Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia

Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia | 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 Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia Hwayeon Cha, Jae Hee Jun, Je Yeong Ko, Ho-Shik Kim, Kyung Hyun Yoo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4911699/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Primary cilia are sensory organelles that regulate various signaling pathways. For this, microtubules work as a highway, and they are tuned with post-translational modifications, such as acetylation. However, the role of acetylation in the regulation of primary cilia remains uncertain. Here, we identified K (lysine) acetyltransferase 2B (Kat2b) as a novel regulator of primary cilia. Kat2b localizes to the cytosol, centrosome, and the cilium basal body. Also, the basal expression of Kat2b gradually increases during ciliogenesis. Kat2b regulates the rate of cilia assembly and the acetylation of α-tubulin via its catalytic activity. The loss of Kat2b reduces the recruitment of intraflagellar transport (IFT) components to the ciliary axoneme and impairs Hedgehog signaling activation. In addition, Kat2b-knockout mice show mild abnormalities and ciliary IFT defect in kidneys. Thus, our new findings establish a link between acetylation regulated by Kat2b and its relevance to ciliary assembly and function. Biological sciences/Genetics Biological sciences/Molecular biology Acetylation Ciliogenesis Intraflagellar transport Kat2b Primary cilia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The primary cilium, also known as non-motile cilia, is a rod-like cellular organelle projecting from the plasma membrane 1 . Most eukaryotic cells contain this highly conserved organelle 2 . Over the decades, the primary cilium has been considered degenerate, however, it was recently reported that loss of primary cilia causes disorders collectively known as ciliopathy 3 . Ciliopathy includes a wide range of diseases, such as polycystic kidney disease (PKD) 4 , nephronophthisis (NPHP), Bardet-Biedl syndrome (BBS), and Joubert syndrome 5 . Moreover, it has been reported that cancer, obesity, and schizophrenia are also related to the primary cilia 6 7 , which acts as a cellular antenna to numerous biological signaling pathways, such as the Hedgehog (Hh), Notch, PCP, and mechanosensing pathways 8 . The structure of primary cilia is composed of the basal body, transition zone, ciliary membrane, ciliary tip, and axoneme 9 . The axoneme is a microtubule-based ciliary backbone with a ‘9 + 0’ arrangement - nine outer doublets and no central microtubules 10 . The components of cilium assembly and maintenance, such as intraflagellar transport (IFT) complex proteins, and the components for signal-transmitting move along with this ciliary backbone 11 12 . Since these proteins are not synthesized at primary cilia, transportation systems are also needed 13 . Thus, microtubules work as a ‘highway’ for these ciliary proteins. The microtubules are subject to diverse post-translational modifications (PTMs) such as acetylation, polyglutamylation, and detyrosination 14 . PTMs affect microtubule dynamics, organization, and interaction with other cellular components 15 , 16 . It is known that the acetylation of microtubules affects the stability of microtubules, including primary cilia 17 . However, more research is needed to reveal how PTMs regulate the formation and functions of primary cilia. K(lysine)acetyltransferase 2b (Kat2b), also known as the p300/CBP-associated factor (PCAF), has been recognized as a histone acetyltransferase (HAT) that promotes transcriptional activation 18 . Recently, it has been reported that HAT and histone deacetylase (HDAC) in the cytosol are involved in novel roles; by contrast, they are known to be transcriptional regulators 19 . Furthermore, in the research field of primary cilia, knockdown of HDAC2 promotes cilium assembly 20 , while HDAC6 overexpression induces rapid cilium disassembly 21 . Also, α-TAT1 is needed for rapid ciliogenesis 22 . However, it has not been well characterized whether acetyltransferase and deacetylase could affect ciliary functions such as IFT components or Hh signaling. For this reason, we wanted to explore the role of acetyltransferase Kat2b in the primary cilia. Here, we determined that Kat2b is needed for the appropriate initiation of primary cilia. Interestingly, the expression level and the proportion of Kat2b localized to the cytosol, centrosome, and basal body increased gradually during cilia assembly. Also, Kat2b interacts with α-tubulin to regulate its acetylation level. Remarkably, Kat2b depletion causes defects in the level of IFT components and Hh signaling activation. In addition, Kat2b-knockout mice show mild abnormalities related to primary cilia defect in kidney. Taken together, our data highlight the novel role of Kat2b in the assembly and function of the primary cilia. 2. Materials and Methods 2.1. Cell culture In this study, mouse embryo fibroblast NIH/3T3 (CRL-1658TM, ATCC) cells obtained from the Korean Cell Line Bank were used. NIH/3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, WELGENE, LM 001–05) supplemented with 10% fetal bovine serum (FBS, Gibco, #26140-079) at 37°C under 5% CO2. All culture media were supplemented with 1% penicillin-streptomycin (P/S, WELGENE, LS 202-02). To induce primary cilia assembly, cells were incubated in serum starvation conditions for 6 to 24 hours. 2.2 Kat2b-Knockout Mice Kat2b-knockout mice (C57BL/6) 23 were kindly gifted by Dr. Ho-Shik Kim (The Catholic University of Korea, Seoul, Republic of Korea). To perform genotyping, mouse genomic DNA was isolated following overnight digestion at 55°C in lysis buffer containing proteinase K. Next day, the mixture was subjected to heat inactivation at 85°C for 30 min. PCR was performed using the primers to distinguish wild-type Kat2b (Oligo F1; 5′-TTCTAGATCTGCCGGTGTCC-3′, Oligo F2; 5′-CTGCCAGACCCTGTTTACAC-3′) from Kat2b-knockout (Oligo F1; 5′-TCGCCTTCTTGACGAGTTCT-3′). In vivo experimental procedures were conducted under review and approval of the IACUC at Sookmyung Women’s University, Seoul, Republic of Korea. Mouse kidney extraction was performed after cervical dislocation. 2.3. Mouse Embryonic Fibroblast (MEF) isolation and culture maintenance To generate Kat2b MEF cells, pregnant female mice were sacrificed at the embryonal stage (E13.5-E15.5). Tails of each embryo were saved for genotyping. The embryo body was minced for 3 min using a blade. Next, minced tissue was incubated for 30–40 min in the 37°C incubator with trypsin. The mixture was harvested with culture media and centrifuged at 1500 rpm for 3 min. The supernatant was aspirated, and the pellet was re-suspended and plated to T75 with culture medium (DMEM with 10% FBS, 1% P/S) 2.4. Antibodies and drugs The primary antibodies used, and their dilutions for western blot (WB), immunocytochemistry/ immunofluorescence (ICC/IF) and immunoprecipitation (IP) are as follows: mouse anti-PCAF(Kat2b) (Santa cruz, sc-13124; 1:500-1:1,000 for WB; for IP), rabbit anti-IFT20 (Proteintech, 13615-1-AP; 1:500-1:1,000 for WB), rabbit anti-IFT25 (Proteintech, 15732-1-AP; 1:500 for WB, 1:200 for ICC/IF), rabbit anti-IFT46 (Abcam, ab122422; 1:1,000 for WB), rabbit anti-IFT52 (Proteintech, 17534-1-AP; 1:1,000 for WB, 1:500 for ICC/IF), rabbit anti-IFT88 (Santa cruz, sc-84318; 1:1,000 for WB), rabbit anti-IFT140 (Proteintech, 17460-1-AP; 1:1,000 for WB, 1:500 for ICC/IF), rabbit anti-GLI1 (Cell Signaling Technologies, #2534, 1:500 for WB), mouse anti-SMO (Santa cruz, sc-166685; 1:50 for ICC/IF), mouse anti-Acetylated- α-tubulin (Sigma, T6793; 1:1,000 for WB and ICC/IF), rabbit anti-Acetylated- α-tubulin (Cell Signaling Technologies, #5335; 1:1,000 for WB), mouse anti-gamma-tubulin (Sigma, T6557; 1:1,000 for ICC/IF), mouse anti- α-tubulin (Cell Signaling Technologies, #3873; 1:1,000 for WB), mouse anti- α-tubulin (Santa cruz, sc-5286; for IP), rabbit anti-Pericentrin (Abcam, ab4448; 1:1,000 for WB and ICC/IF), rabbit cytoskeletal actin antibody (Bethyl Laboratories, A300-491A; 1:14,000 used as loading control for WB), normal mouse anti-IgG (EMD Millipore, 12–371; for IP), and rabbit anti-Flag (Sigma, F7425; 1:2,000 for WB). Secondary antibodies used were goat anti-mouse IgG (Enzo Life Sciences, ADI-SAB-100J; 1:2,500 for WB), goat anti-rabbit IgG (Enzo Life Sciences, ADI-SAB-300J; 1:2,500 for WB), anti-biotin HRP-linked antibody (Cell Signaling Technologies, #7075S; 1:2,500 for WB), and Alexa Fluor ™ 488 goat anti-mouse (Invitrogen, #A11029; 1:1,000 for ICC/IF). The drugs used in this study were as follows: Garcinol was kindly gifted by Dr. Ho-Shik Kim (The Catholic University of Korea) and SAG (Abcam, ab142160). 2.5. siRNA transfection and reagent treatment To regulate target gene expression using small interfering RNA (siRNA), cells were transiently transfected with 30 nM of control siRNA (Santa Cruz, sc-37007) and PCAF siRNA (Santa Cruz, sc-36199) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, #13778150). Plasmid transfection into NIH/3T3 cells was performed using FuGENE® HD Transfection Reagent (Promega) according to the manufacturer’s instructions. 2.6. Quantitative real-time PCR (qRT-PCR) Messenger RNA (mRNA) was extracted from cells using the NucleoSpin® DNA/RNA/Protein kit (Macherey-Nagel), strictly according to the manufacturer’s instructions. 2 µg of mRNA used was reverse-transcribed to cDNA using M-MLV Reverse Transcriptase (Promega, M170B), RNasin® ribonuclease inhibitor (Promega, N211A), 100 nM oligo-dT(C1101, Promega), and 2.5 nM dNTP mixture (Promega, U1205, U1225, U1215, U1235). qRT-PCR was performed using SYBR Green qPCR Master Mix (PB20.15-05, PCR Biosystem, London, UK) and LightCycler® 96 System (Roche). 2.7. Western blot Proteins were extracted from cells and kidney tissues using NucleoSpin® RNA/Protein kit (Macherey-Nagel) following the supplier’s protocol. The protein concentration was quantified using bicinchoninic acid (BCA) solution (Sigma, B9643) and copper (II) sulfate sodium (Sigma, C2284). Next, the protein samples were separated by gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (ATTO, AE-6667-P). The membranes were blocked in 5% skim milk in PBS with 0.1% Tween® 20 (PBST) at room temperature for 1 h and incubated with primary antibodies diluted in 1% skim milk in PBST at 4°C overnight for probing. The next day, the membranes were washed thrice with PBST. Next, the membranes were incubated with HRP-conjugated secondary antibodies and anti-HRP antibody at room temperature for 2 h. Then, the membranes were washed thrice with PBST briefly. The membranes were detected using the chemiluminescence reagent EzWestLumWe Plus (ATTO, WSE-7120) using LAS-3000, LAS-4000 (Fujifilm), and Amersham Imager 600 (GE Healthcare). Using NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, 78833) to separate nucleus and cytosol and incubated at 100℃ after mixing 5X protein sample buffer and quantified protein. We checked that the gels/bots comply with digital image and integrity policies. Additionally, we attached original full-length blots used in figures in the ‘Supplementary Info File’. 2.8. Immunocytochemistry/Immunofluorescence (ICC/IF) For fluorescence staining of cells, cells grown on 18 x18 mm coverslips were washed in PBS and fixed in 4% paraformaldehyde (PFA) at room temperature for 15 min. Alternatively, cells were fixed in cold methanol at -20°C for 5 min. After fixing, cells were blocked and permeabilized with PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA). The coverslips were incubated with diluted primary antibodies at 4°C overnight. Next, the coverslips were washed three times with PBS, incubated with secondary antibodies for 2 h, and stained with DAPI for 5 min at room temperature. Subsequently, the coverslips were washed thrice with PBS. Next, the coverslips were mounted using a mounting solution (DAKO, S3023). For fluorescence staining of paraffin-embedded tissues, the sections were deparaffinized and rehydrated in a graded ethanol. Brog Decloaker RTU (Biocare Medical) was used for heat induced antigen retrieval and the paraffin-embedded sections were blocked for 1h followed by primary antibody incubation at 4℃ overnight. After washing, the slides were incubated with fluorescence-conjugated secondary antibodies (Invitrogen) at room temperature for 2h. Subsequently, the slides were washed three times with PBS and mounted using mounting medium with DAPI (H-1500, Vector Laboratories). Cells and tissues were imaged using confocal microscopy (Carl Zeiss, LSM-700) and Zen software (Carl Zeiss). 2.9. Co-Immunoprecipitation (Co-IP) NIH/3T3 cells were grown to confluence. Subsequently, cells were treated with serum-free medium for 6 h to induce early stage ciliogenesis. Cells were washed briefly in PBS and lysed in buffer containing as follows: 25 mM HEPES, 150 mM NaCl, 1 mM DTT, 0.5% Triton X-100, protease inhibitor (Roche, cOmplete™ Protease inhibitor cocktail, 11697498001), and phosphatase inhibitor (Roche, PhosSTOP™, 4906837001). The amount of protein was measured using the BCA method. Then, 500-1,000 µg of proteins with PCAF (Kat2b) antibody or α-tubulin antibody or mouse IgG antibody was incubated in a rotator at 4°C overnight. The next day, magnetic beads were added and the mixtures were incubated on a rotator at 4°C for 4 h. The samples were washed thrice in wash buffer containing 150 mM NaCl, 50 mM Tris-Cl, protease inhibitor, and phosphatase inhibitor. Next, the samples were boiled at 98°C for 5 min and detected by western blot as described previously. 2.10. Statistical analysis All experiments were repeated more than three times independently. Statistical analysis was conducted using GraphPad Prism 5 software (GraphPad, USA). The data was analyzed by one-tailed Student’s t-test and values are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant. 2.11. Ethics approval The study is reported in accordance with ARRIVE guidelines. All experiments were conducted according to the protocols approved by IACUC at Sookmyung Women’s University, Seoul, Republic of Korea. 3. RESULTS 3.1. Kat2b localized with the cytosol, centrosome, and basal body during cilia assembly According to previous research, it is known that serum withdrawal from culture medium induces the formation of primary cilia 24 . To investigate the basal expression level of Kat2b during cilium assembly, we exposed NIH/3T3 to serum starvation. The time-course analysis revealed that the mRNA levels (Fig. 1 a) and the protein levels (Fig. 1 b) of Kat2b gradually increased. These data indicate that there is a possibility that Kat2b might have a specific role in ciliogenesis. Although it has been recognized that Kat2b predominantly localizes to the nucleus, a recent study showed that Kat2b also localizes to the centrosome 25 . However, Kat2b localization to primary cilia has yet to be characterized. Thus, we examined the subcellular localization of Kat2b during primary cilia biogenesis in order to verify whether kat2b regulates primary cilia as a transcription coactivator in the nucleus or in other cellular locations. NIH/3T3 cells were transfected with expression vectors for DsRed-tagged Kat2b. The next day, the cells were serum-starved for the duration of 24 hours and fixed at each time point. Confocal microscopy data revealed the subcellular localization of Kat2b protein (Fig. 1 c, d). 83.2% of Kat2b localized mainly in the nucleus at the start, and only about 16.8% of Kat2b existed in the cytosol and centrosome. Interestingly, the proportion of the Kat2b localized to the cytosol and centrosome slightly increased at 6 h after serum starvation. Moreover, the proportion increased by approximately 2.5 times as ciliogenesis progressed. Strikingly, we also found that Kat2b localizes to the basal body. Thus, Kat2b is localized in the cytosol, centrosome, and basal body during ciliogenesis. These findings imply a unique role of Kat2b in the context of primary cilia formation 3.2. Loss of Kat2b delays ciliogenesis Next, to test its role in ciliogenesis in vitro , we generated Kat2b-depleted NIH/3T3 cells using short hairpin RNA (shRNA) against Kat2b. The cells were incubated under serum-starved conditions. Subsequently, cells were harvested or fixed in a time-course manner. We confirmed that Kat2b decreased via qRT-PCR and western blot compared with the control group (Fig. 1 e, f). Using immunocytochemistry, we investigated the number of primary cilia assembled at each time point. The ablation of Kat2b proteins led to a decreased percentage of ciliated cells at 6 h and 12 h after serum starvation, indicating that depletion of Kat2b causes retention of ciliogenesis at an early stage (Fig. 1 g, h). Thus, our results indicate that Kat2b is required for the appropriate initiation of cilia formation. 3.3. Kat2b regulates the acetylation of α-tubulin via its acetyltransferase function Acetylated microtubules, such as the axoneme of primary cilia, have been considered as long-lived microtubules 17 . According to a previous study, acetylation of α-tubulin increases the flexibility of microtubules, contributing to better resistance against mechanical stress and consequently making them long-lived 26 . Acetylated-α-tubulin is a cilia marker in the lysine residues of α-tubulin. Thus, we considered α-tubulin as the non-histone target of Kat2b. Also, our data led to us investigate an unreported possibility that Kat2b regulates primary cilia through acetylation of α-tubulin (Fig. 2 a). First, we tested whether Kat2b could affect the level of acetylated tubulin. The western blot data showed that although Kat2b depletion impaired the level of acetylated α-tubulin both early and late serum starvation, the total amount of α-tubulin did not change (Fig. 2 b). To verify whether the regulation of acetylated α-tubulin would be dependent on the existence of Kat2b, we transfected an over-expressed Kat2b flag-tagged plasmid to Kat2b-depleted cells with 6 h and 24 h serum starvation. The data revealed that acetylated α-tubulin levels were rescued in Kat2b-depleted cells when flag-tagged Kat2b was transfected, while the transfection itself did not affect the acetylation level of α-tubulin (Fig. 2 c). These data prompted us to test whether Kat2b regulates the acetylation level of α-tubulin by interacting with it. Our immunoprecipitation results revealed that both Kat2b and α-tubulin existed in the cell lysate and that Kat2b interacted with α-tubulin endogenously (Fig. 2 d). In addition, α-tubulin also interacted with Kat2b and vice versa. According to previous research, garcinol inhibits the acetyltransferase activity of Kat2b 27 . In addition, the cytoplasmic acetylated lysine was decreased in garcinol-treated human U2OS cells 28 . Thus, we treated the NIH/3T3 cells with 500 nM of garcinol and serum-starved for 6 h to induce the early stage of ciliogenesis, which was delayed by Kat2b depletion. Our data indicate that the percentage of ciliated cells in the DMSO-treated group was about 25%, whereas the percentage was significantly decreased in garcinol-treated cells, which showed that approximately 12% of cells were ciliated (Fig. 2 e, f). More importantly, the acetylated α-tubulin level was also decreased in the garcinol-treated group compared to that in the control group (Fig. 2 f). Altogether, we unearthed novel findings that showed α-tubulin as non-histone target of Kat2b. We revealed that Kat2b interacts with α-tubulin endogenously and regulates its acetylation level in a Kat2b-dependent manner. Moreover, our discovery demonstrated that Kat2b hampers the cilium assembly rate and acetylation of α-tubulin via its acetyltransferase function. 3.4. Kat2b is required for the recruitment of IFT proteins and the Hedgehog signaling pathway component to the primary cilia. The microtubules, which have α-tubulin as its subunit, acts as the highway for many molecules, including IFT cargo particles 29 . Previous research has revealed that the stability of microtubules is regulated by acetylation 30 . On the basis of our findings for Kat2b functioning in the acetylation of α-tubulin, we speculated that the weakened microtubule could not break the highway, but could hamper cargo transport along the microtubule. Hence, we tested whether the translational level of IFT components could be affected by Kat2b depletion. In the early stages of primary cilia assembly, our western blot data show that IFT25, IFT46, IFT52, and IFT140, which are involved in cilia assembly, were decreased in the whole lysate of NIH/3T3 cells when Kat2b was silenced (Fig. 3 a and Supplementary Fig. 1a). To exclude the possibility that Kat2b might regulate the level of IFT components through its histone acetyltransferase function, we tested the mRNA levels of IFT components in the whole lysate of NIH/3T3 cells after 6 h of serum starvation. Interestingly, the transcriptional level of IFTs was either a slight increased or not changed at all in Kat2b-depleted cells (Supplementary Fig. 1c). These data prompted us to conduct immunocytochemistry experiments to verify whether the loss of Kat2b could affect ciliary localized IFT components. We used stable Kat2b knockdown cells to verify IFT components localization with or without Kat2b expression (Fig. 3 b). Our confocal microscopy data revealed that IFT25 localized to the basal body, and IFT52 was stained at the basal body, the axoneme, and ciliary tip in shControl NIH/3T3 cells at the early stage of ciliogenesis. Strikingly, the fluorescence intensity of IFT25, and IFT52 decreased in stable Kat2b knockdown cells (Fig. 3 b). However, the intensity of IFT46 and IFT140 was not changed in Kat2b depleted cells (Supplementary Fig. 2a-b). Unexpectedly, our western blot data verified that majority of IFT components also decreased after 24 h of serum starvation, which is at the maturation stage of ciliogenesis, whereas mRNA levels either increased slightly or did not change (Fig. 3 c and Supplementary Fig. 1b and 1d). In this late stage, IFT25 was strongly stained at the basal body. IFT52 was also localized at the basal body and the axoneme. Surprisingly, our immunocytochemistry data clearly showed that the intensity of IFT fluorescence was dramatically decreased in shKat2b cells (Fig. 3 d). This is unexpected because the depletion of Kat2b did not show a decreased percentage of ciliated cells at the time point of 24 h after serum starvation. Our results imply that even though cilia formation was saturated in Kat2b-depleted cells as much as in control, the ciliary function might be damaged. To consolidate our result, we used whole-body Kat2b knock-out mouse embryonic fibroblast (MEF) cell lines. Intriguingly, we could observe the fluorescence intensity of IFT25 and IFT52 is significantly reduced in Kat2b knockout (KO) MEF cells at 6h and 24h serum starvation (Fig. 3 e). Since the loss of IFT25 impairs Hedgehog (Hh) signaling 31 and depletion of Kat2b hampered the Hh signaling in a cancer model 32 , we further tested whether ablation of Kat2b expression could affect the activation of Hh signaling in the context of primary cilia. To test this, we silenced Kat2b and exposed them to serum starvation with 500 nM of SAG (Smo agonist) the following day for 24 h. SAG (Smo agonist) as activator of Hh signaling binds to the heptahelical bundle of Smo protein 33 and induces ciliary translocation of Smo, leading to activation of Hh signaling. Our data showed that a few Smo proteins were recruited to the ciliary axoneme, despite the lack of SAG treatment in Kat2b depleted cells (Fig. 4 a). When SAG was treated, Smo was recruited to the whole axoneme with strong fluorescence intensity in control NIH/3T3 cells, while the fluorescence intensity of Smo protein was significantly decreased in Kat2b depleted cells (Fig. 4 a). Next, we also checked that both the mRNA and protein levels of Gli1, the Hh signaling marker, increased with SAG treated cells but not as much as when Kat2b was depleted (Fig. 4 b, c). Therefore, our data clarifies that Kat2b regulates ciliary localized IFT components bona fide and impairs ciliary function through Hh signaling pathways. Furthermore, the recruitment of Smo protein at ciliary axoneme and Gli1 mRNA level were also impaired in Kat2b knockout MEF cells (Fig. 4 d, e). Together, our data indicate that IFT recruitment and Hh signaling at primary cilium were damaged when Kat2b was absent. 3.5. Function of acetyltransferase Kat2b for ciliogenesis depends upon the localization in cytosol Since Kat2b has an acetyltransferase domain, we hypothesized that Kat2b might regulate the cilium assembly rate and acetylation of α-tubulin through its catalytic function. Thus, we generated an acetyltransferase domain-deleted construct of Kat2b (ΔAT) to elucidate this discovery more concretely (Fig. 5 a). In addition, we created nuclear localization signal domain deleted Kat2b and both domain deleted Kat2b mutant. Through immunocytochemistry experiments, it was confirmed that DsRed tagged ΔNLS-Kat2b and ΔATΔNLS-Kat2b were not located in nucleus (Fig. 5 b). And we also identified localization domain modified Kat2b in perfect working order by separation of nuclear and cytoplasmic fraction experiments (Fig. 5 c). NIH/3T3 cell was transfected with GFP tagged Kat2b construct in 48 h and harvested cells were immediately extracted between cytosolic and nuclear fractions. The immunoblotting band intensities were normalized with each loading controls; α-tubulin, as a cytosol control; Histone H3, as a nuclear control; and the percentage of Kat2b exist in the nucleus versus the cytoplasm was graphically represented. Less than half of NLS domain depleted Kat2b was in nucleus and NLS and AT domain depleted Kat2b was a nearly non-nuclear position. We replaced the tagged vector with GFP in order not to overlap with the fluorescence wavelength that identifies the IFT components, and proceeded with the following experiment. As observed in Fig. 3 D, the ciliary localization of IFT52 in shControl NIH/3T3 cells with empty-vector was situated at both cilia and basal body, and no ciliary distribution but only basal body under Kat2b removed condition (Fig. 5 d). In full-length-Kat2b transfected shKat2b NIH/3T3 cells that had undergone complete recovery, it was observed that the localization proportion of IFT52 with cilia surpassed shControl cells and almost IFT52 was found in ciliary structure of transfected cells. We also transfected ΔAT and ΔATΔNLS construct to stable Kat2b-depleted cells, as expected, proportion of IFT52-existed cilia decreased compared to that in FL-Kat2b transfected cells at maturation stages of primary cilia assembly. ΔATΔNLS -Kat2b in Kat2b-depleted cells, in particular, conducted like as empty vector since the ratio of IFT52 on the cilia and basal bodies was extremely similar. Finally, in the ΔNLS-Kat2b transfected shKat2b cell, it was a quite complete recovery with ciliary localized IFT52 as analogous as shControl cells. These results demonstrated that the function of Kat2b as acetyltransferase was significant for correct localization of IFT components even intact primary cilia structure and cytoplasmic Kat2b alone can play a sufficient role in the localization of IFT components in the cilia. 3.6. Kat2b knockout mice show mild abnormalities related to cilia defect in kidneys. To test whether Kat2b depletion in vivo closely reflects in vitro results, we examined the kidney tissue of a Kat2b knockout (KO) mouse model. This is because primary cilia are found in the apical region of renal epithelial cells 34 , and previous research papers have revealed that IFT defect causes ciliopathy such as cystic kidney disease 35 36 . First, we confirmed that the level of Kat2b deceased in the Kat2b KO mice (Fig. 6 a and 6 b). The value of two kidney weights per total body weight did not change (Fig. 6 c), but Kat2b KO mice showed decreased expression of acetylated alpha tubulin (Fig. 6 d). H&E data revealed that Kat2b KO mice have mild abnormalities in kidneys. They show dilated tubules and loosened glomeruli, while wild-type kidneys showed tight glomeruli (Fig. 6 e). This is interesting because the disruption of the primary cilia component causes glomerular cysts 37 38 . Since our in vitro data (Fig. 3 ) revealed that the loss of Kat2b impairs the recruitment of IFT components to the primary cilia, we conducted immunofluorescence experiment to investigate whether IFT defect also exists in vivo as well. Confocal microscopy data showed that the fluorescence intensity of IFT25 at primary cilia decreased in Kat2b KO, whereas IFT25 distributes over the cilium of renal tubule in WT mice (Fig. 6 f). Therefore, our data demonstrate that the loss of Kat2b shows primary cilia defect in vivo . 4. DISCUSSION In this study, we investigated Kat2b as a novel regulator of primary cilia using in vitro and in vivo experiments. Our data revealed that the basal expression of Kat2b gradually increased and that Kat2b at the cytosol, centrosome, and the basal body also gradually increased during primary cilia assembly. These findings suggest that Kat2b, which functions as a histone acetyltransferase, could be involved in many more diverse roles, including that of the primary cilia. Our work also revealed that the loss of Kat2b causes a delay in ciliogenesis and reduces the acetylation of α-tubulin through its acetyltransferase activity. Importantly, our confocal microscopy data clarifies that Kat2b depletion hampers the recruitment of IFT components to the cilium and decreases the level of Hh signaling activation. It is interesting to note that IFT components are involved in the ciliary formation and ciliary maintenance, such as signaling regulation. In addition, our data revealed that Kat2b KO mice show mild abnormalities related to cilia defect. These data strongly imply a possible relationship between Kat2b and ciliopathy caused by primary cilia defects (Fig. 7 ). Our findings were further supported by two recently published papers covering the relationship between primary cilia and other PTMs, such as polyglutamylation. The authors suggested that polyglutamylation itself does not impair the primary cilia phenotype; however, it regulates the function of primary cilia, via the Hh signaling pathway 39 40 . This supported our hypothesis and results, considering that the cilium assembly compensated at the late stage of ciliogenesis in Kat2b depleted cells, although Kat2b regulates ciliary functions such as IFTs and the Hh signaling pathway. Thus, we can assume that PTMs in the primary cilium, such as acetylation and polyglutamylation, are involved in the functional aspects of primary cilia rather than assembly and having shared roles. Since our data suggest the possibility of regulating the component of microtubules (highways), such as the acetylation of α-tubulin and its effect on IFTs (cargo proteins), identifying exact motor proteins (vehicles) related to this phenomenon merits further investigation. A recently performed proteomic approach, conducted by Fournier et al., to identify the Kat2b-targeted acetylome 25 may give us insight for this. For example, KIF17 and KIF27 could be the target of Kat2b. KIF17 is a member of the kinesin-2 family, which contributes to IFT-B transport 41 42 . KIF27, which is a member of kinesin-4, is located in the basal body 43 . More interestingly, their acetylome data uncovered many proteins related to not only motor proteins but also other primary ciliary proteins could be acetylated by Kat2b; for instance, ALMS1 is an Alstrom syndrome gene, which is ciliopathic 44 . DZIP1L is another ciliopathic gene encoding the ciliary-transition-zone protein and is related to autosomal recessive polycystic kidney disease (ARPKD) 45 . SDCCAG3 has also been reported to localize to the basal body and interact with IFT88 46 . In addition, TTBK2 is a ciliary gene related to the early step of ciliogenesis 47 . These results provide strong evidence for the ciliary role of Kat2b. Further investigation is needed to elucidate the possible relations between Kat2b and other primary cilia proteins. Given previous studies and our results, there is a possibility of therapeutic development for ciliopathy. First, our data revealed that Kat2b regulates primary ciliary function. Second, Kat2b KO mice show mild pathological effects like glomerular abnormalities and dilated tubules in the kidney. Thus, whether Kat2b upregulation could attenuate this pathological effect and recover ciliary function merits further investigation. Altogether, this study highlights the novel role of Kat2b in regulating primary cilium function and its pathological effect in the kidney. Declarations Conflict of Interest The authors declare no competing interests. Additional information Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ . Author Contribution Conceptualization: [J. H. Park], [K. H. Yoo]; Methodology: [H. Cha], [J.Y. Ko], [Ho-S. Kim]; formal analysis and investigation: [H. Cha], [J. H. Jun], [J,Y. Ko], [J. Min]; Writing - original draft preparation; [H. Cha], [J. H. Jun]; Writing - review and editing: [H. Cha], [J. H. Jun], [J. Y. Ko], [J. H. Park]; Funding acquisition: [J. H. Park]; Resources: [Ho-S. Kim]; Supervision: [J. H. Park] Acknowledgements This study was supported by grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (2015M3A9B6027555 and 2016R1A5A1011974). 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Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability. PLoS genetics 14, e1007844, doi: 10.1371/journal.pgen.1007844 (2018). Additional Declarations No competing interests reported. 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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-4911699","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":354166010,"identity":"4041ec37-122a-4d6d-b75e-ff480bce5c17","order_by":0,"name":"Hwayeon Cha","email":"","orcid":"","institution":"Sookmyung Women’s University","correspondingAuthor":false,"prefix":"","firstName":"Hwayeon","middleName":"","lastName":"Cha","suffix":""},{"id":354166011,"identity":"963cbee2-6b0a-4908-af7c-86d1827c5191","order_by":1,"name":"Jae Hee Jun","email":"","orcid":"","institution":"Sookmyung Women’s University","correspondingAuthor":false,"prefix":"","firstName":"Jae","middleName":"Hee","lastName":"Jun","suffix":""},{"id":354166012,"identity":"4b79b77a-2065-476e-abc5-e68374975976","order_by":2,"name":"Je Yeong Ko","email":"","orcid":"","institution":"Sookmyung Women’s University","correspondingAuthor":false,"prefix":"","firstName":"Je","middleName":"Yeong","lastName":"Ko","suffix":""},{"id":354166013,"identity":"2ca35de0-ae10-4b4b-a6a3-c8d89feba4f7","order_by":3,"name":"Ho-Shik Kim","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"prefix":"","firstName":"Ho-Shik","middleName":"","lastName":"Kim","suffix":""},{"id":354166014,"identity":"b44ae178-5f0e-4f6b-9fd2-969592914758","order_by":4,"name":"Kyung Hyun Yoo","email":"","orcid":"","institution":"Sookmyung Women’s University","correspondingAuthor":false,"prefix":"","firstName":"Kyung","middleName":"Hyun","lastName":"Yoo","suffix":""},{"id":354166015,"identity":"55012c6c-e950-4b3a-b924-b5f15682821b","order_by":5,"name":"Jong Hoon Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYHACZgbJBhsgzYYQYmwgrCWNVC2MDYdJ0GLOfvaxgeWO8/b87G2pm3nb7tg1sB9+wDhzD24tlj3pxgmSZ24nzuw5duw2b9uz5AaeNAPGDc9wazE4kMZ8QLLtdoLBjfQ2oJbDyQwMOQyMDw7g0XL+GUjLOXuEFv43BLTcSGNOkGw7wLjhRhrIYYftGCSAtmzAo8VyxjNmA8m2ZJBf0m7OOXc4gU3imcHBGXi0mPOnMUtLttmBQszsxpuyw/b8/MkPH/bgcxgQM0tAOUw8DAyJbUAGHg0QLYwfoBzGHwwM9vhUj4JRMApGwcgEAPc4Vi68TdEBAAAAAElFTkSuQmCC","orcid":"","institution":"Sookmyung Women’s University","correspondingAuthor":true,"prefix":"","firstName":"Jong","middleName":"Hoon","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2024-08-14 07:44:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4911699/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4911699/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-86292-5","type":"published","date":"2025-01-16T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64610863,"identity":"68ee854d-819f-4f34-be85-3bae78e22621","added_by":"auto","created_at":"2024-09-16 14:00:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2019655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of Kat2b located in the cytosol, centrosome, and basal body increased during ciliogenesis, and depletion of Kat2b causes delays in ciliogenesis. (a)\u003c/strong\u003e qRT-PCR data showing that the level of Kat2b transcript gradually increased during primary cilia formation. \u003cstrong\u003e(b)\u003c/strong\u003eWestern blot data showing an increase in Kat2b protein expression during ciliogenesis. \u003cstrong\u003e(c)\u003c/strong\u003eRepresentative figures of Kat2b localization in NIH/3T3 cells by serum starvation time-course. NIH/3T3 cells were transfected with expressing empty vector (DsRed) or vector (DsRed-tagged Kat2b). After 24 h, cells were incubated under serum starvation conditions, followed by staining with endogenous acetylated α-tubulin for primary cilia and γ-tubulin for centrosome and basal body (green). Nuclei were stained with DAPI (blue). Scale bar: 10 μm. \u003cstrong\u003e(d)\u003c/strong\u003e Quantification of the subcellular localization of Kat2b. Counted cell numbers were as follows: serum starvation for 6 h, n=133; 12 h, n=129; and 24 h, n=124. Data were collected from three independent experiments. \u003cstrong\u003e(d)\u003c/strong\u003e Validation of transcriptional Kat2b level reduction in stable Kat2b knockdown NIH/3T3 cell line using qRT-PCR. \u003cstrong\u003e(f)\u003c/strong\u003e Validation of translational Kat2b level reduction by shRNA treatment using western blot analysis. \u003cstrong\u003e(g)\u003c/strong\u003e Representative figures of ciliated NIH/3T3 cells transfected with shRNA targeting Kat2b or control shRNA. After serum-free treatment, cells were fixed and stained using ICC/IF in a time-course manner. Acetylated α-tubulin was stained for primary cilia (green). Nuclei were stained with DAPI (blue). Primary cilia are indicated by white arrows. Scale bar: 10 μm. \u003cstrong\u003e(h)\u003c/strong\u003eQuantification of the percentage of ciliated cells in Kat2b-depleted cells compared to control shRNA cells. Counted cell numbers were as follows: controls with serum starvation for 6 h, n=685; 12 h, n=665; 24 h, n=589. Kat2b-depleted cells with serum starvation for 6 h, n=789; 12 h, n=579; 24 h, n=649. Data were collected from three independent experiments.\u003c/p\u003e","description":"","filename":"Fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4911699/v1/0207c4e8ab7bc4908f12f1f4.jpg"},{"id":64611343,"identity":"dd9423b6-7aac-470c-90d4-9ab7b8506468","added_by":"auto","created_at":"2024-09-16 14:08:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1542733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKat2b interacts with and regulates the acetylation level of α-tubulin\u003c/strong\u003e.\u003cstrong\u003e (a)\u003c/strong\u003e Western blots showing Kat2b and acetylated α-tubulin expression at 0, 6, 12, and 24 h after serum withdrawal. The graphs showed relative western blotting band intensity of Kat2b and acetylated-α-tubulin normalized each loading controls. Data were collected from three independent experiments. \u003cstrong\u003e(b)\u003c/strong\u003e Acetylated α-tubulin expression was decreased in Kat2b silenced NIH/3T3 cells at 6 h and 24 h after serum starvation. The graphs next to immunoblot analysis showed quantified acetylated α-tubulin band intensity. \u003cstrong\u003e(c)\u003c/strong\u003eWestern blot data showed that overexpression of Kat2b restored the decreased acetylation level of α-tubulin in Kat2b silenced cells at 6 h and 24 h after serum starvation. The relative band intensity of α-tubulin acetylation was displayed through the graphs below. \u003cstrong\u003e(d)\u003c/strong\u003eImmunoprecipitation data revealed that endogenous Kat2b interacts with α-tubulin in NIH/3T3 cells. \u003cstrong\u003e(e)\u003c/strong\u003e ICC data indicate that the percentage of ciliated cells decreased when garcinol, the acetyltransferase activity inhibitor of Kat2b, was treated with 500 nM for 6 h with serum starvation. Acetylated α-tubulin and γ-tubulin were stained for primary cilia (green). Nuclei were stained with DAPI (blue). Primary cilia are indicated by white arrows. White arrows indicate primary cilia. Scale bar: 10 μm. The graph showed quantification of the percentage of primary cilia in garcinol-treated cells compared to DMSO-treated NIH/3T3 cells. Counted cell numbers were as follows: DMSO-treated cells n=287, garcinol-treated cells n=248. \u003cstrong\u003e(f)\u003c/strong\u003e Western blot data showed that the acetylation level of α-tubulin decreased in garcinol-treated NIH/3T3 cells. Data collected from three experiments were quantified graphically.\u003c/p\u003e","description":"","filename":"Fig2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4911699/v1/af5dd72765e26a619ee3f9ec.jpg"},{"id":64611841,"identity":"9c27b0ef-e9bb-4d4f-939c-cbfca12df3c3","added_by":"auto","created_at":"2024-09-16 14:16:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1371381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Depletion of Kat2b impairs the recruitment of IFT and Hh components to primary cilia.\u003c/strong\u003e \u003cstrong\u003e(a-b)\u003c/strong\u003e IFT components 6 h after serum starvation (early ciliogenesis). \u003cstrong\u003e(a)\u003c/strong\u003eWestern blot data showed that protein levels of IFT components decreased in Kat2b silenced NIH/3T3 cells. \u003cstrong\u003e(b)\u003c/strong\u003eImmunocytochemistry data revealed that the fluorescence intensity of IFT components located in primary cilia was decreased in the stable Kat2b knockdown cell line. Scale bar: 2 μm \u003cstrong\u003e(c-d)\u003c/strong\u003e IFT components 24 h after serum starvation (maturated stage of ciliogenesis). \u003cstrong\u003e(c)\u003c/strong\u003e Western blot data show that protein levels of IFT components decreased in Kat2b silenced NIH/3T3 cells. \u003cstrong\u003e(d)\u003c/strong\u003e Immunocytochemistry data revealed that fluorescence intensity of IFT components located in primary cilia was decreased in the stable Kat2b knockdown cell line. Scale bar: 2 μm \u003cstrong\u003e(e)\u003c/strong\u003e Immunocytochemistry data display that the fluorescence intensity of IFT25 and IFT52 (green) at primary cilia (red) was decreased in Kat2b KO MEF cells at 6h and 24 h after serum starvation, respectively. Scale bar: 2 μm.\u003c/p\u003e","description":"","filename":"Fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4911699/v1/97e1f41204818c5d8d7088e4.jpg"},{"id":64610868,"identity":"ae6a42cc-7060-48f0-bbef-565e4101de1c","added_by":"auto","created_at":"2024-09-16 14:00:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":818045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Kat2b knocked down impairs the recruitment of Hh components to primary cilia.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Immunocytochemistry data confirmed that the fluorescence intensity of Smo (green) was decreased in Kat2b silenced cell by siRNA transfection. Acetylated α-tubulin and γ-tubulin were stained for primary cilia and basal body (red). Nuclei were stained with DAPI (blue). Scale bar: 2 μm \u003cstrong\u003e(b, c)\u003c/strong\u003e qRT-PCR and western data showed that mRNA and protein levels of Gli1 expression are decreased when Kat2b was depleted by siRNA transfection. Gli1 is used as an activation marker of the Hh signaling pathway. NIH/3T3 cells were treated with serum starvation for 24 h and 500 nM of SAG for activation of Hh signaling. \u003cstrong\u003e(d)\u003c/strong\u003e Immunocytochemistry data of SMO protein (green) at primary cilia (red) in Kat2b KO MEF cells with serum starvation for 24 h and 400 nM of SAG. Scale bar: 2 μm. \u003cstrong\u003e(e)\u003c/strong\u003e qRT-PCR data showing that transcriptional Gli1 expression is decreased in Kat2b KO MEF cells.\u003c/p\u003e","description":"","filename":"Fig4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4911699/v1/a18725db035f9bf9b654c77d.jpg"},{"id":64611344,"identity":"36d94f5d-d333-43cf-88d5-0aee0b2e568e","added_by":"auto","created_at":"2024-09-16 14:08:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1817625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe acetyltransferase domain of Kat2b in cytosol is significant for IFT components localization at primary cilia\u003c/strong\u003e. \u003cstrong\u003e(a)\u003c/strong\u003e Graphical view of domains of Kat2b and designed mutant Kat2b. \u003cstrong\u003e(b)\u003c/strong\u003e Immunocytochemistry images confirmed the localization of designed mutant Kat2b construct. DsRed tagged Kat2b constructs were investigated vector transfected cell and nuclear localization signal domain deleted Kat2b positioned only cytosol. \u003cstrong\u003e(c)\u003c/strong\u003eImmunoblotting analysis verified cytosolic and nucleic distribution under transfection with Kat2b constructs in NIH/3T3 cell. α-tubulin represented as cytosol marker and histone H3 as nuclear marker. \u003cstrong\u003e(d)\u003c/strong\u003e Immunofluorescence images showed that IFT 52 localization depending on Kat2b existence in the nucleus and function of acetylation. shControl NIH/3T3 cells transfected by empty vector, and stable Kat2b depleted NIH/3T3 cells, as shKat2b, transfected by empty vector and mutant Kat2b constructs. Nuclei were stained with DAPI (blue), transfected vector constructs were EGFP (green), acetylated α-tubulin and γ-tubulin were stained for primary cilia (red), and IFTs were indicated yellow. White arrows indicated IFT components located in the cilia axoneme. Below graphs displayed the proportion of IFTs distribution by categories; cilia axoneme, basal body and not on the cilia. Scale bar: 2 μm\u003c/p\u003e","description":"","filename":"Fig5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4911699/v1/3f641de79eda52652f415f67.jpg"},{"id":64610871,"identity":"76f7c2d4-ec2d-4496-b86b-6c7d0a9be13f","added_by":"auto","created_at":"2024-09-16 14:00:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2389351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKat2b knockout mice show decreased IFT proteins, Hh signaling, and renal abnormalities.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003eKat2b mRNA expression data using qRT-PCR validated that the level of Kat2b was decreased in the kidney of Kat2b KO mice. \u003cstrong\u003e(b)\u003c/strong\u003eWestern blots also showed decreased Kat2b expression in KO mice. \u003cstrong\u003e(c)\u003c/strong\u003e Two kidney weights per total body weight demonstrate that there are no significant differences between Kat2b KO mice and wild-type mice. \u003cstrong\u003e(d)\u003c/strong\u003e Western blot data show that acetylated α-tubulin level decreased in the renal tissue of Kat2b KO mice. \u003cstrong\u003e(e)\u003c/strong\u003e H\u0026amp;E staining of kidney paraffin sections from 16 weeks Kat2b knockout mice shows glomerular cyst, loosen glomeruli and dilated tubules. Black arrowheads show glomeruli. The Black dotted line defines the area of the dilated tubule. Scale bar: 100 μm. \u003cstrong\u003e(f)\u003c/strong\u003e Confocal microscopy analysis revealed that the fluorescence intensity of IFT25 (red) at primary cilia decreased in the renal tubule from 16 weeks Kat2b knockout mice compared to the wild-type. Acetylated α-tubulin and γ-tubulin were stained for primary cilia (green). Nuclei were stained with DAPI (blue). The White dotted line defines the area of the renal tubule. Scale bar: 20 μm.\u003c/p\u003e","description":"","filename":"Fig6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4911699/v1/f3dc6db8d69a6e35c3eded27.jpg"},{"id":64610866,"identity":"7a232eb1-b9b8-4bf3-b9fd-549cbbe204c2","added_by":"auto","created_at":"2024-09-16 14:00:06","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1269419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary illustration of this study.\u003c/strong\u003e Red ovals indicate KAT2B; green circles indicate IFT-B particles; blue circles indicate SMO; light grey circles indicate α-tubulin; and purple rectangles with rounded corners indicate GLI1. The color intensity indicates the abundance. \u003cstrong\u003e(a-b)\u003c/strong\u003eDuring ciliogenesis, the basal expression level and subcellular localization of Kat2b in the cytosol, centrosome, and basal body gradually increased. Kat2b interacts with α-tubulin and regulates the acetylation level of α-tubulin via its catalytic activity. When Kat2b is depleted, the rate of cilium assembly is delayed, causing the acetylation level of α-tubulin to decrease, and the recruitment of IFT to be impaired. \u003cstrong\u003e(c)\u003c/strong\u003eWhen the Hh signaling pathway is activated using SAG treatment, the recruitment of SMO along the cilium and the level of GLI1, components of Hh signaling, were reduced.\u003c/p\u003e","description":"","filename":"Fig7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4911699/v1/336f351b39c3fd481f1022ca.jpg"},{"id":74285580,"identity":"617cbae1-bb27-436e-8626-79e6700b14fb","added_by":"auto","created_at":"2025-01-20 16:14:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12450672,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4911699/v1/1c6dfe0a-03e9-44a2-9d0e-6d987a747d7c.pdf"},{"id":64610870,"identity":"4fb892f9-2347-4559-b4bf-05ff53c4bf79","added_by":"auto","created_at":"2024-09-16 14:00:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1503484,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaldataScientificreports.docx","url":"https://assets-eu.researchsquare.com/files/rs-4911699/v1/b7e51ebf0e620252f37c9ed6.docx"},{"id":64610872,"identity":"9e3fcdad-232d-4a01-ac98-926c645fa87f","added_by":"auto","created_at":"2024-09-16 14:00:06","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2547653,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInfoFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4911699/v1/3a002bac3c276106e6cb29c9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe primary cilium, also known as non-motile cilia, is a rod-like cellular organelle projecting from the plasma membrane \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Most eukaryotic cells contain this highly conserved organelle \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Over the decades, the primary cilium has been considered degenerate, however, it was recently reported that loss of primary cilia causes disorders collectively known as ciliopathy \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Ciliopathy includes a wide range of diseases, such as polycystic kidney disease (PKD) \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, nephronophthisis (NPHP), Bardet-Biedl syndrome (BBS), and Joubert syndrome \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Moreover, it has been reported that cancer, obesity, and schizophrenia are also related to the primary cilia \u003csup\u003e6 7\u003c/sup\u003e, which acts as a cellular antenna to numerous biological signaling pathways, such as the Hedgehog (Hh), Notch, PCP, and mechanosensing pathways \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe structure of primary cilia is composed of the basal body, transition zone, ciliary membrane, ciliary tip, and axoneme \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The axoneme is a microtubule-based ciliary backbone with a \u0026lsquo;9\u0026thinsp;+\u0026thinsp;0\u0026rsquo; arrangement - nine outer doublets and no central microtubules \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The components of cilium assembly and maintenance, such as intraflagellar transport (IFT) complex proteins, and the components for signal-transmitting move along with this ciliary backbone \u003csup\u003e11 12\u003c/sup\u003e. Since these proteins are not synthesized at primary cilia, transportation systems are also needed \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Thus, microtubules work as a \u0026lsquo;highway\u0026rsquo; for these ciliary proteins.\u003c/p\u003e \u003cp\u003eThe microtubules are subject to diverse post-translational modifications (PTMs) such as acetylation, polyglutamylation, and detyrosination \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. PTMs affect microtubule dynamics, organization, and interaction with other cellular components \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It is known that the acetylation of microtubules affects the stability of microtubules, including primary cilia \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, more research is needed to reveal how PTMs regulate the formation and functions of primary cilia.\u003c/p\u003e \u003cp\u003eK(lysine)acetyltransferase 2b (Kat2b), also known as the p300/CBP-associated factor (PCAF), has been recognized as a histone acetyltransferase (HAT) that promotes transcriptional activation \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Recently, it has been reported that HAT and histone deacetylase (HDAC) in the cytosol are involved in novel roles; by contrast, they are known to be transcriptional regulators \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Furthermore, in the research field of primary cilia, knockdown of HDAC2 promotes cilium assembly \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, while HDAC6 overexpression induces rapid cilium disassembly \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Also, α-TAT1 is needed for rapid ciliogenesis \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, it has not been well characterized whether acetyltransferase and deacetylase could affect ciliary functions such as IFT components or Hh signaling. For this reason, we wanted to explore the role of acetyltransferase Kat2b in the primary cilia.\u003c/p\u003e \u003cp\u003eHere, we determined that Kat2b is needed for the appropriate initiation of primary cilia. Interestingly, the expression level and the proportion of Kat2b localized to the cytosol, centrosome, and basal body increased gradually during cilia assembly. Also, Kat2b interacts with α-tubulin to regulate its acetylation level. Remarkably, Kat2b depletion causes defects in the level of IFT components and Hh signaling activation. In addition, Kat2b-knockout mice show mild abnormalities related to primary cilia defect in kidney. Taken together, our data highlight the novel role of Kat2b in the assembly and function of the primary cilia.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell culture\u003c/h2\u003e \u003cp\u003eIn this study, mouse embryo fibroblast NIH/3T3 (CRL-1658TM, ATCC) cells obtained from the Korean Cell Line Bank were used. NIH/3T3 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, WELGENE, LM 001\u0026ndash;05) supplemented with 10% fetal bovine serum (FBS, Gibco, #26140-079) at 37\u0026deg;C under 5% CO2. All culture media were supplemented with 1% penicillin-streptomycin (P/S, WELGENE, LS 202-02). To induce primary cilia assembly, cells were incubated in serum starvation conditions for 6 to 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Kat2b-Knockout Mice\u003c/h2\u003e \u003cp\u003eKat2b-knockout mice (C57BL/6) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e were kindly gifted by Dr. Ho-Shik Kim (The Catholic University of Korea, Seoul, Republic of Korea). To perform genotyping, mouse genomic DNA was isolated following overnight digestion at 55\u0026deg;C in lysis buffer containing proteinase K. Next day, the mixture was subjected to heat inactivation at 85\u0026deg;C for 30 min. PCR was performed using the primers to distinguish wild-type Kat2b (Oligo F1; 5\u0026prime;-TTCTAGATCTGCCGGTGTCC-3\u0026prime;, Oligo F2; 5\u0026prime;-CTGCCAGACCCTGTTTACAC-3\u0026prime;) from Kat2b-knockout (Oligo F1; 5\u0026prime;-TCGCCTTCTTGACGAGTTCT-3\u0026prime;). \u003cem\u003eIn vivo\u003c/em\u003e experimental procedures were conducted under review and approval of the IACUC at Sookmyung Women\u0026rsquo;s University, Seoul, Republic of Korea. Mouse kidney extraction was performed after cervical dislocation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Mouse Embryonic Fibroblast (MEF) isolation and culture maintenance\u003c/h2\u003e \u003cp\u003eTo generate Kat2b MEF cells, pregnant female mice were sacrificed at the embryonal stage (E13.5-E15.5). Tails of each embryo were saved for genotyping. The embryo body was minced for 3 min using a blade. Next, minced tissue was incubated for 30\u0026ndash;40 min in the 37\u0026deg;C incubator with trypsin. The mixture was harvested with culture media and centrifuged at 1500 rpm for 3 min. The supernatant was aspirated, and the pellet was re-suspended and plated to T75 with culture medium (DMEM with 10% FBS, 1% P/S)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Antibodies and drugs\u003c/h2\u003e \u003cp\u003eThe primary antibodies used, and their dilutions for western blot (WB), immunocytochemistry/ immunofluorescence (ICC/IF) and immunoprecipitation (IP) are as follows: mouse anti-PCAF(Kat2b) (Santa cruz, sc-13124; 1:500-1:1,000 for WB; for IP), rabbit anti-IFT20 (Proteintech, 13615-1-AP; 1:500-1:1,000 for WB), rabbit anti-IFT25 (Proteintech, 15732-1-AP; 1:500 for WB, 1:200 for ICC/IF), rabbit anti-IFT46 (Abcam, ab122422; 1:1,000 for WB), rabbit anti-IFT52 (Proteintech, 17534-1-AP; 1:1,000 for WB, 1:500 for ICC/IF), rabbit anti-IFT88 (Santa cruz, sc-84318; 1:1,000 for WB), rabbit anti-IFT140 (Proteintech, 17460-1-AP; 1:1,000 for WB, 1:500 for ICC/IF), rabbit anti-GLI1 (Cell Signaling Technologies, #2534, 1:500 for WB), mouse anti-SMO (Santa cruz, sc-166685; 1:50 for ICC/IF), mouse anti-Acetylated- α-tubulin (Sigma, T6793; 1:1,000 for WB and ICC/IF), rabbit anti-Acetylated- α-tubulin (Cell Signaling Technologies, #5335; 1:1,000 for WB), mouse anti-gamma-tubulin (Sigma, T6557; 1:1,000 for ICC/IF), mouse anti- α-tubulin (Cell Signaling Technologies, #3873; 1:1,000 for WB), mouse anti- α-tubulin (Santa cruz, sc-5286; for IP), rabbit anti-Pericentrin (Abcam, ab4448; 1:1,000 for WB and ICC/IF), rabbit cytoskeletal actin antibody (Bethyl Laboratories, A300-491A; 1:14,000 used as loading control for WB), normal mouse anti-IgG (EMD Millipore, 12\u0026ndash;371; for IP), and rabbit anti-Flag (Sigma, F7425; 1:2,000 for WB). Secondary antibodies used were goat anti-mouse IgG (Enzo Life Sciences, ADI-SAB-100J; 1:2,500 for WB), goat anti-rabbit IgG (Enzo Life Sciences, ADI-SAB-300J; 1:2,500 for WB), anti-biotin HRP-linked antibody (Cell Signaling Technologies, #7075S; 1:2,500 for WB), and Alexa Fluor \u0026trade; 488 goat anti-mouse (Invitrogen, #A11029; 1:1,000 for ICC/IF). The drugs used in this study were as follows: Garcinol was kindly gifted by Dr. Ho-Shik Kim (The Catholic University of Korea) and SAG (Abcam, ab142160).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. siRNA transfection and reagent treatment\u003c/h2\u003e \u003cp\u003eTo regulate target gene expression using small interfering RNA (siRNA), cells were transiently transfected with 30 nM of control siRNA (Santa Cruz, sc-37007) and PCAF siRNA (Santa Cruz, sc-36199) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, #13778150). Plasmid transfection into NIH/3T3 cells was performed using FuGENE\u0026reg; HD Transfection Reagent (Promega) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eMessenger RNA (mRNA) was extracted from cells using the NucleoSpin\u0026reg; DNA/RNA/Protein kit (Macherey-Nagel), strictly according to the manufacturer\u0026rsquo;s instructions. 2 \u0026micro;g of mRNA used was reverse-transcribed to cDNA using M-MLV Reverse Transcriptase (Promega, M170B), RNasin\u0026reg; ribonuclease inhibitor (Promega, N211A), 100 nM oligo-dT(C1101, Promega), and 2.5 nM dNTP mixture (Promega, U1205, U1225, U1215, U1235). qRT-PCR was performed using SYBR Green qPCR Master Mix (PB20.15-05, PCR Biosystem, London, UK) and LightCycler\u0026reg; 96 System (Roche).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Western blot\u003c/h2\u003e \u003cp\u003eProteins were extracted from cells and kidney tissues using NucleoSpin\u0026reg; RNA/Protein kit (Macherey-Nagel) following the supplier\u0026rsquo;s protocol. The protein concentration was quantified using bicinchoninic acid (BCA) solution (Sigma, B9643) and copper (II) sulfate sodium (Sigma, C2284). Next, the protein samples were separated by gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (ATTO, AE-6667-P). The membranes were blocked in 5% skim milk in PBS with 0.1% Tween\u0026reg; 20 (PBST) at room temperature for 1 h and incubated with primary antibodies diluted in 1% skim milk in PBST at 4\u0026deg;C overnight for probing. The next day, the membranes were washed thrice with PBST. Next, the membranes were incubated with HRP-conjugated secondary antibodies and anti-HRP antibody at room temperature for 2 h. Then, the membranes were washed thrice with PBST briefly. The membranes were detected using the chemiluminescence reagent EzWestLumWe Plus (ATTO, WSE-7120) using LAS-3000, LAS-4000 (Fujifilm), and Amersham Imager 600 (GE Healthcare). Using NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, 78833) to separate nucleus and cytosol and incubated at 100℃ after mixing 5X protein sample buffer and quantified protein. We checked that the gels/bots comply with digital image and integrity policies. Additionally, we attached original full-length blots used in figures in the \u0026lsquo;Supplementary Info File\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Immunocytochemistry/Immunofluorescence (ICC/IF)\u003c/h2\u003e \u003cp\u003eFor fluorescence staining of cells, cells grown on 18 x18 mm coverslips were washed in PBS and fixed in 4% paraformaldehyde (PFA) at room temperature for 15 min. Alternatively, cells were fixed in cold methanol at -20\u0026deg;C for 5 min. After fixing, cells were blocked and permeabilized with PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA). The coverslips were incubated with diluted primary antibodies at 4\u0026deg;C overnight. Next, the coverslips were washed three times with PBS, incubated with secondary antibodies for 2 h, and stained with DAPI for 5 min at room temperature. Subsequently, the coverslips were washed thrice with PBS. Next, the coverslips were mounted using a mounting solution (DAKO, S3023). For fluorescence staining of paraffin-embedded tissues, the sections were deparaffinized and rehydrated in a graded ethanol. Brog Decloaker RTU (Biocare Medical) was used for heat induced antigen retrieval and the paraffin-embedded sections were blocked for 1h followed by primary antibody incubation at 4℃ overnight. After washing, the slides were incubated with fluorescence-conjugated secondary antibodies (Invitrogen) at room temperature for 2h. Subsequently, the slides were washed three times with PBS and mounted using mounting medium with DAPI (H-1500, Vector Laboratories). Cells and tissues were imaged using confocal microscopy (Carl Zeiss, LSM-700) and Zen software (Carl Zeiss).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Co-Immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eNIH/3T3 cells were grown to confluence. Subsequently, cells were treated with serum-free medium for 6 h to induce early stage ciliogenesis. Cells were washed briefly in PBS and lysed in buffer containing as follows: 25 mM HEPES, 150 mM NaCl, 1 mM DTT, 0.5% Triton X-100, protease inhibitor (Roche, cOmplete\u0026trade; Protease inhibitor cocktail, 11697498001), and phosphatase inhibitor (Roche, PhosSTOP\u0026trade;, 4906837001). The amount of protein was measured using the BCA method. Then, 500-1,000 \u0026micro;g of proteins with PCAF (Kat2b) antibody or α-tubulin antibody or mouse IgG antibody was incubated in a rotator at 4\u0026deg;C overnight. The next day, magnetic beads were added and the mixtures were incubated on a rotator at 4\u0026deg;C for 4 h. The samples were washed thrice in wash buffer containing 150 mM NaCl, 50 mM Tris-Cl, protease inhibitor, and phosphatase inhibitor. Next, the samples were boiled at 98\u0026deg;C for 5 min and detected by western blot as described previously.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were repeated more than three times independently. Statistical analysis was conducted using GraphPad Prism 5 software (GraphPad, USA). The data was analyzed by one-tailed Student\u0026rsquo;s t-test and values are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, n.s., not significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Ethics approval\u003c/h2\u003e \u003cp\u003eThe study is reported in accordance with ARRIVE guidelines. All experiments were conducted according to the protocols approved by IACUC at Sookmyung Women\u0026rsquo;s University, Seoul, Republic of Korea.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Kat2b localized with the cytosol, centrosome, and basal body during cilia assembly\u003c/h2\u003e \u003cp\u003eAccording to previous research, it is known that serum withdrawal from culture medium induces the formation of primary cilia \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. To investigate the basal expression level of Kat2b during cilium assembly, we exposed NIH/3T3 to serum starvation. The time-course analysis revealed that the mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and the protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) of Kat2b gradually increased. These data indicate that there is a possibility that Kat2b might have a specific role in ciliogenesis. Although it has been recognized that Kat2b predominantly localizes to the nucleus, a recent study showed that Kat2b also localizes to the centrosome \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, Kat2b localization to primary cilia has yet to be characterized. Thus, we examined the subcellular localization of Kat2b during primary cilia biogenesis in order to verify whether kat2b regulates primary cilia as a transcription coactivator in the nucleus or in other cellular locations. NIH/3T3 cells were transfected with expression vectors for DsRed-tagged Kat2b. The next day, the cells were serum-starved for the duration of 24 hours and fixed at each time point. Confocal microscopy data revealed the subcellular localization of Kat2b protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). 83.2% of Kat2b localized mainly in the nucleus at the start, and only about 16.8% of Kat2b existed in the cytosol and centrosome. Interestingly, the proportion of the Kat2b localized to the cytosol and centrosome slightly increased at 6 h after serum starvation. Moreover, the proportion increased by approximately 2.5 times as ciliogenesis progressed. Strikingly, we also found that Kat2b localizes to the basal body. Thus, Kat2b is localized in the cytosol, centrosome, and basal body during ciliogenesis. These findings imply a unique role of Kat2b in the context of primary cilia formation\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Loss of Kat2b delays ciliogenesis\u003c/h2\u003e \u003cp\u003eNext, to test its role in ciliogenesis \u003cem\u003ein vitro\u003c/em\u003e, we generated Kat2b-depleted NIH/3T3 cells using short hairpin RNA (shRNA) against Kat2b. The cells were incubated under serum-starved conditions. Subsequently, cells were harvested or fixed in a time-course manner. We confirmed that Kat2b decreased via qRT-PCR and western blot compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). Using immunocytochemistry, we investigated the number of primary cilia assembled at each time point. The ablation of Kat2b proteins led to a decreased percentage of ciliated cells at 6 h and 12 h after serum starvation, indicating that depletion of Kat2b causes retention of ciliogenesis at an early stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, h). Thus, our results indicate that Kat2b is required for the appropriate initiation of cilia formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Kat2b regulates the acetylation of α-tubulin via its acetyltransferase function\u003c/h2\u003e \u003cp\u003eAcetylated microtubules, such as the axoneme of primary cilia, have been considered as long-lived microtubules \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. According to a previous study, acetylation of α-tubulin increases the flexibility of microtubules, contributing to better resistance against mechanical stress and consequently making them long-lived \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Acetylated-α-tubulin is a cilia marker in the lysine residues of α-tubulin. Thus, we considered α-tubulin as the non-histone target of Kat2b. Also, our data led to us investigate an unreported possibility that Kat2b regulates primary cilia through acetylation of α-tubulin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst, we tested whether Kat2b could affect the level of acetylated tubulin. The western blot data showed that although Kat2b depletion impaired the level of acetylated α-tubulin both early and late serum starvation, the total amount of α-tubulin did not change (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). To verify whether the regulation of acetylated α-tubulin would be dependent on the existence of Kat2b, we transfected an over-expressed Kat2b flag-tagged plasmid to Kat2b-depleted cells with 6 h and 24 h serum starvation. The data revealed that acetylated α-tubulin levels were rescued in Kat2b-depleted cells when flag-tagged Kat2b was transfected, while the transfection itself did not affect the acetylation level of α-tubulin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). These data prompted us to test whether Kat2b regulates the acetylation level of α-tubulin by interacting with it. Our immunoprecipitation results revealed that both Kat2b and α-tubulin existed in the cell lysate and that Kat2b interacted with α-tubulin endogenously (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In addition, α-tubulin also interacted with Kat2b and vice versa.\u003c/p\u003e \u003cp\u003eAccording to previous research, garcinol inhibits the acetyltransferase activity of Kat2b \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In addition, the cytoplasmic acetylated lysine was decreased in garcinol-treated human U2OS cells \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Thus, we treated the NIH/3T3 cells with 500 nM of garcinol and serum-starved for 6 h to induce the early stage of ciliogenesis, which was delayed by Kat2b depletion. Our data indicate that the percentage of ciliated cells in the DMSO-treated group was about 25%, whereas the percentage was significantly decreased in garcinol-treated cells, which showed that approximately 12% of cells were ciliated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). More importantly, the acetylated α-tubulin level was also decreased in the garcinol-treated group compared to that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eAltogether, we unearthed novel findings that showed α-tubulin as non-histone target of Kat2b. We revealed that Kat2b interacts with α-tubulin endogenously and regulates its acetylation level in a Kat2b-dependent manner. Moreover, our discovery demonstrated that Kat2b hampers the cilium assembly rate and acetylation of α-tubulin via its acetyltransferase function.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4. Kat2b is required for the recruitment of IFT proteins and the Hedgehog signaling pathway component to the primary cilia.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe microtubules, which have α-tubulin as its subunit, acts as the highway for many molecules, including IFT cargo particles \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Previous research has revealed that the stability of microtubules is regulated by acetylation \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. On the basis of our findings for Kat2b functioning in the acetylation of α-tubulin, we speculated that the weakened microtubule could not break the highway, but could hamper cargo transport along the microtubule.\u003c/p\u003e \u003cp\u003eHence, we tested whether the translational level of IFT components could be affected by Kat2b depletion. In the early stages of primary cilia assembly, our western blot data show that IFT25, IFT46, IFT52, and IFT140, which are involved in cilia assembly, were decreased in the whole lysate of NIH/3T3 cells when Kat2b was silenced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;1a). To exclude the possibility that Kat2b might regulate the level of IFT components through its histone acetyltransferase function, we tested the mRNA levels of IFT components in the whole lysate of NIH/3T3 cells after 6 h of serum starvation. Interestingly, the transcriptional level of IFTs was either a slight increased or not changed at all in Kat2b-depleted cells (Supplementary Fig.\u0026nbsp;1c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese data prompted us to conduct immunocytochemistry experiments to verify whether the loss of Kat2b could affect ciliary localized IFT components. We used stable Kat2b knockdown cells to verify IFT components localization with or without Kat2b expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Our confocal microscopy data revealed that IFT25 localized to the basal body, and IFT52 was stained at the basal body, the axoneme, and ciliary tip in shControl NIH/3T3 cells at the early stage of ciliogenesis. Strikingly, the fluorescence intensity of IFT25, and IFT52 decreased in stable Kat2b knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). However, the intensity of IFT46 and IFT140 was not changed in Kat2b depleted cells (Supplementary Fig.\u0026nbsp;2a-b).\u003c/p\u003e \u003cp\u003eUnexpectedly, our western blot data verified that majority of IFT components also decreased after 24 h of serum starvation, which is at the maturation stage of ciliogenesis, whereas mRNA levels either increased slightly or did not change (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and Supplementary Fig.\u0026nbsp;1b and 1d). In this late stage, IFT25 was strongly stained at the basal body. IFT52 was also localized at the basal body and the axoneme. Surprisingly, our immunocytochemistry data clearly showed that the intensity of IFT fluorescence was dramatically decreased in shKat2b cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). This is unexpected because the depletion of Kat2b did not show a decreased percentage of ciliated cells at the time point of 24 h after serum starvation. Our results imply that even though cilia formation was saturated in Kat2b-depleted cells as much as in control, the ciliary function might be damaged. To consolidate our result, we used whole-body Kat2b knock-out mouse embryonic fibroblast (MEF) cell lines. Intriguingly, we could observe the fluorescence intensity of IFT25 and IFT52 is significantly reduced in Kat2b knockout (KO) MEF cells at 6h and 24h serum starvation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eSince the loss of IFT25 impairs Hedgehog (Hh) signaling \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and depletion of Kat2b hampered the Hh signaling in a cancer model \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, we further tested whether ablation of Kat2b expression could affect the activation of Hh signaling in the context of primary cilia. To test this, we silenced Kat2b and exposed them to serum starvation with 500 nM of SAG (Smo agonist) the following day for 24 h. SAG (Smo agonist) as activator of Hh signaling binds to the heptahelical bundle of Smo protein \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and induces ciliary translocation of Smo, leading to activation of Hh signaling. Our data showed that a few Smo proteins were recruited to the ciliary axoneme, despite the lack of SAG treatment in Kat2b depleted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). When SAG was treated, Smo was recruited to the whole axoneme with strong fluorescence intensity in control NIH/3T3 cells, while the fluorescence intensity of Smo protein was significantly decreased in Kat2b depleted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Next, we also checked that both the mRNA and protein levels of Gli1, the Hh signaling marker, increased with SAG treated cells but not as much as when Kat2b was depleted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). Therefore, our data clarifies that Kat2b regulates ciliary localized IFT components bona fide and impairs ciliary function through Hh signaling pathways. Furthermore, the recruitment of Smo protein at ciliary axoneme and Gli1 mRNA level were also impaired in Kat2b knockout MEF cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). Together, our data indicate that IFT recruitment and Hh signaling at primary cilium were damaged when Kat2b was absent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Function of acetyltransferase Kat2b for ciliogenesis depends upon the localization in cytosol\u003c/h2\u003e \u003cp\u003eSince Kat2b has an acetyltransferase domain, we hypothesized that Kat2b might regulate the cilium assembly rate and acetylation of α-tubulin through its catalytic function. Thus, we generated an acetyltransferase domain-deleted construct of Kat2b (ΔAT) to elucidate this discovery more concretely (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In addition, we created nuclear localization signal domain deleted Kat2b and both domain deleted Kat2b mutant. Through immunocytochemistry experiments, it was confirmed that DsRed tagged ΔNLS-Kat2b and ΔATΔNLS-Kat2b were not located in nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). And we also identified localization domain modified Kat2b in perfect working order by separation of nuclear and cytoplasmic fraction experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). NIH/3T3 cell was transfected with GFP tagged Kat2b construct in 48 h and harvested cells were immediately extracted between cytosolic and nuclear fractions. The immunoblotting band intensities were normalized with each loading controls; α-tubulin, as a cytosol control; Histone H3, as a nuclear control; and the percentage of Kat2b exist in the nucleus versus the cytoplasm was graphically represented. Less than half of NLS domain depleted Kat2b was in nucleus and NLS and AT domain depleted Kat2b was a nearly non-nuclear position. We replaced the tagged vector with GFP in order not to overlap with the fluorescence wavelength that identifies the IFT components, and proceeded with the following experiment. As observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, the ciliary localization of IFT52 in shControl NIH/3T3 cells with empty-vector was situated at both cilia and basal body, and no ciliary distribution but only basal body under Kat2b removed condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). In full-length-Kat2b transfected shKat2b NIH/3T3 cells that had undergone complete recovery, it was observed that the localization proportion of IFT52 with cilia surpassed shControl cells and almost IFT52 was found in ciliary structure of transfected cells. We also transfected ΔAT and ΔATΔNLS construct to stable Kat2b-depleted cells, as expected, proportion of IFT52-existed cilia decreased compared to that in FL-Kat2b transfected cells at maturation stages of primary cilia assembly. ΔATΔNLS -Kat2b in Kat2b-depleted cells, in particular, conducted like as empty vector since the ratio of IFT52 on the cilia and basal bodies was extremely similar. Finally, in the ΔNLS-Kat2b transfected shKat2b cell, it was a quite complete recovery with ciliary localized IFT52 as analogous as shControl cells. These results demonstrated that the function of Kat2b as acetyltransferase was significant for correct localization of IFT components even intact primary cilia structure and cytoplasmic Kat2b alone can play a sufficient role in the localization of IFT components in the cilia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Kat2b knockout mice show mild abnormalities related to cilia defect in kidneys.\u003c/h2\u003e \u003cp\u003eTo test whether Kat2b depletion \u003cem\u003ein vivo\u003c/em\u003e closely reflects \u003cem\u003ein vitro\u003c/em\u003e results, we examined the kidney tissue of a Kat2b knockout (KO) mouse model. This is because primary cilia are found in the apical region of renal epithelial cells \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and previous research papers have revealed that IFT defect causes ciliopathy such as cystic kidney disease \u003csup\u003e35 36\u003c/sup\u003e. First, we confirmed that the level of Kat2b deceased in the Kat2b KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The value of two kidney weights per total body weight did not change (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), but Kat2b KO mice showed decreased expression of acetylated alpha tubulin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). H\u0026amp;E data revealed that Kat2b KO mice have mild abnormalities in kidneys. They show dilated tubules and loosened glomeruli, while wild-type kidneys showed tight glomeruli (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). This is interesting because the disruption of the primary cilia component causes glomerular cysts \u003csup\u003e37 38\u003c/sup\u003e. Since our \u003cem\u003ein vitro\u003c/em\u003e data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) revealed that the loss of Kat2b impairs the recruitment of IFT components to the primary cilia, we conducted immunofluorescence experiment to investigate whether IFT defect also exists \u003cem\u003ein vivo\u003c/em\u003e as well. Confocal microscopy data showed that the fluorescence intensity of IFT25 at primary cilia decreased in Kat2b KO, whereas IFT25 distributes over the cilium of renal tubule in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Therefore, our data demonstrate that the loss of Kat2b shows primary cilia defect \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eIn this study, we investigated Kat2b as a novel regulator of primary cilia using \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments. Our data revealed that the basal expression of Kat2b gradually increased and that Kat2b at the cytosol, centrosome, and the basal body also gradually increased during primary cilia assembly. These findings suggest that Kat2b, which functions as a histone acetyltransferase, could be involved in many more diverse roles, including that of the primary cilia. Our work also revealed that the loss of Kat2b causes a delay in ciliogenesis and reduces the acetylation of α-tubulin through its acetyltransferase activity. Importantly, our confocal microscopy data clarifies that Kat2b depletion hampers the recruitment of IFT components to the cilium and decreases the level of Hh signaling activation. It is interesting to note that IFT components are involved in the ciliary formation and ciliary maintenance, such as signaling regulation. In addition, our data revealed that Kat2b KO mice show mild abnormalities related to cilia defect. These data strongly imply a possible relationship between Kat2b and ciliopathy caused by primary cilia defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur findings were further supported by two recently published papers covering the relationship between primary cilia and other PTMs, such as polyglutamylation. The authors suggested that polyglutamylation itself does not impair the primary cilia phenotype; however, it regulates the function of primary cilia, via the Hh signaling pathway \u003csup\u003e39 40\u003c/sup\u003e. This supported our hypothesis and results, considering that the cilium assembly compensated at the late stage of ciliogenesis in Kat2b depleted cells, although Kat2b regulates ciliary functions such as IFTs and the Hh signaling pathway. Thus, we can assume that PTMs in the primary cilium, such as acetylation and polyglutamylation, are involved in the functional aspects of primary cilia rather than assembly and having shared roles.\u003c/p\u003e \u003cp\u003eSince our data suggest the possibility of regulating the component of microtubules (highways), such as the acetylation of α-tubulin and its effect on IFTs (cargo proteins), identifying exact motor proteins (vehicles) related to this phenomenon merits further investigation. A recently performed proteomic approach, conducted by Fournier et al., to identify the Kat2b-targeted acetylome \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e may give us insight for this. For example, KIF17 and KIF27 could be the target of Kat2b. KIF17 is a member of the kinesin-2 family, which contributes to IFT-B transport \u003csup\u003e41 42\u003c/sup\u003e. KIF27, which is a member of kinesin-4, is located in the basal body \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. More interestingly, their acetylome data uncovered many proteins related to not only motor proteins but also other primary ciliary proteins could be acetylated by Kat2b; for instance, ALMS1 is an Alstrom syndrome gene, which is ciliopathic \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. DZIP1L is another ciliopathic gene encoding the ciliary-transition-zone protein and is related to autosomal recessive polycystic kidney disease (ARPKD) \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. SDCCAG3 has also been reported to localize to the basal body and interact with IFT88 \u003csup\u003e46\u003c/sup\u003e. In addition, TTBK2 is a ciliary gene related to the early step of ciliogenesis \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. These results provide strong evidence for the ciliary role of Kat2b. Further investigation is needed to elucidate the possible relations between Kat2b and other primary cilia proteins.\u003c/p\u003e \u003cp\u003eGiven previous studies and our results, there is a possibility of therapeutic development for ciliopathy. First, our data revealed that Kat2b regulates primary ciliary function. Second, Kat2b KO mice show mild pathological effects like glomerular abnormalities and dilated tubules in the kidney. Thus, whether Kat2b upregulation could attenuate this pathological effect and recover ciliary function merits further investigation. Altogether, this study highlights the novel role of Kat2b in regulating primary cilium function and its pathological effect in the kidney.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAdditional information\u003c/h2\u003e \u003cp\u003ePublisher's note\u003c/p\u003e \u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRights and permissions\u003c/strong\u003e \u003cp\u003eOpen Access\u003c/p\u003e \u003cp\u003eThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://creativecommons.org/licenses/by/4.0/\u003c/span\u003e\u003cspan address=\"http://creativecommons.org/licenses/by/4.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: [J. H. Park], [K. H. Yoo]; Methodology: [H. Cha], [J.Y. Ko], [Ho-S. Kim]; formal analysis and investigation: [H. Cha], [J. H. Jun], [J,Y. Ko], [J. Min]; Writing - original draft preparation; [H. Cha], [J. H. Jun]; Writing - review and editing: [H. Cha], [J. H. Jun], [J. Y. Ko], [J. H. Park]; Funding acquisition: [J. H. Park]; Resources: [Ho-S. Kim]; Supervision: [J. H. Park]\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis study was supported by grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (2015M3A9B6027555 and 2016R1A5A1011974).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated and/or analyzed during this study are available from the corresponding author upon reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSatir, P., Pedersen, L. B. \u0026amp; Christensen, S. T. The primary cilium at a glance. 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PLoS genetics 14, e1007844, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pgen.1007844\u003c/span\u003e\u003cspan address=\"10.1371/journal.pgen.1007844\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Acetylation, Ciliogenesis, Intraflagellar transport, Kat2b, Primary cilia","lastPublishedDoi":"10.21203/rs.3.rs-4911699/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4911699/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePrimary cilia are sensory organelles that regulate various signaling pathways. For this, microtubules work as a highway, and they are tuned with post-translational modifications, such as acetylation. However, the role of acetylation in the regulation of primary cilia remains uncertain. Here, we identified K (lysine) acetyltransferase 2B (Kat2b) as a novel regulator of primary cilia. Kat2b localizes to the cytosol, centrosome, and the cilium basal body. Also, the basal expression of Kat2b gradually increases during ciliogenesis. Kat2b regulates the rate of cilia assembly and the acetylation of α-tubulin via its catalytic activity. The loss of Kat2b reduces the recruitment of intraflagellar transport (IFT) components to the ciliary axoneme and impairs Hedgehog signaling activation. In addition, Kat2b-knockout mice show mild abnormalities and ciliary IFT defect in kidneys. Thus, our new findings establish a link between acetylation regulated by Kat2b and its relevance to ciliary assembly and function.\u003c/p\u003e","manuscriptTitle":"Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-16 14:00:01","doi":"10.21203/rs.3.rs-4911699/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-25T05:27:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-19T13:53:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-30T04:13:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-27T16:52:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315466269248439548040863317982690808560","date":"2024-08-24T05:00:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330737491682291232825123292602365887353","date":"2024-08-24T04:00:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248083864590285839970032727226179068803","date":"2024-08-22T06:33:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-22T03:37:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-22T03:29:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-08-22T03:24:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-19T09:14:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-08-14T07:42:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6c71986f-4270-4b3b-85ec-139fcbcab1d8","owner":[],"postedDate":"September 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":37638215,"name":"Biological sciences/Genetics"},{"id":37638216,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-01-20T16:09:18+00:00","versionOfRecord":{"articleIdentity":"rs-4911699","link":"https://doi.org/10.1038/s41598-025-86292-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-01-16 15:57:23","publishedOnDateReadable":"January 16th, 2025"},"versionCreatedAt":"2024-09-16 14:00:01","video":"","vorDoi":"10.1038/s41598-025-86292-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-86292-5","workflowStages":[]},"version":"v1","identity":"rs-4911699","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4911699","identity":"rs-4911699","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}