minutes at 4\u0026deg;C, cells were washed with PBS containing 0.5% Igepal and sonicated (40% amplitude, 4 seconds on/6 seconds off, ~\u0026thinsp;60 cycles) to shear chromatin. A 10% aliquot of the supernatant was saved as the input sample.\u003c/p\u003e\u003cp\u003eFor immunoprecipitation, 50 \u0026micro;g of sonicated chromatin was incubated overnight at 4\u0026deg;C with 2 \u0026micro;g of anti-NAT10 antibody (Santa Cruz, B-4, sc-271770). Immunocomplexes were captured with ChIP Grade Protein A/G Plus Agarose (Thermo Fisher Scientific, 26159), extensive washed, eluted, and de-crosslinking at 65\u0026deg;C overnight. Samples were treated RNase A and proteinase K, and DNA was purified according to the manufacturer\u0026rsquo;s protocol (Active Motif, 53008).\u003c/p\u003e\u003cp\u003ePurified DNA was eluted in 30 \u0026micro;l of distilled water and prepared for sequencing by adding Illumina adapter. Libraries were sequenced on the HiSeq4000 platform (Illumina), generating\u0026thinsp;\u0026gt;\u0026thinsp;50\u0026nbsp;million paired-end reads (200\u0026ndash;300 bp) per sample.\u003c/p\u003e\u003cp\u003e\u003cb\u003eATAC-seq\u003c/b\u003e\u003c/p\u003e\u003cp\u003eATAC sequencing service was provided by Cloud-Seq Biotech (Shanghai, China) with GenSeq\u0026reg; ATAC kit (GenSeq Inc.). Briefly, cells were lysed using cold lysis buffer. After lysis, nuclei were collected by centrifuging at 500 g for 10 min at 4℃. The pellet was transposed with transposome and add tags on the both sides of fragmented genomic DNA according to the manufacturer\u0026rsquo;s instruction. After tagmentation, the purified tagged DNA was used for PCR amplification with Genseq\u0026reg; 2\u0026times;HiFi PCR Mix (GenSeq Inc.). The libraries were quantified using Qubit fluorometric assay (ThermoFisher) and then sequenced. in a NovaSeq platform (Illumina).\u003c/p\u003e\u003cp\u003eATAC-Seq high throughput sequencing and subsequent bioinformatics analysis were all done by Cloud-Seq Biotech (Shanghai, China). Briefly, raw data were generated after sequencing, image analysis, base calling and quality filtering on sequencer. Use fastp software (v 0.23.4) to remove joints and low quality reads to obtain high quality clean reads; And clean reads alignment to the reference genome using bowtie2 software (v2.2.4); The reads aligned to the chrM were removed using the samtools software (v1.9), PCR duplicates were removed using getk-picard software (v4); open chromatin regions (Peak Calling) using MACS2 software (v2.1.1); differential enriched regions were identified using diffReps software; and enrichment peaks were annotated using ChIPseeker software (v1.2.6). GO and KEGG pathway analysis were performed for genes associated with differentially enriched peaks.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantification and Statistical Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eData were analyzed using GraphPad InStat software (Version 5.01, GraphPad Prism, GraphPad Software Inc., San Diego, CA) and SPSS 21.0 software. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from at least three independent biological replicate experiments. Statistical significance was determined using the Student\u0026rsquo;s t test or one-way ANOVA, with p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e\u003cp\u003eThe project was supported partially by the National Natural Science Foundation of China (Grant No. 32371313) and the Fundamental Research Funds for the Central Universities (2024CDJXY-016) to Wenhui Zhao.\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTION\u003c/h2\u003e\u003cp\u003eConceptualization, W.Z.; Methodology, Y.S., R.Z., S.J. and W.Z.; Formal Analysis, Y.S., R.Z., S.J. and W.Z.; Investigation, Y.S., R.Z., S.J. and W. Z.; Resources, Y.S., R.Z., S. J., K.L., Z.F., S.J.., S.L., and W.Z.; Data Curation and Visualization, Y.S., R.Z., S.J. and W.Z.; Funding Acquisition, W.Z.; Supervision, W.Z.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e\u003cp\u003eThanks to Drs. Haibo Wu, Chuan Huang, Zhenghong Lin, Shanshan Pang, Wei Deng, Zhong Luo and Zhengguo Li for critical discussion and critical reading the manuscript. We would like to thank Dr. Yingming Zhao at the University of Chicago, Dr. Wei Gu at Columbia University, Dr. Qinglian Liu at Virginia Commonwealth University and Dr. Liang Ce at Hebei Medical University for technical help. We appreciate Dr. Zhongyi Cheng (Jingjie PTM BioLab, Co. Ltd, Hangzhou, China) for provding β-hydroxybutyrylation histones antibodies. We thank Cloud-Seq Biotech (Shanghai, China) for providing ATAC sequencing and analysis service. Wenhui Zhao was partial supported by National Natural Science Foundation of China (Grant No. 32371313) and the Fundamental Research Funds for the Central Universities (2024CDJXY-016).\u003c/p\u003e\u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e\u003cp\u003eThe ChIP-seq data has been deposited in Gene Expression Omnibus (GEO) #GSE175731. The ATAC-seq data has been deposited in Gene Expression Omnibus (GEO) #GSE292952. All data are available on \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE175731\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE175731\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and acc\u0026thinsp;=\u0026thinsp;GSE292952.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKlein, K. 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Here, we observe NAT10 is a β-hydroxybutyryl-transerase and NAT10-mediated β-hydroxybutyrylation (Kbhb) of histones that appears to affect replication fork progression without significantly impacting origin firing, potentially to reduce replication stress and to help maintain genomic stability. DNA fiber analyses show β-hydroxybutyrate (BHB) treatment enhances replication efficiency while maintaining fork symmetry, effects abolished by NAT10 depletion or inhibition. BrdU/EdU labeling, and EdU-FACS analyses reveal that NAT10-mediated Kbhb accelerates replication fork velocity and shortens S-phase duration. LC-MS/MS profiling shows no significant changes in origin firing following BHB treatment. Assessment of replication stress markers, including γH2AX foci, non-denaturing BrdU incorporation, RPA2 foci, S317-CHK1 phosphorylation, and levels of γH2AX and RPA2 on chromatin, suggests that NAT10-mediated Kbhb reduces replication stress. Evaluation of genomic instability, measured by micronuclei formation, sister chromatid bridges, and chromatid breaks/gaps during mitosis, indicates that NAT10-mediated Kbhb also reduces genomic instability. Mechanistically, NAT10-mediated Kbhb modulates chromatin association, thereby modulating chromatin accessibility to establish a replication-permissive environment. This epigenetic remodeling serves to moderate replication stress markers and genomic instability. Conserved effects in transformed and primary cell models position NAT10 as a metabolic-epigenetic nexus linking nutrient signaling to replication fidelity. Our findings suggest targeting Kbhb signaling as a potential therapeutic strategy against replication stress-associated pathologies.\u003c/p\u003e","manuscriptTitle":"NAT10-mediated β-hydroxybutyrylation Affects DNA Replication","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 13:33:38","doi":"10.21203/rs.3.rs-7110451/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ac68a894-87ff-4d30-a139-0f05d3ef4393","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51889426,"name":"Biological sciences/Biochemistry/Proteins/DNA-binding proteins"},{"id":51889427,"name":"Biological sciences/Molecular biology/Chromatin/Histone post-translational modifications"}],"tags":[],"updatedAt":"2025-10-28T17:06:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-24 13:33:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7110451","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7110451","identity":"rs-7110451","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}