Epigenetic Reshaping through Damage: Promoting Cell Fate Transition by BrdU and IdU incorportion

Epigenetic Reshaping through Damage: Promoting Cell Fate Transition by BrdU and IdU incorportion | 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 Research Article Epigenetic Reshaping through Damage: Promoting Cell Fate Transition by BrdU and IdU incorportion chuang li, Xiaoduo Xu, Shuyan Chen, Anchun Xu, Tongxing Guan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3520658/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2024 Read the published version in Cell & Bioscience → Version 1 posted 5 You are reading this latest preprint version Abstract Background: Thymidine analogs have long been recognized for their ability to randomly incorporate into DNA. However, the precise mechanisms through which thymidine analogs facilitate cell fate transition remains unclear. Results: Here, we discovered a strong correlation between the dosage dependence of thymidine analogs and their ability to overcome reprogramming barrier. Meanwhile, we found that homologous recombination repair (HRR) pathway causes an overall epigenetic reshaping of cells and enabling them to overcome greater barriers. Moreover, extraembryonic endoderm (XEN) state appears to be a selective response behavior of cells to DNA damage repair (DDR), providing a shortcut for cells to overcome reprogramming barriers, creates a hypomethylated environment that promotes cell fate transition in multiple reprogramming systems. We term this mechanism as Epigenetic Reshaping through Damage (ERD). Conclustion: Overall, our study sheds light on the dynamic interplay between thymidine analogs, DDR, and epigenetic modifications, providing valuable insights into the mechanisms underlying cell fate transition. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Thymidine analogs are often considered to incorporate randomly into DNA sequences, introducing inherent unpredictability and randomness in their mechanisms of action( 1 ). Despite this inherent nature, they play a critical role in the precise regulation of cell fate( 2 – 7 ). In a study conducted by Xie et al., the utilization of BrdU, widely employed to label proliferating cells in vivo, has demonstrated its significant potential in facilitating chemical induction of pluripotency( 3 ). The study by Cao et al., sheds light on how BrdU can impact the reorganization of nuclear architecture and influence the cell fate decisions( 5 ). IdU, which is also a thymidine analog was reported to cause stochastic fluctuations in gene expression to facilitate cellular reprogramming( 8 ). However, the precise mechanisms through which BrdU/IdU facilitates cell reprogramming are not yet fully understood. BrdU/IdU may be involved in cell reprogramming through mechanisms beyond transcriptional fluctuations. This could include the impact of thymidine analogs on DNA structure, repair mechanisms, or other cellular processes, providing a potential shortcut for cells to overcome reprogramming barriers. As a result, thymidine analogs may play a more crucial and multifaceted role in regulating cell fate, extending beyond the induction of transcriptional fluctuations alone. This study links DNA damage repair with genome, epigenetics and cell fate regulation, providing us with new understanding and helping to advance the field of cell fate decision and regenerative medicine. Results BrdU and IdU: Essential in overcoming two barriers during chemical induction of pluripotency To determine the optimal duration of BrdU treatment for chemical induction of pluripotency (CIP), a series of time windows were optimized and observed for their impact on CIP (Fig. 1 A). Two main barriers were found in the CIP reprogramming process, and BrdU was found to play a crucial role in overcoming these barriers. BrdU dropout experiments showed that dropout of BrdU at barrier I resulted in a failure to form colonies at Day 22, while dropout at barrier II between Day 12–22 resulted in a failure to form GFP + colonies at Day 40 (Fig. 1 B). Meanwhile, we have established a time gradient for each barrier (Figure S1A), and breaking through the barrier requires at least 6 days of BrdU incorporation, which must be added within the first 4 days (Figure S1B). Increasing the duration of BrdU treatment led to an increase in the number of GFP + colonies, with a 0–22 days treatment resulting in over 240 GFP + colonies at Day 40 from a starting population of 20,000 cells, achieving an efficiency of 1.2%. In contrast, a 0–12 days treatment only resulted in about 20 GFP + colonies (Fig. 1 C and S1 C). Furthermore, we attempted to use three base analogs, namely IdU, EdU, and 5Aza, but only IdU was found to be capable of replacing the function of BrdU during CIP reprogramming (Fig. 1 D and S1 D). The combination of both thymidine analogs can increase the selectivity of cells, so that only cells that have been correctly reprogrammed can survive; hence accelerated the reprogramming process (Fig. 1 D,E and S1 E). Thus, it can be concluded that BrdU and IdU are essential in overcoming two main barriers during CIP. Gene expression dynamics and chromatin accessibility dynamics during CIP with or without BrdU/IdU To identify the two barriers in which BrdU and IdU are involved during CIP reprogramming, RNA-seq and ATAC-seq was performed on MEFs undergoing CIP with or without BrdU, IdU and I + B (IdU + BrdU) at D8, 14, 20, 26 and 40 (Fig. 2 A). It was found that loss of BrdU and IdU at barrier I resulted in incomplete reprogramming of MEFs to XEN-like cells (Fig. 2 B). The expression of XEN-like genes such as Gata4 , Sox17 and Aqp8 increased significantly with increasing duration of thymidine analogs treatment and reached a peak at D20. Conversely, dropout of BrdU resulted in a failure to express XEN-like genes (Figure S2A,C), suggesting that MEFs undergoing reprogramming without BrdU /IdU follow a different fate path. Previous studies have reported that CIP goes through an XEN-like intermediate stage( 2 ). Additionally, loss of BrdU and IdU at barrier II resulted in incomplete reprogramming of XEN-like cells to CiPSCs. Pluripotency genes such as Oct4 , Esrrb, Tfcp2l1 , Nanog and Sox2 were highly activated in a BrdU/IdU dependent manner during stage 2. In addition, the incorporation of I + B showed a faster transition towards to iPSCs compared to BrdU/IdU treatment at the RNA level (Figure S2B,D). The function state of a cell is determined by its genome architecture. To investigate the role of BrdU/IdU in CIP, we mapped chromatin accessibility dynamics (CADs) during CIP with or without BrdU/IdU and found that many loci opened during CIP failed to open without BrdU/IdU. Moreover, detailed analysis of the ATAC-seq datasets revealed many similarities between BrdU and IdU treatments. Chromatin accessibility significantly increased when MEFs undergoing CIP were treated with I + B compared to only BrdU or IdU treatment (Fig. 2 C). In Fig. 2 D, we analyzed the loci near fibroblast marker gene Twist2 and Fbn1 found that they were more open in DMSO treatment compared to BrdU/IdU treatment. Conversely, loci near XEN marker genes such as Gata4 and Sall4 were only open in BrdU/IdU treatment. Loci near the pluripotency marker gene Oct4 and Tfcp2l1 were also only open in BrdU/IdU treatment, and the chromatin accessibility was much higher when samples were treated with both I + B. We compared the peaks at each locus between these samples and classified them into three categories: closed in MEFs but open in ESCs (CO), open in MEFs but closed in ESCs (OC), and permanent open (PO). Further, we divided the CO and CO peaks into subgroups based on the day of reprogramming to illustrate the progression of cellular reprogramming associated CADs, as shown in Fig. 2 E. To further investigate the molecular mechanism of BrdU/IdU during CIP, we performed motif analysis, as illustrated in Fig. 2 F. Loci containing motifs for OCT2, 4, 6, and 11 gradually opened, peaking at CO6 when treated with BrdU/IdU. Loci with motifs for GATA1, 2, 4, 6, KLF3-6, and SOX2-4, 6, 9, 10, 15, 17 opened from CO1-6 when treated with BrdU/IdU. However, the TF motifs profile was similar with or without BrdU/IdU in OC loci, indicating that BrdU/IdU treatment tended to open up loci that are enriched with motifs binding to TFs from the GATA, KLF, and SOX families. We then statistically analyzed the number of total peaks in CO1-6/OC1-6, respectively, and presented the results in the form of a Venn diagram, as shown in Fig. 2 G,H. The diagram showed that BrdU/IdU/I + B shared 25.2% (8240/32655) in CO peaks, but only 19.3% (7243/37575) in OC peaks. Figure S2E illustrates that a larger fraction of peaks in CO are located in the promoter regions. In conclusion, the function of BrdU/IdU is more biased towards opening chromatin and activating gene expression. Incorporation of BrdU/IdU leads to DNA damage repair According to the RNA-seq analysis we obtained in Fig. 2 , we then performed Venn plots and enriched GO functions (Fig. 3 A,B and Figure S3A,B). Interestingly, Venn plots for overlapping genes in the upregulated groups between BrdU, IdU, and I + B versus the control (without thymidine analogs) showed that DNA repair-related GO functions were highly enriched when MEFs were treated with BrdU/IdU during CIP (Fig. 3 A,B). Specifically, DNA repair-related gene expression was highly upregulated in all three groups. The main repair pathways are base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination repair (HRR) and non-homologous end joining repair (NHEJ). In our study, we observed that Apex1 , a gene related to BER pathway, was significantly upregulated in the early stages of reprogramming after treatment with BrdU/IdU, but gradually decreased with longer exposure. The HRR related genes Brca1 and Brca2 were upregulated as Apex1 expression decreased (Figure S3C). Additionally, a combination of both thymidine analogs (I + B) accelerated the DNA repair process, as shown by the early upregulation of DNA repair genes expression at D8 in this group compared to the BrdU or IdU only groups (Fig. 3 C). To verify the relationship between thymidine analogs and DNA damage repair, alkaline comet assays were performed to compare the olive tail moment difference between samples with or without thymidine analogs treatment (Fig. 3 D,E). Interestingly, the BrdU treatment showed an increased olive tail moment indicating significant DNA damage. Furthermore, the degree of DNA damage was further promoted by a combination of BrdU and IdU. Figure 3 F shows two DNA damage peaks during the CIP process at D14 and D24, which is coherent with the time window of the two main barriers discussed in the previous sections. This evidence illustrates a close association between DNA damage/repair and thymidine analogues assisting CIP through two barriers. To verify whether BrdU treatment directly causes DNA damage, immunofluorescent analysis was performed to observe the spatial colocalization of BrdU (in red) and γ-H2AX (in green), a novel biomarker for DNA double-strand breaks (Fig. 3 G). The dropout of BrdU showed a much weaker γ-H2AX signal, leading us to conclude that the accumulation of DNA damage during the CIP process is directly caused by BrdU binding to the DNA. Using CHIP-seq analysis targeting the DNA double-strand damage marker γ-H2AX, a correlation was found between its level and the ATAC-seq for Gata4 and Sox17 (Fig. 3 H). This suggests that the activation of the Gata 4 and Sox 17 genes is mediated through the DNA damage repair pathway. Mechanism of DDR in promoting somatic cell reprogramming ATM (Ataxia Telangiectasia Mutated) is a protein kinase that plays a critical role in the cellular response to DNA damage, particularly double-strand breaks (DSBs). When DSBs occur, the MRN complex (Mre11-Rad50-Nbs1) recognizes and binds to the site of break, recruiting ATM. Subsequently, ATM undergoes autophosphorylation, resulting in its full activation. Once activated, ATM phosphorylates several downstream targets involved in cell cycle checkpoint control, DNA repair, and apoptosis. Additionally, ATM activation is sustained by a phosphorylation-acetylation cascade. This cascade helps to maintain ATM activity and promote efficient DNA repair. Overall, ATM serves as a critical player in the cellular response to DNA damage, and its activation and downstream signaling are tightly regulated to ensure proper DNA repair and maintenance of genomic integrity( 9 ). In order to understand how DDR is involved in regulating somatic cell reprogramming, we hypothesize that DDR can upregulate the acetylation levels of genes at the site of DNA damage, thereby regulating gene expression, as shown in Fig. 4 A. Firstly, we found that inhibiting the ATM signaling pathway with an ATM inhibitor KU-55933 prevented BrdU from functioning (Fig. 4 B,C). Furthermore, we revealed that Gata4 , Sox17 and Sall4 's H3KC27ac and H3K9ac were significantly upregulated under the treatment of BrdU and I + B (Fig. 4 D). To further verify this hypothesis, we increased the concentration of VPA (a histone deacetylase inhibitor) to further promote acetylation accumulation. We found that this significantly accelerated the process of chemical reprogramming, and its acceleration effect depended on the addition of BrdU (Fig. 4 E,F). Therefore, we believe that BrdU/IdU induces DNA damage by incorporating into the genome, and recruits ATM to the site of damage. Subsequently, the acetylation levels of gene loci at the site of DNA damage is upregulated through a series of phosphorylation and acetylation cascades, thereby altering chromatin accessibility and activating gene expression. Combined with the results of RNA-seq, ATAC-seq, motif, γ-H2AX, H3K9ac, and H3K27ac analysis, we found that Gata4 has a high degree of specificity under the treatment of BrdU/IdU (Fig. 2 D, 2 F, 3 H, 4 D, Figure S2A). Therefore, GATA4 may be one of the specific downstream factors of BrdU/IdU involved in somatic cell reprogramming. Further Cut & tag analysis showed that the binding sites of GATA4 were significantly increased under the treatment of BrdU/IdU, and the sites enriched with GATA4 were accompanied by higher chromatin accessibility (Fig. 4 G), including pluripotency gene loci such as Oct4 , Sall4 as illustrated in Fig. 4 H. This indicates that GATA4 can respond to DDR, thereby promoting somatic cell reprogramming. BrdU/IdU creates a more open hypomethylated environment for the transformation of cell fate BrdU and IdU have been demonstrated to play a crucial role in chemical reprogramming, exhibiting two distinct stages of action. Hence we postulate that BrdU not only activates intermediate XEN genes such as Gata4 , but also potentially induces DNA demethylation, where DNA methylation is a major regulatory factor that limits GATA4 function( 10 ). To confirm this, we measured the methylation levels of GATA4 downstream gene Aqp8 and XEN gene Pth1r. We observed a significant decrease in DNA methylation levels with BrdU/IdU treatment (Fig. 5 A), indicating that BrdU/IdU not only activates gene expression but also induces DNA demethylation, thus creating a conducive environment for pluripotency network activation. We also integrated the aspect of DNA damage repair and hypothesized that BrdU and IdU act through double-stranded break excision and new chain synthesis during the process of DNA damage repair. Since new chain synthesis occurs without DNA methylation marks, it leads to DNA demethylation. To validate this hypothesis, we inhibited DNMT using CM272 and SGI1027 to prevent the re-methylation of new chains. The results indicated that this approach significantly accelerated the process of chemical reprogramming (Figs. 5 B and C), and this effect was dependent on the presence of BrdU. To summarize, as the degree of BrdU damage increases, the frequency of double-stranded breakage and synthesis also increases, which leads to DNA demethylation, thereby creating a more open hypomethylated environment for the transformation of cell fate. In the global genome methylation analysis using Gm-seq, we found that the methylation levels of CpG sites after treatment with BrdU, IdU, and I + B were mostly concentrated below 50%, while the distribution was more even in the untreated (DMSO) group, and in MEF it was biased towards above 50% (Fig. 5 D). The average percentage of CpG sites with a methylated C base was below 30% after treatment with BrdU, IdU, and IB, 50% in the DMSO group, and 60% in MEF (Figure S4A). Furthermore, we used a circos plot to show the distribution of methylation density on chromosomes. The size of each bin was 1,000,000, and the number of methylated Cytosine in each bin with different sequence environments (CpG, CHG, CHH) was counted. The distribution density of CpG sites in different sequence environments throughout the genome showed that after treatment with BrdU/IdU, a significant global hypomethylation state was observed (Figure S4B). We performed methylation differential analysis between treated and untreated cells with thymidine analogs. Interestingly, we found that the pluripotency related gene Sall4, Oct4, and Prdm14 loci exhibited significant hypomethylation after thymidine analog treatment (Fig. 5 E,G). This further confirms that BrdU/IdU creates a more open hypomethylated environment for the transformation of cell fate. In addition, we found that IdU exhibited a more widespread differential methylation distribution, followed by I + B, and BrdU exhibited the least, which may be related to its atomic size, where the atomic size of IdU > I + B > BrdU (Fig. 5 F). Differences in atomic size lead to variations in the degree of DNA damage. Using IdU alone would cause severe damage, while using BrdU alone wouldn’t have a significant impact. Therefore, I + B might be able to compensate for each other and generate a synergistic effect, BrdU/IdU can participate in cell fate regulation with negligible mutations As the substitute of thymidine, the safety of BrdU has always been a concern. Therefore, we performed mutation analysis on the samples treated with or without thymidine analogs and MEFs in our system, including SNP (Single Nucleotide Polymorphism), InDel (insertion-deletion), CNV (Copy Number Variant), and SV (Structural variation). We found that the mutation level of BrdU/IdU-treated samples was almost the same as that of MEFs (Fig. 6 A-E), while no treatment with thymidine analogs led to more mutations. In addition, we found that IdU had some insertion mutations. Overall, this suggests that the rational use of BrdU/IdU can participate in cell fate regulation with negligible mutations. BrdU and IdU have functions in various reprogramming systems To investigate whether the effects of BrdU and IdU are applicable in other reprogramming systems, we found that BrdU significantly improves reprogramming efficiency in the OKS system (Figure S5A-D, Fig. 7AB). Additionally, we found that BrdU can substitute for OCT4 and play a critical role in the KS system (Figure S5E,F,G, Fig. 7 A,B). Incorporation of BrdU not only improves reprogramming efficiency, but also accelerates reprogramming speed (Fig. 7 C). Interestingly, by comparing the effects of BrdU on OKS, KS, and CIP (Fig. 7 A,B), we found that their dependence on BrdU treatment time gradually increases. Specially, in OKS system, GFP + colonies reach their peak when BrdU is treated for 2 days. In KS system, GFP + colonies reach their peak when BrdU is treated for 6 days. Furthermore, in CIP system, 22 days of BrdU treatment is required to reach maximum number of GFP + colonies, as illustrated in Fig. 7 B. This suggests that different degrees of DNA damage may be required to overcome various barriers to reprogramming. We discovered that the addition of BrdU in the KS reprogramming system can specifically activate Gata4 , Sox17 , and certain developmental stem cell pluripotency regulating genes, as shown in Fig. 7 D, Additionally, we observed that the most significant difference occurred at D12, which was precisely the time point when genes such as Gata4 were highly expressed. We found that inhibiting the ATM signaling pathway with an ATM inhibitor also prevented BrdU from functioning in KS system (Fig. 7 E). By using GATA4 Cut & tag, we discovered that GATA4-enriched peaks were accompanied by ATAC opening (Fig. 7 F). By overexpressing Gata4 in the KS system under the presence of BrdU, the reprogramming process can be accelerated and the cell state can be improved (Fig. 7 G). Remarkably, our results bear a resemblance to the phenomenon observed in chemical reprogramming. Discussion In summary, we found that the use of the thymidine analogs BrdU/IdU causes epigenetic reshaping. This potentiates cells to enter a plastic state and accelerates the fate transition of cells by activating DNA damage repair and causing significant H3K27ac, H3K9ac and DNA demethylation. Additionally, the study discovered that the XEN state can be specifically activated after BrdU/IdU treatment. This suggest that XEN state may be a selective response behavior of cells to DNA damage repair, providing a shortcut for cells to overcome reprogramming barriers, and that different degree of DNA damage may be required to overcome various barriers to reprogramming. Furthermore, rational doesage of BrdU/IdU can participate in cell fate transition with negligible mutantations. In our study, through gene expression patterns, immunofluorescence of gH2AX (a marker for DNA double-strand breaks), ChIP-seq, and Comet assays, we confirmed that prolonged incorporation of BrdU/IdU causes a more severe form of DNA damage. Importantly, we did not observe any significant mutations, ruling out a role for non-homologous end joining (NHEJ). Therefore, we concluded that the homologous recombination repair (HRR) pathway becomes more dominant with prolonged incorporation of BrdU/IdU. DNA single-strand break causes “Discordant Transcription through Repair (DiThR)”( 8 ), while DNA double-strand break results in “Epigenetic Reshaping through Damage (ERD)”. However, it is worth to investigated the roles of other DNA repair pathways, such as MMR, NHRJ and NER, in the process of cellular fate transition. Additionally, not all of thymidine analogs are equally effective in somatic reprogramming, and their specific mechanisms of action remain to be fully explored. Interestingly, although BrdU and IdU are structurally very similar, with only minor differences in their chemical structure, the difference in atomic size may lead to a more pronounced impact of IdU on the entire genome. This difference in impact may also explain why other thymidine analogs are not effective. It is worth to consider that the XEN-like intermediate state is not unique to chemical reprogramming, as cells may require multiple intermediate states to reach their final fate during the process of fate transition. Reprogramming barriers may limit the process of fate transition, and thus cells may choose different intermediate states as shortcuts. Gata4 and Sox17 , among others, are representative of XEN states that have been selected as transitional states that facilitate cell fate transition because they promote both dedifferentiation and redifferentiation of cells, and play essential roles in embryonic development and cell regeneration. Moreover, DNA damage repair may also drive cells into a plastic state. When DNA damage occurs, cells need to repair the damage to maintain genome integrity. DNA repair processes may lead to reprogramming events, resulting in cells entering a plastic state and providing an alternative pathway for cell fate transition. Declarations Ethics approval and consent to participate Not applicable for that section. Consent for publication Not applicable for that section. Availability of data and materials The datasets generated during the current study are available in the National Center for Biotechnology Information under the accession number PRJCA019276 Competing interests The authors declare no competing financial interests. F unding This research was supported by grants from the National Key R&D Program of Chi-na (2018YFE0204800 [J.L.]), National Natural Science Foundation of China (32022019 [J.L.], U20A2013 [C.Z.], 31970681 [J.L.], 32100594 [C.Z.], 32200695 [S.Y.]), Guangdong Basic and Applied Basic Research Foundation (2020A1515110122 [L.W.], 2021A1515111156 [S.Y.], 2022A1515012267 [S.Y.]), Science and Technology Planning Project of Guangdong Province (2020B1212060052 [J.L.], 2022B1212010010 [Y.L.]), Health@InnoHK Program launched by the Innovation Technology Commission of the Hong Kong SAR, P.R. China, and the China Postdoctoral Science Foundation (2022M713172 [S.Y.]). Author contributions J.L. and C.L. initiated the project and designed the experiments. C.L. performed the chemical reprogramming experiments. C.L conducted and performed ATAC-seq, RNA-seq, Cut & tag library construction with X.X. S.C and Chen Li preformed bioinformatics analysis. X.X.performed OKS and KS reprogramming experiments with C.L. X.X performed bisulfite sequencing PCR. C.L performed GM-seq with Gene plus company. J.L. D.P. and H.W supervised the whole study. H.W supervised the whole study and wrote the manuscript. J.L conceived the whole study and approved the final version. Acknowledgements We would like to thank the support from the Guangzhou Regional Center of Bioscience Instrument of the Chinese Academy of Sciences. References B, G. The effects of incorporation of 5-halogenated deoxyuridines into the DNA of eukaryotic cells. Pharmacol Rev 29 , 249-272 (1977). Zhao, Y. et al. A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming. Cell 163 , 1678-1691 (2015). Long, Y. et al. Bromodeoxyuridine promotes full-chemical induction of mouse pluripotent stem cells. Cell Research 25 , 1171-1174 (2015). Ping, W. et al. 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Hutchins, A.P., Jauch, R., Dyla, M. & Miranda-Saavedra, D. glbase: a framework for combining, analyzing and displaying heterogeneous genomic and high-throughput sequencing data. Cell Regen 3 , 1 (2014). Chen, X. et al. Identification of DNA methylation and genetic alteration simultaneously from a single blood biopsy. Genes Genomics 45 , 627-635 (2023). Supplementary Files GraphicalAbstract.jpg SupplementaryFile.docx Cite Share Download PDF Status: Published Journal Publication published 16 Jan, 2024 Read the published version in Cell & Bioscience → Version 1 posted Editorial decision: Major revision 19 Dec, 2023 Reviewers invited by journal 20 Nov, 2023 Reviewers agreed at journal 14 Nov, 2023 Editor assigned by journal 06 Nov, 2023 First submitted to journal 02 Nov, 2023 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. 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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-3520658","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":249772703,"identity":"ee3efbb7-d0eb-4487-a56e-4968e3e7279a","order_by":0,"name":"chuang li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYBACPmYGBmYwi72B4TBjAxFa2OBaeA4Qq4UBpkUigYGZOC3sDIyfC9vs8uQjn188XLiDQZ6/gfnZAwIOY5ae2ZZcbHg7p+DwzDMMhjMOsJkbENDCxszbxpy4cXZOwmHeNgbGDQw8bBJEaKlP3DjzDFiLPbFaDifOl2A/ANKSSIwWZmmec8cTN/DkMBye2SaRPOMwmxleLfz8Bxg/85RVJ85vP/4YGHQ2tv3tzc/wagFq+gCmDA7wgMJJAhZNRAD5BvYHxKodBaNgFIyCEQYAAcA+EUtxfn0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4984-5066","institution":"USTC: University of Science and Technology of China","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"chuang","middleName":"","lastName":"li","suffix":""},{"id":249772704,"identity":"30ba9849-8140-449c-98bd-e94225331a6c","order_by":1,"name":"Xiaoduo Xu","email":"","orcid":"","institution":"USTC: University of Science and Technology of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoduo","middleName":"","lastName":"Xu","suffix":""},{"id":249772705,"identity":"da16c88b-f478-4787-9c12-70c91234f540","order_by":2,"name":"Shuyan Chen","email":"","orcid":"","institution":"GIBH: Guangzhou Institutes of Biomedicine and Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuyan","middleName":"","lastName":"Chen","suffix":""},{"id":249772706,"identity":"6f7f46db-5889-4e64-aed0-73bf08fd96fb","order_by":3,"name":"Anchun Xu","email":"","orcid":"","institution":"USTC: University of Science and Technology of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anchun","middleName":"","lastName":"Xu","suffix":""},{"id":249772707,"identity":"27ed88cb-28d4-4baf-bb8d-2395a05582cf","order_by":4,"name":"Tongxing Guan","email":"","orcid":"","institution":"USTC: University of Science and Technology of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tongxing","middleName":"","lastName":"Guan","suffix":""},{"id":249772708,"identity":"d7f43eda-0800-4d89-881c-b74f638614c1","order_by":5,"name":"haokaifeng wu","email":"","orcid":"","institution":"Chinese Academy of Sciences Hong Kong Institute of Science and Innovation Centre for Regenerative Medicine and Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"haokaifeng","middleName":"","lastName":"wu","suffix":""},{"id":249772709,"identity":"66540b47-565d-4ca7-bc36-584c8b6dbbd9","order_by":6,"name":"duanqing pei","email":"","orcid":"","institution":"USTC: University of Science and Technology of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"duanqing","middleName":"","lastName":"pei","suffix":""},{"id":249772710,"identity":"173c0f22-4c32-4711-820e-6042bdd3a267","order_by":7,"name":"Jing Liu","email":"","orcid":"https://orcid.org/0000-0003-1600-7744","institution":"GIBH: Guangzhou Institutes of Biomedicine and Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2023-10-31 07:10:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3520658/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3520658/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13578-024-01192-x","type":"published","date":"2024-01-16T15:00:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":46664752,"identity":"224e07fc-3612-4e0f-9758-c76ec28f9ed1","added_by":"auto","created_at":"2023-11-17 16:54:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":84788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBrdU and IdU: Essential in overcoming two barriers during chemical induction of pluripotency.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Schematic diagram of the induction of CiPSCs from MEFs. (\u003cstrong\u003eB\u003c/strong\u003e) Morphological changes at two distinct barriers during induction of CiPSCs. Scale bar 100 μm. (\u003cstrong\u003eC\u003c/strong\u003e) Number of Oct4-GFP+CiPSC colonies generated under indicated conditions. (\u003cstrong\u003eD\u003c/strong\u003e) Morphological changes at distinct time points during induction of CiPSCs treated with BrdU or IdU. Scale bar 100 \u003cem\u003eμ\u003c/em\u003em. (\u003cstrong\u003eE\u003c/strong\u003e) Number of Oct4-GFP+CiPSC colonies generated under different treatment conditions.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3520658/v1/405693f7e6fcaaaf447bf581.jpg"},{"id":46665808,"identity":"3a240b7b-27b8-412d-8628-1af1672c690d","added_by":"auto","created_at":"2023-11-17 17:02:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":204177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression dynamics and chromatin accessibility dynamics during CIP with or without BrdU/IdU.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Schematic diagram of the induction of CiPSCs from MEFs. (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap of RNA-seq for CIP at D8,14,20 and 26 with or without BrdU or IdU. (\u003cstrong\u003eC\u003c/strong\u003e)The global chromatin status of CO/OC and PO for CIP at D8,14,20,26 and 40 with or without BrdU or IdU. (\u003cstrong\u003eD\u003c/strong\u003e) Representative fibroblast genes, XEN genes and pluripotency genes peaks from ATAC-seq for CIP at D8,14,20 and 26 with or without BrdU or IdU. (\u003cstrong\u003eE\u003c/strong\u003e) The global chromatin status of CO/OC and PO arranged as groups for CIP at D8,14,20,26 and 40 with or without BrdU or IdU. (\u003cstrong\u003eF\u003c/strong\u003e) TF motifs enriched at least 2 fold for CO/OC loci defined by ATAC-seq peaks with or without BrdU or IdU. TF families are indicated on the right of the heatmap. (\u003cstrong\u003eG\u003c/strong\u003e) Statistical analysis of number of total peaks in CO1-6/OC1-6. (\u003cstrong\u003eH\u003c/strong\u003e) Venn diagrams of OC1-6/CO1-6 peaks between samples with or without BrdU or IdU.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3520658/v1/9a2f0b86c90a4f0af2c2a8b5.jpg"},{"id":46664756,"identity":"efd767cd-419e-4891-a5d3-a23a7aeb4ca5","added_by":"auto","created_at":"2023-11-17 16:54:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncorporation of BrdU/IdU leads to DNA damage repair.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Venn plots for overlapping genes in the upregulated groups between BrdU, IdU, and BrdU+IdU versus the control. (\u003cstrong\u003eB\u003c/strong\u003e) Enriched GO functions in upregulated groups. (\u003cstrong\u003eC\u003c/strong\u003e) Heatmap of DNA-repair genes related RNA-seq for CIP at D8,14,20 and 26 with or without BrdU or IdU. (\u003cstrong\u003eD\u003c/strong\u003e) Alkaline comet assays of samples with or without BrdU or IdU. Scale bar 50 μm. (\u003cstrong\u003eE\u003c/strong\u003e) Statistical analysis of olive tailmomer from (\u003cstrong\u003eD\u003c/strong\u003e). (\u003cstrong\u003eF\u003c/strong\u003e) Statistical analysis of mean tailmomer from (\u003cstrong\u003eE\u003c/strong\u003e). (\u003cstrong\u003eG\u003c/strong\u003e) Immunofluorescent analysis to observe the spatial colocalization of BrdU (in red) and γ-H2AX (in green). Scale bar 40 μm. (\u003cstrong\u003eH\u003c/strong\u003e) Representative peaks from ATAC-seq aligned with γ-H2AX Cut \u0026amp; tag signals for Gata4 and Sox17 with or without BrdU or IdU.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3520658/v1/30daa92446cf7e0e1f38a210.jpg"},{"id":46665805,"identity":"822ba4dd-0112-4e69-a004-39ee393c6b12","added_by":"auto","created_at":"2023-11-17 17:02:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":117831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanism of DDR in promoting somatic cell reprogramming. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eA model for DNA damage repair (DDR) mechanism during BrdU/IdU incorporation. (\u003cstrong\u003eB\u003c/strong\u003e) Morphological changes during induction of CiPSCs treated with or without ATM inhibitor. Scale bar 100 μm. (\u003cstrong\u003eC\u003c/strong\u003e) Number of Oct4-GFP+CiPSC colonies generated under different treatment conditions. (\u003cstrong\u003eD\u003c/strong\u003e) Representative Gata4, Sox17 and Sall4 peaks from H3K27ac Cut \u0026amp; tag aligned with H3K9ac Cut \u0026amp; tag signals for CIP at D20 with or without BrdU or IdU. (\u003cstrong\u003eE\u003c/strong\u003e) Morphological changes during induction of CiPSCs treated with different concentration of VPA. Scale bar 100 μm. (\u003cstrong\u003eF\u003c/strong\u003e) Number of Oct4-GFP+CiPSC colonies generated under different treatment conditions. (\u003cstrong\u003eG\u003c/strong\u003e) GATA4 Cut \u0026amp; tag analysis and ATAC-seq analysis for CIP at D20 and D26 under different treatment conditions. (\u003cstrong\u003eH\u003c/strong\u003e) Representative Oct4, Sall4 and Aqp8 peaks from ATAC-seq aligned with H3K27ac Cut \u0026amp; tag and GATA4 Cut \u0026amp; tag for CIP at D20 with or without BrdU or IdU.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3520658/v1/0a4cb08f9ffecc1183f44e9e.jpg"},{"id":46664751,"identity":"14684634-0533-4fc4-931b-332d0a10843f","added_by":"auto","created_at":"2023-11-17 16:54:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":470086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBrdU/IdU creates a more open hypomethylated environment for the transformation of cell fate. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) The methylation patterns of Aqp8 and Pthr1 when treated with or without BrdU or IdU. (\u003cstrong\u003eB\u003c/strong\u003e)Morphological changes at D30 during induction of CiPSCs treated with DNMT inhibitor CM272 and SGI1027. (\u003cstrong\u003eC\u003c/strong\u003e) FSC analysis of Oct4-GFP colonies generated under different treatment conditions. (\u003cstrong\u003eD\u003c/strong\u003e) Histogram of percentage CpG methylation generated under different treatment conditions. (\u003cstrong\u003eE\u003c/strong\u003e) Methylation differential analysis between treated and untreated cells with IdU/BrdU. (\u003cstrong\u003eF\u003c/strong\u003e) Genomic panorama differential methylation regional distribution. (\u003cstrong\u003eG\u003c/strong\u003e) Differential DNA methylation gene and annotations.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3520658/v1/67c5f36b02289710e2ea0312.jpg"},{"id":46665806,"identity":"5ddaaacf-5be5-4a2e-b776-d1d1bdd66d0a","added_by":"auto","created_at":"2023-11-17 17:02:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":169929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBrdU/IdU can participate in cell fate regulation with negligible mutations. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Mutation analysis-SNP (Single Nucleotide Polymorphism) on the samples treated with or without BrdU/IdU and MEFs. (\u003cstrong\u003eB\u003c/strong\u003e) InDel (insertion-deletion) analysis. (\u003cstrong\u003eC\u003c/strong\u003e) CNV (Copy Number Variant) analysis. (\u003cstrong\u003eD\u003c/strong\u003e) SV (Structural variation) analysis. (\u003cstrong\u003eE\u003c/strong\u003e) Genomic panorama variation analysis.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3520658/v1/34e105347d7de3595ce97735.jpg"},{"id":46664759,"identity":"a8821ccb-2c19-42cb-93c6-646d50b6718f","added_by":"auto","created_at":"2023-11-17 16:54:20","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":142860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBrdU and IdU have functions in various reprogramming systems. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Images of GFP+ colonies taken by fluorescence microscope in situ. Scale bar 5mm. (\u003cstrong\u003eB\u003c/strong\u003e) Number of Oct4-GFP colonies generated under indicated conditions. (\u003cstrong\u003eC\u003c/strong\u003e) FSC analysis of Oct4-GFP colonies generated under different treatment conditions. (\u003cstrong\u003eD\u003c/strong\u003e) Heatmap of RNA-seq for KS at D4,8,12 and 16 with or without BrdU or IdU. (\u003cstrong\u003eE\u003c/strong\u003e) Morphological changes during induction of KS treated with ATM inhibitor and corresponding statistical analysis. Scale bar 50 μm. (\u003cstrong\u003eF\u003c/strong\u003e) GATA4 Cut \u0026amp; tag analysis and ATAC-seq analysis for CIP at D4,8,12 and 16 under different treatment conditions. (\u003cstrong\u003eG\u003c/strong\u003e) Morphological changes during induction of KS when overexpressing Gata4 in the system. Scale bar 50 μm\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3520658/v1/912d03792d1d56424a4eab77.jpg"},{"id":49978415,"identity":"3e0aca7c-57ba-4cc3-b41e-41c8d01d5ce1","added_by":"auto","created_at":"2024-01-22 15:03:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1269993,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3520658/v1/513122ec-cfb4-4d8e-90fd-7bfa6a71ecb7.pdf"},{"id":46665999,"identity":"7c94e637-0a37-43d9-ad89-7ad6b47071a0","added_by":"auto","created_at":"2023-11-17 17:10:20","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":78339,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3520658/v1/02a26c6832d82d6b948239cf.jpg"},{"id":46664758,"identity":"d3eb7601-2954-4130-9257-6e882bc13877","added_by":"auto","created_at":"2023-11-17 16:54:20","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2690195,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-3520658/v1/755273b908cb82227471bb91.docx"}],"financialInterests":"","formattedTitle":"Epigenetic Reshaping through Damage: Promoting Cell Fate Transition by BrdU and IdU incorportion","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThymidine analogs are often considered to incorporate randomly into DNA sequences, introducing inherent unpredictability and randomness in their mechanisms of action(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Despite this inherent nature, they play a critical role in the precise regulation of cell fate(\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In a study conducted by Xie et al., the utilization of BrdU, widely employed to label proliferating cells in vivo, has demonstrated its significant potential in facilitating chemical induction of pluripotency(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The study by Cao et al., sheds light on how BrdU can impact the reorganization of nuclear architecture and influence the cell fate decisions(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). IdU, which is also a thymidine analog was reported to cause stochastic fluctuations in gene expression to facilitate cellular reprogramming(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, the precise mechanisms through which BrdU/IdU facilitates cell reprogramming are not yet fully understood. BrdU/IdU may be involved in cell reprogramming through mechanisms beyond transcriptional fluctuations. This could include the impact of thymidine analogs on DNA structure, repair mechanisms, or other cellular processes, providing a potential shortcut for cells to overcome reprogramming barriers. As a result, thymidine analogs may play a more crucial and multifaceted role in regulating cell fate, extending beyond the induction of transcriptional fluctuations alone. This study links DNA damage repair with genome, epigenetics and cell fate regulation, providing us with new understanding and helping to advance the field of cell fate decision and regenerative medicine.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBrdU and IdU: Essential in overcoming two barriers during chemical induction of pluripotency\u003c/h2\u003e \u003cp\u003eTo determine the optimal duration of BrdU treatment for chemical induction of pluripotency (CIP), a series of time windows were optimized and observed for their impact on CIP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Two main barriers were found in the CIP reprogramming process, and BrdU was found to play a crucial role in overcoming these barriers. BrdU dropout experiments showed that dropout of BrdU at barrier I resulted in a failure to form colonies at Day 22, while dropout at barrier II between Day 12\u0026ndash;22 resulted in a failure to form GFP\u003csup\u003e+\u003c/sup\u003e colonies at Day 40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Meanwhile, we have established a time gradient for each barrier (Figure S1A), and breaking through the barrier requires at least 6 days of BrdU incorporation, which must be added within the first 4 days (Figure S1B). Increasing the duration of BrdU treatment led to an increase in the number of GFP\u003csup\u003e+\u003c/sup\u003e colonies, with a 0\u0026ndash;22 days treatment resulting in over 240 GFP\u003csup\u003e+\u003c/sup\u003e colonies at Day 40 from a starting population of 20,000 cells, achieving an efficiency of 1.2%. In contrast, a 0\u0026ndash;12 days treatment only resulted in about 20 GFP\u003csup\u003e+\u003c/sup\u003e colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Furthermore, we attempted to use three base analogs, namely IdU, EdU, and 5Aza, but only IdU was found to be capable of replacing the function of BrdU during CIP reprogramming (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). The combination of both thymidine analogs can increase the selectivity of cells, so that only cells that have been correctly reprogrammed can survive; hence accelerated the reprogramming process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD,E and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). Thus, it can be concluded that BrdU and IdU are essential in overcoming two main barriers during CIP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGene expression dynamics and chromatin accessibility dynamics during CIP with or without BrdU/IdU\u003c/h2\u003e \u003cp\u003eTo identify the two barriers in which BrdU and IdU are involved during CIP reprogramming, RNA-seq and ATAC-seq was performed on MEFs undergoing CIP with or without BrdU, IdU and I\u0026thinsp;+\u0026thinsp;B (IdU\u0026thinsp;+\u0026thinsp;BrdU) at D8, 14, 20, 26 and 40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). It was found that loss of BrdU and IdU at barrier I resulted in incomplete reprogramming of MEFs to XEN-like cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The expression of XEN-like genes such as \u003cem\u003eGata4\u003c/em\u003e, \u003cem\u003eSox17\u003c/em\u003e and \u003cem\u003eAqp8\u003c/em\u003e increased significantly with increasing duration of thymidine analogs treatment and reached a peak at D20. Conversely, dropout of BrdU resulted in a failure to express XEN-like genes (Figure S2A,C), suggesting that MEFs undergoing reprogramming without BrdU /IdU follow a different fate path. Previous studies have reported that CIP goes through an XEN-like intermediate stage(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Additionally, loss of BrdU and IdU at barrier II resulted in incomplete reprogramming of XEN-like cells to CiPSCs. Pluripotency genes such as \u003cem\u003eOct4\u003c/em\u003e, \u003cem\u003eEsrrb, Tfcp2l1\u003c/em\u003e, \u003cem\u003eNanog\u003c/em\u003e and \u003cem\u003eSox2\u003c/em\u003e were highly activated in a BrdU/IdU dependent manner during stage 2. In addition, the incorporation of I\u0026thinsp;+\u0026thinsp;B showed a faster transition towards to iPSCs compared to BrdU/IdU treatment at the RNA level (Figure S2B,D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe function state of a cell is determined by its genome architecture. To investigate the role of BrdU/IdU in CIP, we mapped chromatin accessibility dynamics (CADs) during CIP with or without BrdU/IdU and found that many loci opened during CIP failed to open without BrdU/IdU. Moreover, detailed analysis of the ATAC-seq datasets revealed many similarities between BrdU and IdU treatments. Chromatin accessibility significantly increased when MEFs undergoing CIP were treated with I\u0026thinsp;+\u0026thinsp;B compared to only BrdU or IdU treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, we analyzed the loci near fibroblast marker gene \u003cem\u003eTwist2\u003c/em\u003e and \u003cem\u003eFbn1\u003c/em\u003e found that they were more open in DMSO treatment compared to BrdU/IdU treatment. Conversely, loci near XEN marker genes such as \u003cem\u003eGata4\u003c/em\u003e and \u003cem\u003eSall4\u003c/em\u003e were only open in BrdU/IdU treatment. Loci near the pluripotency marker gene \u003cem\u003eOct4\u003c/em\u003e and \u003cem\u003eTfcp2l1\u003c/em\u003e were also only open in BrdU/IdU treatment, and the chromatin accessibility was much higher when samples were treated with both I\u0026thinsp;+\u0026thinsp;B.\u003c/p\u003e \u003cp\u003eWe compared the peaks at each locus between these samples and classified them into three categories: closed in MEFs but open in ESCs (CO), open in MEFs but closed in ESCs (OC), and permanent open (PO). Further, we divided the CO and CO peaks into subgroups based on the day of reprogramming to illustrate the progression of cellular reprogramming associated CADs, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE. To further investigate the molecular mechanism of BrdU/IdU during CIP, we performed motif analysis, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF. Loci containing motifs for OCT2, 4, 6, and 11 gradually opened, peaking at CO6 when treated with BrdU/IdU. Loci with motifs for GATA1, 2, 4, 6, KLF3-6, and SOX2-4, 6, 9, 10, 15, 17 opened from CO1-6 when treated with BrdU/IdU. However, the TF motifs profile was similar with or without BrdU/IdU in OC loci, indicating that BrdU/IdU treatment tended to open up loci that are enriched with motifs binding to TFs from the GATA, KLF, and SOX families.\u003c/p\u003e \u003cp\u003eWe then statistically analyzed the number of total peaks in CO1-6/OC1-6, respectively, and presented the results in the form of a Venn diagram, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eG,H. The diagram showed that BrdU/IdU/I\u0026thinsp;+\u0026thinsp;B shared 25.2% (8240/32655) in CO peaks, but only 19.3% (7243/37575) in OC peaks. Figure S2E illustrates that a larger fraction of peaks in CO are located in the promoter regions. In conclusion, the function of BrdU/IdU is more biased towards opening chromatin and activating gene expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIncorporation of BrdU/IdU leads to DNA damage repair\u003c/h2\u003e \u003cp\u003eAccording to the RNA-seq analysis we obtained in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e, we then performed Venn plots and enriched GO functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B and Figure S3A,B). Interestingly, Venn plots for overlapping genes in the upregulated groups between BrdU, IdU, and I\u0026thinsp;+\u0026thinsp;B versus the control (without thymidine analogs) showed that DNA repair-related GO functions were highly enriched when MEFs were treated with BrdU/IdU during CIP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B). Specifically, DNA repair-related gene expression was highly upregulated in all three groups. The main repair pathways are base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination repair (HRR) and non-homologous end joining repair (NHEJ). In our study, we observed that \u003cem\u003eApex1\u003c/em\u003e, a gene related to BER pathway, was significantly upregulated in the early stages of reprogramming after treatment with BrdU/IdU, but gradually decreased with longer exposure. The HRR related genes \u003cem\u003eBrca1\u003c/em\u003e and \u003cem\u003eBrca2\u003c/em\u003e were upregulated as \u003cem\u003eApex1\u003c/em\u003e expression decreased (Figure S3C). Additionally, a combination of both thymidine analogs (I\u0026thinsp;+\u0026thinsp;B) accelerated the DNA repair process, as shown by the early upregulation of DNA repair genes expression at D8 in this group compared to the BrdU or IdU only groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify the relationship between thymidine analogs and DNA damage repair, alkaline comet assays were performed to compare the olive tail moment difference between samples with or without thymidine analogs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD,E). Interestingly, the BrdU treatment showed an increased olive tail moment indicating significant DNA damage. Furthermore, the degree of DNA damage was further promoted by a combination of BrdU and IdU. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eF shows two DNA damage peaks during the CIP process at D14 and D24, which is coherent with the time window of the two main barriers discussed in the previous sections. This evidence illustrates a close association between DNA damage/repair and thymidine analogues assisting CIP through two barriers.\u003c/p\u003e \u003cp\u003eTo verify whether BrdU treatment directly causes DNA damage, immunofluorescent analysis was performed to observe the spatial colocalization of BrdU (in red) and γ-H2AX (in green), a novel biomarker for DNA double-strand breaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). The dropout of BrdU showed a much weaker γ-H2AX signal, leading us to conclude that the accumulation of DNA damage during the CIP process is directly caused by BrdU binding to the DNA. Using CHIP-seq analysis targeting the DNA double-strand damage marker γ-H2AX, a correlation was found between its level and the ATAC-seq for \u003cem\u003eGata4\u003c/em\u003e and \u003cem\u003eSox17\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). This suggests that the activation of the \u003cem\u003eGata\u003c/em\u003e4 and \u003cem\u003eSox\u003c/em\u003e17 genes is mediated through the DNA damage repair pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMechanism of DDR in promoting somatic cell reprogramming\u003c/h2\u003e \u003cp\u003eATM (Ataxia Telangiectasia Mutated) is a protein kinase that plays a critical role in the cellular response to DNA damage, particularly double-strand breaks (DSBs). When DSBs occur, the MRN complex (Mre11-Rad50-Nbs1) recognizes and binds to the site of break, recruiting ATM. Subsequently, ATM undergoes autophosphorylation, resulting in its full activation. Once activated, ATM phosphorylates several downstream targets involved in cell cycle checkpoint control, DNA repair, and apoptosis. Additionally, ATM activation is sustained by a phosphorylation-acetylation cascade. This cascade helps to maintain ATM activity and promote efficient DNA repair. Overall, ATM serves as a critical player in the cellular response to DNA damage, and its activation and downstream signaling are tightly regulated to ensure proper DNA repair and maintenance of genomic integrity(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn order to understand how DDR is involved in regulating somatic cell reprogramming, we hypothesize that DDR can upregulate the acetylation levels of genes at the site of DNA damage, thereby regulating gene expression, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Firstly, we found that inhibiting the ATM signaling pathway with an ATM inhibitor KU-55933 prevented BrdU from functioning (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,C). Furthermore, we revealed that \u003cem\u003eGata4\u003c/em\u003e, \u003cem\u003eSox17\u003c/em\u003e and \u003cem\u003eSall4\u003c/em\u003e's H3KC27ac and H3K9ac were significantly upregulated under the treatment of BrdU and I\u0026thinsp;+\u0026thinsp;B (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). To further verify this hypothesis, we increased the concentration of VPA (a histone deacetylase inhibitor) to further promote acetylation accumulation. We found that this significantly accelerated the process of chemical reprogramming, and its acceleration effect depended on the addition of BrdU (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE,F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTherefore, we believe that BrdU/IdU induces DNA damage by incorporating into the genome, and recruits ATM to the site of damage. Subsequently, the acetylation levels of gene loci at the site of DNA damage is upregulated through a series of phosphorylation and acetylation cascades, thereby altering chromatin accessibility and activating gene expression.\u003c/p\u003e \u003cp\u003eCombined with the results of RNA-seq, ATAC-seq, motif, γ-H2AX, H3K9ac, and H3K27ac analysis, we found that \u003cem\u003eGata4\u003c/em\u003e has a high degree of specificity under the treatment of BrdU/IdU (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eH,\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, Figure S2A). Therefore, GATA4 may be one of the specific downstream factors of BrdU/IdU involved in somatic cell reprogramming. Further Cut \u0026amp; tag analysis showed that the binding sites of GATA4 were significantly increased under the treatment of BrdU/IdU, and the sites enriched with GATA4 were accompanied by higher chromatin accessibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), including pluripotency gene loci such as \u003cem\u003eOct4\u003c/em\u003e, \u003cem\u003eSall4\u003c/em\u003e as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eH. This indicates that GATA4 can respond to DDR, thereby promoting somatic cell reprogramming.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBrdU/IdU creates a more open hypomethylated environment for the transformation of cell fate\u003c/h2\u003e \u003cp\u003eBrdU and IdU have been demonstrated to play a crucial role in chemical reprogramming, exhibiting two distinct stages of action. Hence we postulate that BrdU not only activates intermediate XEN genes such as \u003cem\u003eGata4\u003c/em\u003e, but also potentially induces DNA demethylation, where DNA methylation is a major regulatory factor that limits GATA4 function(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). To confirm this, we measured the methylation levels of GATA4 downstream gene \u003cem\u003eAqp8\u003c/em\u003e and XEN gene \u003cem\u003ePth1r.\u003c/em\u003e We observed a significant decrease in DNA methylation levels with BrdU/IdU treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), indicating that BrdU/IdU not only activates gene expression but also induces DNA demethylation, thus creating a conducive environment for pluripotency network activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also integrated the aspect of DNA damage repair and hypothesized that BrdU and IdU act through double-stranded break excision and new chain synthesis during the process of DNA damage repair. Since new chain synthesis occurs without DNA methylation marks, it leads to DNA demethylation. To validate this hypothesis, we inhibited DNMT using CM272 and SGI1027 to prevent the re-methylation of new chains. The results indicated that this approach significantly accelerated the process of chemical reprogramming (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and C), and this effect was dependent on the presence of BrdU. To summarize, as the degree of BrdU damage increases, the frequency of double-stranded breakage and synthesis also increases, which leads to DNA demethylation, thereby creating a more open hypomethylated environment for the transformation of cell fate.\u003c/p\u003e \u003cp\u003eIn the global genome methylation analysis using Gm-seq, we found that the methylation levels of CpG sites after treatment with BrdU, IdU, and I\u0026thinsp;+\u0026thinsp;B were mostly concentrated below 50%, while the distribution was more even in the untreated (DMSO) group, and in MEF it was biased towards above 50% (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The average percentage of CpG sites with a methylated C base was below 30% after treatment with BrdU, IdU, and IB, 50% in the DMSO group, and 60% in MEF (Figure S4A). Furthermore, we used a circos plot to show the distribution of methylation density on chromosomes. The size of each bin was 1,000,000, and the number of methylated Cytosine in each bin with different sequence environments (CpG, CHG, CHH) was counted. The distribution density of CpG sites in different sequence environments throughout the genome showed that after treatment with BrdU/IdU, a significant global hypomethylation state was observed (Figure S4B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe performed methylation differential analysis between treated and untreated cells with thymidine analogs. Interestingly, we found that the pluripotency related gene Sall4, Oct4, and Prdm14 loci exhibited significant hypomethylation after thymidine analog treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eE,G). This further confirms that BrdU/IdU creates a more open hypomethylated environment for the transformation of cell fate. In addition, we found that IdU exhibited a more widespread differential methylation distribution, followed by I\u0026thinsp;+\u0026thinsp;B, and BrdU exhibited the least, which may be related to its atomic size, where the atomic size of IdU\u0026thinsp;\u0026gt;\u0026thinsp;I\u0026thinsp;+\u0026thinsp;B\u0026thinsp;\u0026gt;\u0026thinsp;BrdU (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Differences in atomic size lead to variations in the degree of DNA damage. Using IdU alone would cause severe damage, while using BrdU alone wouldn\u0026rsquo;t have a significant impact. Therefore, I\u0026thinsp;+\u0026thinsp;B might be able to compensate for each other and generate a synergistic effect,\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBrdU/IdU can participate in cell fate regulation with negligible mutations\u003c/h2\u003e \u003cp\u003eAs the substitute of thymidine, the safety of BrdU has always been a concern. Therefore, we performed mutation analysis on the samples treated with or without thymidine analogs and MEFs in our system, including SNP (Single Nucleotide Polymorphism), InDel (insertion-deletion), CNV (Copy Number Variant), and SV (Structural variation). We found that the mutation level of BrdU/IdU-treated samples was almost the same as that of MEFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-E), while no treatment with thymidine analogs led to more mutations. In addition, we found that IdU had some insertion mutations. Overall, this suggests that the rational use of BrdU/IdU can participate in cell fate regulation with negligible mutations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBrdU and IdU have functions in various reprogramming systems\u003c/h2\u003e \u003cp\u003eTo investigate whether the effects of BrdU and IdU are applicable in other reprogramming systems, we found that BrdU significantly improves reprogramming efficiency in the OKS system (Figure S5A-D, Fig.\u0026nbsp;7AB). Additionally, we found that BrdU can substitute for OCT4 and play a critical role in the KS system (Figure S5E,F,G, Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,B). Incorporation of BrdU not only improves reprogramming efficiency, but also accelerates reprogramming speed (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Interestingly, by comparing the effects of BrdU on OKS, KS, and CIP (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,B), we found that their dependence on BrdU treatment time gradually increases. Specially, in OKS system, GFP\u003csup\u003e+\u003c/sup\u003e colonies reach their peak when BrdU is treated for 2 days. In KS system, GFP\u003csup\u003e+\u003c/sup\u003e colonies reach their peak when BrdU is treated for 6 days. Furthermore, in CIP system, 22 days of BrdU treatment is required to reach maximum number of GFP\u003csup\u003e+\u003c/sup\u003e colonies, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. This suggests that different degrees of DNA damage may be required to overcome various barriers to reprogramming.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe discovered that the addition of BrdU in the KS reprogramming system can specifically activate \u003cem\u003eGata4\u003c/em\u003e, \u003cem\u003eSox17\u003c/em\u003e, and certain developmental stem cell pluripotency regulating genes, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, Additionally, we observed that the most significant difference occurred at D12, which was precisely the time point when genes such as \u003cem\u003eGata4\u003c/em\u003e were highly expressed. We found that inhibiting the ATM signaling pathway with an ATM inhibitor also prevented BrdU from functioning in KS system (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). By using GATA4 Cut \u0026amp; tag, we discovered that GATA4-enriched peaks were accompanied by ATAC opening (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). By overexpressing \u003cem\u003eGata4\u003c/em\u003e in the KS system under the presence of BrdU, the reprogramming process can be accelerated and the cell state can be improved (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Remarkably, our results bear a resemblance to the phenomenon observed in chemical reprogramming.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn summary, we found that the use of the thymidine analogs BrdU/IdU causes epigenetic reshaping. This potentiates cells to enter a plastic state and accelerates the fate transition of cells by activating DNA damage repair and causing significant H3K27ac, H3K9ac and DNA demethylation. Additionally, the study discovered that the XEN state can be specifically activated after BrdU/IdU treatment. This suggest that XEN state may be a selective response behavior of cells to DNA damage repair, providing a shortcut for cells to overcome reprogramming barriers, and that different degree of DNA damage may be required to overcome various barriers to reprogramming. Furthermore, rational doesage of BrdU/IdU can participate in cell fate transition with negligible mutantations.\u003c/p\u003e \u003cp\u003eIn our study, through gene expression patterns, immunofluorescence of gH2AX (a marker for DNA double-strand breaks), ChIP-seq, and Comet assays, we confirmed that prolonged incorporation of BrdU/IdU causes a more severe form of DNA damage. Importantly, we did not observe any significant mutations, ruling out a role for non-homologous end joining (NHEJ). Therefore, we concluded that the homologous recombination repair (HRR) pathway becomes more dominant with prolonged incorporation of BrdU/IdU. DNA single-strand break causes \u0026ldquo;Discordant Transcription through Repair (DiThR)\u0026rdquo;(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), while DNA double-strand break results in \u0026ldquo;Epigenetic Reshaping through Damage (ERD)\u0026rdquo;. However, it is worth to investigated the roles of other DNA repair pathways, such as MMR, NHRJ and NER, in the process of cellular fate transition. Additionally, not all of thymidine analogs are equally effective in somatic reprogramming, and their specific mechanisms of action remain to be fully explored. Interestingly, although BrdU and IdU are structurally very similar, with only minor differences in their chemical structure, the difference in atomic size may lead to a more pronounced impact of IdU on the entire genome. This difference in impact may also explain why other thymidine analogs are not effective.\u003c/p\u003e \u003cp\u003eIt is worth to consider that the XEN-like intermediate state is not unique to chemical reprogramming, as cells may require multiple intermediate states to reach their final fate during the process of fate transition. Reprogramming barriers may limit the process of fate transition, and thus cells may choose different intermediate states as shortcuts. \u003cem\u003eGata4\u003c/em\u003e and \u003cem\u003eSox17\u003c/em\u003e, among others, are representative of XEN states that have been selected as transitional states that facilitate cell fate transition because they promote both dedifferentiation and redifferentiation of cells, and play essential roles in embryonic development and cell regeneration. Moreover, DNA damage repair may also drive cells into a plastic state. When DNA damage occurs, cells need to repair the damage to maintain genome integrity. DNA repair processes may lead to reprogramming events, resulting in cells entering a plastic state and providing an alternative pathway for cell fate transition.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable for that section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable for that section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available in the National Center for Biotechnology Information under the accession number PRJCA019276\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003cstrong\u003eunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the National Key R\u0026amp;D Program of Chi-na (2018YFE0204800 [J.L.]), National Natural Science Foundation of China (32022019 [J.L.], U20A2013 [C.Z.], 31970681 [J.L.], 32100594 [C.Z.], 32200695 [S.Y.]), Guangdong Basic and Applied Basic Research Foundation (2020A1515110122 [L.W.], 2021A1515111156 [S.Y.], 2022A1515012267 [S.Y.]), Science and Technology Planning Project of Guangdong Province (2020B1212060052 [J.L.], 2022B1212010010 [Y.L.]), Health@InnoHK Program launched by the Innovation Technology Commission of the Hong Kong SAR, P.R. China, and the China Postdoctoral Science Foundation (2022M713172 [S.Y.]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.L. and C.L. initiated the project and designed the experiments. C.L. performed the chemical reprogramming experiments. C.L conducted and performed ATAC-seq, RNA-seq, Cut \u0026amp; tag library construction with X.X. S.C and Chen Li preformed bioinformatics analysis. X.X.performed OKS and KS reprogramming experiments with C.L. X.X performed bisulfite sequencing PCR. C.L performed GM-seq with Gene plus company. J.L. D.P. and H.W supervised the whole study. H.W supervised the whole study and wrote the manuscript. J.L conceived the whole study and approved the final version.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the support from the Guangzhou Regional Center of Bioscience Instrument of the Chinese Academy of Sciences.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eB, G. The effects of incorporation of 5-halogenated deoxyuridines into the DNA of eukaryotic cells. \u003cem\u003ePharmacol Rev\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 249-272 (1977).\u003c/li\u003e\n\u003cli\u003eZhao, Y. et al. A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e163\u003c/strong\u003e, 1678-1691 (2015).\u003c/li\u003e\n\u003cli\u003eLong, Y. et al. 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Identification of DNA methylation and genetic alteration simultaneously from a single blood biopsy. \u003cem\u003eGenes Genomics\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 627-635 (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-bioscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbio","sideBox":"Learn more about [Cell \u0026 Bioscience](http://cellandbioscience.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cbio/default.aspx","title":"Cell \u0026 Bioscience","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3520658/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3520658/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003eThymidine analogs have long been recognized for their ability to randomly incorporate into DNA. However, the precise mechanisms through which thymidine analogs facilitate cell fate transition remains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eHere, we discovered a strong correlation between the dosage dependence of thymidine analogs and their ability to overcome reprogramming barrier. Meanwhile, we found that homologous recombination repair (HRR) pathway causes an overall epigenetic reshaping of cells and enabling them to overcome greater barriers. Moreover, extraembryonic endoderm (XEN) state appears to be a selective response behavior of cells to DNA damage repair (DDR), providing a shortcut for cells to overcome reprogramming barriers, creates a hypomethylated environment that promotes cell fate transition in multiple reprogramming systems. We term this mechanism as Epigenetic Reshaping through Damage (ERD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclustion:\u003c/strong\u003eOverall, our study sheds light on the dynamic interplay between thymidine analogs, DDR, and epigenetic modifications, providing valuable insights into the mechanisms underlying cell fate transition.\u003c/p\u003e","manuscriptTitle":"Epigenetic Reshaping through Damage: Promoting Cell Fate Transition by BrdU and IdU incorportion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-17 16:54:15","doi":"10.21203/rs.3.rs-3520658/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-12-19T16:38:34+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-11-20T18:44:39+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-11-14T17:02:54+00:00","index":0,"fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-06T13:41:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell \u0026 Bioscience","date":"2023-11-03T03:36:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-bioscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbio","sideBox":"Learn more about [Cell \u0026 Bioscience](http://cellandbioscience.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cbio/default.aspx","title":"Cell \u0026 Bioscience","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a05cd046-516f-11e9-9e20-12b504df345a","owner":[],"postedDate":"November 17th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-01-22T15:02:01+00:00","versionOfRecord":{"articleIdentity":"rs-3520658","link":"https://doi.org/10.1186/s13578-024-01192-x","journal":{"identity":"cell-and-bioscience","isVorOnly":false,"title":"Cell \u0026 Bioscience"},"publishedOn":"2024-01-16 15:00:34","publishedOnDateReadable":"January 16th, 2024"},"versionCreatedAt":"2023-11-17 16:54:15","video":"","vorDoi":"10.1186/s13578-024-01192-x","vorDoiUrl":"https://doi.org/10.1186/s13578-024-01192-x","workflowStages":[]},"version":"v1","identity":"rs-3520658","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3520658","identity":"rs-3520658","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}