Reversible acetylation of HDAC8 regulates cell cycle

HDAC8, a member of class I HDACs, plays a pivotal role in cell cycle regulation by deacetylating the cohesin subunit SMC3. While cyclins and CDKs are well-established cell cycle regulators, our knowledge of other regulators remains limited. Here we reveal the acetylation of K202 in HDAC8 as a key cell cycle regulator responsive to stress. K202 acetylation in HDAC8, primarily catalyzed by Tip60, restricts HDAC8 activity, leading to increased SMC3 acetylation and cell cycle arrest. Furthermore, cells expressing the mutant form of HDAC8 mimicking K202 acetylation display significant alterations in gene expression, potentially linked to changes in 3D genome structure, including enhanced chromatid loop interactions. K202 acetylation impairs cell cycle progression by disrupting the expression of cell cycle-related genes and sister chromatid cohesion, resulting in G2/M phase arrest. These findings indicate the reversible acetylation of HDAC8 as a cell cycle regulator, expanding our understanding of stress-responsive cell cycle dynamics.

Figure 1.The authors provide a set of in vivo and invitro data demonstrating that HDAC8 is acetylated at lysine K202.Based on these they concluded that acetylation of residue K202 has the effect of inhibiting deacetylation of Smc3 by HDAC8.However, they did not determine clearly the impact of the K202 mutation on Smc3 acetylation in human cells.The authors performed CRISPR mutations in the HDAC8 gene but surprisingly did not study the impact of these mutations on Smc3 acetylation.To make sure that K202 acetylation inhibits HDAC8 mediated Smc3 de-acetylation in living cells it is necessary to analyze the impact of these mutations on Smc3 acetylation in a revised version of the article.
Response: We appreciate the valuable suggestion from the reviewer.Following your guidance, we conducted Western blot analysis on Knock-in HDAC8 K202R and K202Q cells (New Figure 4b).Our findings reveal a noteworthy increase in the acetylation level of SMC3 in HeLa cells subsequent to the introduction of HDAC8 K202R and K202Q mutations.
New Figure 4b Figure 2C.Based on the fact that Tip60 overexpression induces an increase in Smc3 and HDAC8 acetylation, the authors claim that Tip60 is the acetylase that induces HADC8 acetylation.However, it is conceivable that Tip60 overexpression causes cell arrest in G2/M, which in turn induces increased acetylation of Smc3 and HADC8.It is therefore important to monitor the effect of Tip60 overexpression on the cell cycle.The authors should also monitor the impact of Tip60 overexpression on the cellular amount of HDAC8 in their western blot.
Response: We thank the reviewer for this suggestion.Following your suggestions, we ectopically overexpressed Tip60 in HeLa cells with approximately 30-40% confluence for 24h, and then analyzed cell cycle stages by FACS as well as monitored intracellular HDAC8 amount by immunoblotting.In the figure below, the impact of Tip60 overexpression on the cell cycle was obvious (Supplementary Figure 2b), and the intracellular HDAC8 amount remained generally stable while the acetylation levels of HDAC8 and SMC3 were significantly increased (Supplementary Figure 2c).These findings suggested that Tip60 could affect the cell cycle through the acetylation of HDAC8.
However, several of our additional experiments have provided evidence that Tip60 is capable of acetylating HDAC8.As depicted in Figure 2a, b, it clearly demonstrated the interaction between Tip60 and HDAC8.Through knockdown Tip60 by using siRNA, we established Tip60's ability to deacetylate HDAC8 (Figure 2d).Further experiments indicated that acetylation of HDAC8-K202 are dependent on Tip60, regardless of the presence or absence of stress stimulation (Supplementary Figure 3d).Figure for referee with unpublished data has been removed upon request by the authors.

New Supplementary Figure 2b-2c
Figure for referee with unpublished data has been removed upon request by the authors.
K202 of HDAC8 throughout the cell cycle progression.Therefore, we were unable to detect the K202Q/R mutants within the endogenous cell population.Minor comment: The Hi-C maps should be square and not rectangular.The color code must be added next to the Hi-C maps.

Original Figure 3d
Response: We appreciate the reviewer's suggestion and have corrected these in the revised version.

New Figure 4i
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Figure for referee with unpublished data has been removed upon request by the authors.
Referee #2: HDAC8 is a class I deacetylase that deacetylates the cohesin complex subunit SMC3.How HDAC8's deacetylase activity is controlled is poorly understood.Sang et al. find that HDAC8 is regulated by TIP60-mediated acetylation of K202 on HDAC8.K202 is positioned close to the active site and they find that acetylation of this lysine inhibits HDAC8's deacetylase activity.They show that K202 acetylation is dynamic during the cell cycle.Using mutants that either mimick the acetylated lysine, or the non-acetylated lysine, they find that acetylation of this site alters gene expression and regulates the 3D genome.The findings presented in this manuscript are cleary interesting, but further support is required for the model put forward by the authors to be suitable for publication in EMBO Reports.
We thank the reviewer for the positive comments and insightful suggestions.Please find our point-to-point response below.
Comments Sang et al.
-The authors generated cell lines harbouring acetylation mimick and non-acetylatable mutants for K202, to assess the effect on gene expression and the 3D genome.When preparing their samples for Hi-C analyses, they comment that these cell lines have a similar cell cycle stage and can therefore be compared.However, in the next figure they show that mutation of K202 leads to an arrest in G2/M-phase and changes in gene expression.It would therefore be important to perform Hi-C analyses in synchronized cells, preferably G1-phase to prevent any potential influence by changes in cohesin complexes holding the sister chromatids together.It would also be important to check the levels of HDAC8 in these cells and assess whether the acetylation of SMC3 is affected.
Response: We appreciate the reviewer's valuable suggestion.The analysis of unsynchronized cells with the K202Q mutation for Hi-C experiments, as illustrated in the supplementary Figure 4b, revealed a slightly increased proportion of cells in the G2/M phase in K202Q cells, consistent with the statistical findings presented in Figure 5f.
However, we considered this difference acceptable and emphasized that the cell samples had comparable cell cycle stages.While using synchronized G1 phase cells for Hi-C experiments would be an ideal choice, our attempts to synchronize cells to the G1 phase using thymidine arrest, especially in the K202Q mutant cells (Results of attempted synchronization to G1 phase), proved challenging due to varied proliferation statuses among mutant cells (Supplementary figure 5c).Achieving strictly synchronized G1-phase cells may be impractical.Moreover, the integrated analysis of RNA-seq and Hi-C results on unsynchronized cells allows for linking chromatin loops to gene expression changes, providing comprehensive insights into regulatory mechanisms.Additionally, in response to the reviewer's suggestion, we conducted a basic immunoblotting examination of the knock-in cells (Figure 4b).As expected, the SMC3 acetylation level was significantly elevated in the K202Q cells, while the HDAC8 expression level remained essentially unchanged compared to WT cells.This further supports our findings and strengthens the correlation between HDAC8 activity and chromatin loop dynamics.

New Supplementary Figure 4b
Results of attempted synchronization to G1 phase New Supplementary Figure 5c New Figure 4b -The authors should include replicates for the Hi-C analyses, preferably in independent clones.
They should also provide information on the amount of reads in the methods and whether these are similar between genotypes.The differences observed in for example figure 4e could (at least partially) be explained by a difference in sequencing depth.
Response: We appreciate the reviewer's constructive suggestion.In response to your guidance, we conducted a replicative Hi-C experiment using an additional site mutant clone.
To mitigate potential biases arising from sequencing depth, we normalized valid contacts to ensure consistency among the three samples.-It appears to me that the changes depicted in figure 4g and 4h are rather small.Is this considered a strong correlation?The main text describing these panels is rather limited and could use further explanation.
Response: We appreciate the reviewer's comments.Gene expression is a highly intricate process, and the spatiotemporal aspects of genomic structure are increasingly acknowledged as crucial for understanding eukaryotic gene expression.Nevertheless, the mechanistic underpinnings and causal links between structure and gene expression remain poorly understood.The development and application of Hi-C technology have provided valuable insights into such studies.In our study, we employed multi-omics analysis to investigate the relationship between chromatin interactions and gene expression.Initially, we selected differentially expressed genes (DEGs) with more than 2fold changes between K202Q/K202R with WT cells from the RNA-seq data.Meanwhile, we obtained normalized contact frequencies of chromatin loops from Hi-C data.By defining the ±1kb regions around the transcription start site (TSS) of genes as the approximate promoter regions, we overlapped the promoters of DEGs with chromatin loops to identify loops associated with these genes (generated .csvfile had been submitted as source data with the title as shown below).Subsequently, we calculated the Pearson correlation Figure for referee with unpublished data has been removed upon request by the authors.

coefficient (PCC) and the corresponding p-value between the changes in gene expression
and the changes in normalized contact frequency in chromatin loops (Figure 4g, h).
Through the overall analysis, we discovered that the normalized contact frequency of interactions exhibited a significant correlation with gene expression levels at the distal elements of promoters.Our findings underscore the pivotal role of HDAC8-mediated chromatin interactions in regulating gene expression.Given the intricate nature of gene expression regulation, the significant correlation shown in our overall analysis provides robust support for our conclusions.Also, following the comments, we have corrected the "strong correlation" to "significant correlation" and added further explanation in the revised manuscript.

Figure 4g-4h
-The finding that K202Q mutant cells display a strong cohesion defect is interesting.To draw meaningful conclusions, it would be important to score the severity of the cohesion defect and provide example pictures of the different categories.
Response: We appreciate the reviewer's suggestion, and in response, we have implemented additional improvements to our study.Specifically, we further classified the observed cohesion phenotypes into four categories: normal, mild, moderate, and severe, as illustrated in Figure 5e.Additionally, we conducted a chromosome spread assay with siRad21 as a positive control, clearly demonstrating the knockdown effect of Rad21, as depicted in Supplementary Figure 5b.Results were shown in the Figure 5d.These refinements aim to provide a more detailed and comprehensive analysis of cohesion phenotypes and strengthen the overall robustness of our experimental results.

New Figure 5e
New Supplementary Figure 5b New Figure 5d -The authors show in figure 2e and 3b that several stressors lead to a G2/M arrest.However, the extent to which this arrest occurs is very different between these two panels.Could the authors explain these differences?
Response: Thank you for providing clarification regarding the differences in experimental conditions between Figure 2e and Figure 3b.The distinction in the synchronization status of HeLa cells, with Figure 2e involving cells synchronized to the early S phase and Figure 3b utilizing unsynchronized cells, explains the observed variations between these two panels.We have added this information into figure legends to help the interpretation of the results presented in the figures.
-In figure 3c the authors show that cells can recover from these stressors and that both the acetylation of HDAC8 and SMC3 is rescued to normal wild type unperturbed levels.However, it is difficult to compare these settings, as the western blot samples are harvested from cells that are from different cell cycle phases, and SMC3 acetylation is known to change throughout the cell cycle.The increase in acetylated SMC3 observed in stressed cells could therefore be at least partially explained by the enrichment of cells in G2 phase.It would be important to compare the acetylation between e.g.only G1 cells or G2 cells.In addition, the authors could test whether the increase in acetylated SMC3 in stressed cells is dependent on HDAC8 acetylation by simultaneously depleting Tip60, and whether this rescues the cell cycle defect.

Figure for referee with unpublished data has been removed upon request by the authors.
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Response: We thank the reviewer's insightful suggestion.As shown in Figure 3d, we conducted overexpression of Flag-HDAC8 in HeLa cells followed by stress treatment.
Subsequently, we enriched the HDAC8 protein using Flag beads and measured the enzyme activity in vitro, confirming a significant reduction in HDAC8 enzyme activity induced by stress.Extensive research has established SMC3 as an unequivocal substrate of HDAC8, and inhibition of HDAC8 has been linked to elevated intracellular SMC3 acetylation levels.Furthermore, in our experiments, cells treated with stress were not restricted to G1/G2 phases post-cell cycle synchronization.Although stress-induced G2/M phase arrest was statistically significant, the overall alteration was relatively minor (Figure 3b).Moreover, the duration of TSA treatment and glucose starvation (24-36 hours) exceeded the typical cell cycle duration, rendering it challenging to obtain G1/G2 cells postcell cycle synchronization following stress.In summary, we posit that the observed increase in SMC3 acetylation levels following stress treatment primarily arises from the reduction in HDAC8 enzyme activity.

New Figure 3d
Figure 3b Following your recommendations, we proceeded with Tip60 knockdown in stressstimulated cells and subsequently evaluated the cell cycle stage using FACS analysis.
However, despite the Tip60 knockdown, we observed no rescue of the cell cycle defect (Supplementary Figure 3e).Additionally, we examined acetylated K202-HDAC8 levels in glucose-starved cells with Tip60 knockdown (Supplementary Figure 3d).Notably, while Tip60 protein levels increased under glucose-free stimulation (line 3 compared to line 1), the acetylated K202-HDAC8 returned to normal levels under Tip60 knockdown conditions despite glucose starvation (line 4 compared to line 2).These findings suggest that the stimulations triggering increased acetylation of K202-HDAC8 are dependent on Tip60.

New Supplementary Figure 3e
New Supplementary Figure 3d -The finding that Tip60 can interact with HDAC8 and acetylate it, is clear from figure 2. Is this acetyl transferase only active when stressors are present, and/or is Tip60 also cell cycle regulated?It would be useful for the reader if Tip60 is better introduced in the text.
Response: We appreciate the valuable suggestion from the reviewer.In line with your recommendation, we investigated the expression of Tip60 throughout the cell cycle, revealing that the expression level of Tip60 remained unchanged in synchronized cells (Figure 3e).This finding suggests that other regulatory mechanisms, such as posttranslational modifications, may govern Tip60 activity during the cell cycle.

New Figure 3e
Furthermore, we investigated the alterations in Tip60 expression under various glucose concentration treatments (Supplementary Figure 3c).Our results demonstrated an increase in Tip60 expression under conditions of decreased glucose concentration, consistent with previous findings (2019 Cell Rep, PMID: 30699357).This observation suggests that glucose starvation may upregulate Tip60 expression, thereby influencing HDAC8 acetylation.

New Supplementary Figure 3c
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-The finding that K202R also shows a decrease in its deacetylation is surprising.The authors hypothesise that this K202 might be important for the catalytic activity.Could they further elaborate on how K202 might do so?
Response: We have consolidated our responses to this and the subsequent comment.
Please refer to the response provided for the next question.
- Mutation of D233 to G233 results in a significant decrease in protein thermostability ( Tm = -6.8°C)and only 49% residual activity.Crystal structure analysis reveals that the G233 mutation induces minor structural changes but disrupts the hydrogen bond with K202 and weakens the remaining hydrogen bond between S276 and K202 (the right figure in Decroos et al. 2015).Further molecular dynamics (MD) simulations demonstrate increased root mean square (rms) fluctuations of K202 and adjacent residues upon the G233 mutation.Given that K202 resides at the end of the -strand, S276 is positioned in the L7 loop, and D233 is located in the L6 loop, the hydrogen bond network involving D233-K202-S276 is deemed critical for stabilizing the HDAC8 structure.In our study, we observed that the K202R mutation disrupted the hydrogen bond with S276 and weakened the bond with D233, while the K202Q mutation led to the simultaneous loss of hydrogen bonds with both S276 and D233 (Supplementary Figure 1f).These structural alterations may account for the differential residual activities observed for the K202R and K202Q mutants, approximately 58% and 12%, respectively (Supplementary Figure 1e).Overall, our findings suggest that mutations or acetylation of K202 may impair HDAC8 activity by disrupting the formation of the hydrogen bond network.

Decroos et al. 2015 (Left) (Right)
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New Supplementary Figure 1f
Supplementary Figure 1e Furthermore, we conducted molecular docking assays between HDAC8 and its substrate, SMC3 (Supplementary Figure 1g).Consistent with existing knowledge, we observed that the hydrophobic pocket of HDAC8 (WT) was occupied by the acetylated lysines (K105 and K106) of the substrate SMC3 during the deacetylation reaction.Notably, when docking mutants of HDAC8 to SMC3 in a similar manner, we discovered that both the K202R and K202Q mutations interfered with the accessibility of SMC3's acetylated lysines to the pocket.Intriguingly, the interference caused by the K202Q mutation was more pronounced.
To validate these findings, we overexpressed Flag-HDAC8 or its mutants in 293T cells and subsequently performed immunoprecipitation.The results revealed that the HDAC8 K202R/K202Q mutants exhibited progressively weaker binding to the substrate SMC3 (Supplementary Figure 1h).These outcomes imply that K202R or K202Q mutations of HDAC8 may affect the structural stability by disrupting the hydrogen bond network, resulting in a weaker binding to substrates and thus a lower catalytic activity.

New Supplementary Figure 1g
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-It is unclear to me how the authors assessed the total amount of acetylation on HDAC8 in figure 2d.Did the authors first enrich for HDAC8 with a pulldown?Or does that this antibody recognize multiple acetylated residues on HDAC8?
Response: Thank you for your inquiry.In our experimental setup, we conducted Tip60 knockdown in cells overexpressing HDAC8 tagged with FLAG.Subsequently, we utilized anti-FLAG beads to immunoprecipitate HDAC8 and performed corresponding detection (Figure 2d).We have revised the respective figure accordingly.

New Figure 2d
-It would be informative if the authors include Hos1, the S. cerevisiae cohesin deacetylase, in their evolutionary conservation alignments.
Response: Thank you and we have added the Hos1 into our evolutionary alignments.Noting the change in the corresponding residue sequence of Hos1 in S. cerevisiae to leucine instead of lysine is an important clarification.
-The authors should include loading controls for all their western blot analyses.
-The authors should always show both the input and the pulldown for the IP experiments they perform.E.g. in figure 1g they only show the pulldown, but not the levels of AcK202, AcK, and FLAG in the input.In figure 2a and 2b, please show both the signal for FLAG and HA in the input.
Response: We thank the reviewer's suggestion.Following your recommendation, we have revised the corresponding figure and included the corresponding input data.Figure for referee with unpublished data has been removed upon request by the authors.

Figure for referee with unpublished data has been removed upon request by the authors.
Referee #3: This is a paper that attempts to find the acetylation of HDAC8 and investigate its physiological function.Hdac8 is a cohesin deacetylase that is important for regulation of cohesin dynamics, and thus regulates cohesin function in cell cycle and transcription.I think this paper has an impact, but I am concerned about many points as follows.
1) For example, in Fig 1f, they used a system in which HDAC8 expressed in E. coli is acetylated in E. coli, and it shows that the activity is reduced by K202ac.It certainly shows acetylation in K202, but acetylation of other residues must also be occurring.To prove that acetylation of K202ac reduces the activity of HDAC8, it is necessary to perform the same experiment with K202R.
Response: We appreciate the reviewer's concern, and we would like to provide an overview of the site-specific incorporation assay for inducing the acetylated modification in Figure 1f.This methodology was initially introduced by Neumann et al. in 2018 in Nature Chemical Biology (PMID: 18278036, cited by 702).To create a homogeneously K202acetylated HDAC8 construct, we utilized a three-plasmid system (TEV-8, pCDFpylT-1, and pAcKRS), detailed below.Wild-type HDAC8 was cloned into pTEV-8, yielding a C-terminal His6-tagged construct, and an amber codon was introduced at lysine 202 (AAG to TAG through site-directed mutagenesis).The amber construct was overexpressed in LB with spectinomycin (50 mg/ml), kanamycin (50 mg/ml), and ampicillin (150 mg/ml), along with 2mM N-acetyl-lysine and 20 mM nicotinamide to inhibit E. coli deacetylase activity during induction.The procedures for cell culture, expression, and purification were consistent with those outlined for recombinant human HDAC8.This system selectively yields total acetylated-K202 in HDAC8, preventing acetylation at other lysines.It has been successfully employed in purifying two distinct site-specific acetylated proteins in our prior research (Wei et al. 2018, PMID: 30755608, andWan et al. 2020, PMID: 32783943).Additionally, we introduced the K202R mutation, mimicking the deacetylation status in HDAC8, to investigate its activity.The K202R variant exhibited a significantly reduced activity compared to wildtype HDAC8 (Supplemental Figure 1e), highlighting the critical role of K202 in HDAC8 activity and suggesting that acetylation at K202 may impact its deacetylase activity.

Figure 1f New Supplementary Figure 1e
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2) Fig4: They have data showing that the interaction in the TAD is stronger in cells heterozygous for K202Q (acetyl mimic) and that the loop gets also stronger.This mutation reduces the activity of HDAC8 (Fig1c).This means that the cohesin pool for loop extrusion is reduced.As far as I understand from other papers, this is strange.To resolve this discrepancy, .This analysis revealed a significant elevation in SMC3 acetylation levels in the K202Q cells, while the HDAC8 expression level remained essentially unchanged compared to WT cells.These results further strengthen our assertion that acetylated HDAC8 plays a crucial role in modulating chromatin loop dynamics.

Figure 4a
New Figure 4b 3) Fig5, d&e; the K202Q mutant cell line has a cohesion defect of over 70%.I wonder if such a high level of cohesion defects can cause death.I think that authors should perform a cohesion assay with a control such as siRad21 in parallel to see if there are any qualitative or quantitative differences.
Response: We appreciate the valuable suggestion from the reviewer.In response to your guidance, we conducted a chromosome spread assay with siRad21 as a positive control,

Figures for referee with unpublished data have been removed upon request by the authors.
clearly demonstrating the knockdown effect of Rad21 in the new Supplementary Figure 5b.
To provide a comprehensive understanding of the observed cohesion phenotypes, we categorized them into four distinct levels: normal, mild, moderate, and severe, as illustrated in the new Figure 5e.Statistical analysis revealed a significant increase in the proportion of cohesion defects in K202Q mutant cells, reaching approximately 67%.Notably, these defects were predominantly mild and moderate, with severe cohesion defects primarily observed in cells subjected to siRad21 treatment, in the new Figure 5d.This discrepancy can be attributed to the direct involvement of Rad21, a core subunit of the cohesion complex, in sister chromatid cohesion.In contrast, HDAC8 indirectly influences cohesion through the deacetylation modification of SMC3, another core subunit of cohesin.
Additionally, we observed significant changes in the proliferation rates of different cell populations, as shown in the new Supplementary Figure 5c.As a positive control, cells treated with siRad21 exhibited pronounced growth inhibition, with the inhibitory effects appearing more prominent within 48 hours, aligning with the characteristics of transient transfection.In comparison to WT cells, the proliferation of K202R cells showed a modest decrease, while the proliferation inhibition observed in K202Q cells was more pronounced.
These findings contribute to a more comprehensive understanding of the impact of HDAC8 mutations on cell cohesion and proliferation dynamics.

New Supplementary Figure 5b
New Figure 5e New Figure 5d New Supplementary Figure 5c I raised here the most important points I feel that should be addressed at least.Overall the data contains lots of roughness.Therefore I could not recommend this paper to be published in any journal.
Response: In addition to the aforementioned points, it is imperative to further elaborate on  1f).These structural modifications may elucidate the differential residual activities observed for the K202R and K202Q mutants, approximately 58% and 12%, respectively (Supplementary figure 1e).In summary, our findings underscore that mutations or acetylation of K202 may compromise HDAC8 activity by disrupting the formation of the hydrogen bond network.
Furthermore, we conducted molecular docking assays between HDAC8 and its substrate, SMC3 (Supplementary Figure 1g).Consistent with existing knowledge, we observed that the hydrophobic pocket of HDAC8 (WT) was occupied by the acetylated lysines (K105 and K106) of the substrate SMC3 during the deacetylation reaction.Notably, when docking mutants of HDAC8 to SMC3 in a similar manner, we discovered that both the K202R and K202Q mutations interfered with the accessibility of SMC3's acetylated lysines to the pocket.Intriguingly, the interference caused by the K202Q mutation was more pronounced.
To validate these findings, we overexpressed Flag-HDAC8 or its mutants in 293T cells and subsequently performed immunoprecipitation.The results revealed that the HDAC8 K202R/K202Q mutants exhibited progressively weaker binding to the substrate SMC3 (Supplementary Figure 1h).These outcomes imply that K202R or K202Q mutations of HDAC8 may affect the structural stability by disrupting the hydrogen bond network, resulting in a weaker binding to substrates and thus a lower catalytic activity.

New Supplementary Figure 1f Supplementary Figure 1e
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Figures for referee with unpublished data have been removed upon request by the authors.
Figures for referee with unpublished data have been removed upon request by the authors.
We have added a description of the variability of Hi-C experiments of K202R mutant cells in the Results and Discussion sections of the revised manuscript, respectively.

Discussion
I also have some remaining textual comments.The authors describe the structures presented in supplemental Figure 1F as actual structures, and discuss how mutation affects the H-bonds in the catalytic site.However, I am missing information on how these structures are obtained in the methods.
Are these new crystal structures, or are these predictions of how mutation of K202 in the published HDAC8 crystal structure (1W22) might affect the Hbonds?The authors should describe this more accurately in the text and methods.If these are indeed predictions, they should also rewrite their conclusions about these mutant forms of HDAC8.Similarly, the authors describe in supplemental Figure 1G that they provide a crystal structure of the SMC3-HDAC8 interaction, while this actually is a predicted structure that uses the crystal structure of HDAC8 and cryo-EM structure of SMC3.They don't describe how they obtained the HDAC8 mutant structures, which should be included in the methods.
Response: We thank the reviewer for this suggestion.As noted, the K202R and K202Q mutations were indeed predicted using the software PyMOL.
Given that the crystal structure of HDAC8-WT has been determined and published (PDB accession code 1W22), utilizing the mutagenesis function of PyMOL based on the HDAC8-WT crystal structure to predict the structure of point mutations is a viable approach.Following the reviewer's suggestion, we have described this process more accurately in the manuscript and figure legends of the latest revised version to facilitate a better and more precise understanding of our study.

Latest revised version (Manuscript) Latest revised version (Figure legends)
Dear Prof. Yu, Before we can accept the manuscript, the following remaining points need to be addressed: -Please add a discussion point into the text acknowledging the variability in the chromatid loop strength of K202R mutant cells (as pointed out by referee #3).
-Please add a scale bar to Figure 5e and define its length in the figure legends.
-Please specify the nature of the replicates stated in the figure legends (i.e.biological, technical).
Many thanks.
Your paper has been placed back in the Author Approval Folder where you may access via the following link: *Link Unavailable* Please make the correction(s) as specified above and resubmit your paper following the same steps as before.
Should you have any queries, please do not hesitate to contact us.

Kind regards, Bojana
Bojana Perkucin Editorial Assistant EMBO Press 25th Jun Additional Correspondence from the Editor -Please add a discussion point into the text acknowledging the variability in the chromatid loop strength of K202R mutant cells (as pointed out by referee #3).

Response:
We have already done so in the revised manuscript, as you requested.
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Figure 2a-b

Figure 3D .
Figure 3D.The auteurs should repeat experiment Figure 3D in a context where cells express K202Q and K202R mutations.

Figure 4 .
Figure 4.It would have been interesting to compare the effect of the K202 mutations with that induced by inactivation of the HDAC8 gene.Rowland's Benjamin laboratory recently studied the effect of the absence of HDAC8 on genome organization in HAP1 cells.Would it be possible for the authors to use this same type of cell to study the effect of their mutations on genome organisation?This would also make it possible to compare the effects of HDAC8 point mutations with those induced by the absence of HDAC8.

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Could the authors elaborate on how the acetylation of K202 on HDAC8 reduces the deacetylase activity?Does acetylation of K202 e.g.change the folding of this part of HDAC8?Does it prevent entry of acetylated lysines into the deacetylation pocket?Response: According to Decroos et al. (2015, Biochemistry, PMID: 26463496), K202 plays a crucial role in establishing a hydrogen bond network (D233-K202-S276) essential for HDAC8 activity.Specifically, in wild-type (WT) HDAC8, the side chain of K202 forms hydrogen bonds concurrently with S276 and D233 (the left figure in Decroos et al. 2015).

Figure
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I
Figure 4b.This analysis revealed a significant elevation in SMC3 acetylation levels in the

Response:-
We have added a description of the variability of Hi-C experiments of K202R mutant cells in the Results and Discussion sections of the revised manuscript, respectively.Results Discussion -Please add a scale bar to Figure 5e and define its length in the figure legends.Response: We have already done so in the revised manuscript, as you requested.Please specify the nature of the replicates stated in the figure legends (i.e.biological, technical).
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(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)If collected and within the bounds of privacy constraints report on age, sex and gender or ethnicity for all study participants.

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(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) ). the assay(s) and method(s) used to carry out the reported observations and measurements.anexplicit mention of the biological and chemical entity(ies) that are being measured.anexplicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.ideally,figure panels should include only measurements that are directly comparable to each other and obtained with the same assay.plotsinclude clearly labeled error bars for independent experiments and sample sizes.Unless justified, error bars should not be shown for technical the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).a statement of how many times the experiment shown was independently replicated in the laboratory.Journal Submitted to: EMBO Reports This checklist is adapted from Materials Design Analysis Reporting (MDAR) Checklist for Authors.MDAR establishes a minimum set of requirements in transparent reporting in the life sciences (see Statement of Task:

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) If study protocol has been pre-registered, provide DOI in the manuscript.For clinical trials, provide the trial registration number OR cite DOI.(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

definition and in-laboratory replication Information included in the manuscript? In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)In the figure legends: state number of times the experiment was replicated in laboratory.

In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)Studies involving human participants: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Not ApplicableStudies involving human participants: Include a statement confirming that informed consent was obtained from all subjects and that the experiments Not ApplicableStudies involving human participants: For publication of patient photos, include a statement confirming that consent to publish was obtained.

Use Research of Concern (DURC) Information included in the manuscript? In which section is the information available?
(Reagents and ToolsTable, Materials and Methods, Figures, Data Availability Section) Could your study fall under dual use research restrictions?Please check biosecurity documents and list of select agents and toxins (CDC): https://www.selectagents.gov/sat/list.htmNot Applicable If you used a select agent, is the security level of the lab appropriate and reported in the manuscript?Not Applicable If a study is subject to dual use research of concern regulations, is the name of the authority

granting approval and reference number for
the regulatory approval provided in the manuscript?

and III randomized controlled trials
Table, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.Not Applicable For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines., please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.