Article Figures & Data
Figures
- Figure 1. H3K9ac levels during mitosis.
(A) Schematic representation of the synchronization strategy by double thymidine block method. (B) Immunofluorescence of HeLa-S3 cells enriched for mitotic stages stained for H3K9ac (green) and DNA (blue). Representative pictures from the major mitosis stages are shown. Quantification of the immunofluorescence results presented below the pictures. Intensity values represent the mean ± SEM of at least 20 cells for every stage. (C) Schematic representation of the synchronization strategy by kinesin-5 inhibitor S-trityl-L-cysteine (STC). (D) Immunofluorescence of STC arrested HeLa-S3 cells stained for DNA (blue) and H3K9ac (green). The cells were either not treated (NT) or treated with TSA (150 nM); nicotinamide (NAM, 10 mM); RGFP966 (40 μM); EX-527 (5 nM). Quantification of the immunofluorescence results are shown below the pictures. Intensity values represent the mean ± SEM of at least 25 cells for every condition. (D, E) All treatments significantly (P < 10−23; one sided t test) increased H3K9ac levels (E) Immunoblot showing the H3K9ac signal for synchronized HeLa-S3 cells treated with the indicated inhibitors (same concentration as in D), total histone H3 serves as a loading control. Scale bars: 5 μm.
- Figure S1. FACS analysis of double thymidine block (DTB) synchronized Hela S3.
(A)The cell cycle distribution of cells synchronized by double thymidine block was monitored by FACS. The number of mitotic cells (arbitrary units) is plotted against DNA content for time points after release. (B) Bar graph showing the percentage of cells in mitosis at various time points after release from the DTB. (C) Pie chart showing the percentage of cells at different mitotic stages at the 8.5 h time point.
- Figure 2. HDAC inhibition shows involvement of specific HDACs in modulating the dynamics of H3K9ac during mitosis.
(A) Immunofluorescence of HeLa-S3 cells enriched for mitotic stages stained for H3K9ac (green) and DNA (blue). Representative pictures from the major mitosis stages are shown. The cells were either not treated (NT) or treated with TSA (150 nM); nicotinamide (NAM, 10 mM); RGFP966 (40 μM); chidamide (10 μM); EX-527 (5 nM); CAY10683 (1 μM); MS-275 (1 μM). Below: quantification of the immunofluorescence results. Intensity values represent the mean ± SEM of at least 20 cells for every stage. (B) Immunofluorescence of MEFs cells enriched for mitotic stages stained for H3K9ac (green) and DNA (blue). Representative pictures from the interphase, prophase, and metaphase stages are shown. The cells were either not treated (NT) or treated with TSA (150 nM); nicotinamide (NAM, 10 mM); RGFP966 (40 μM); chidamide (10 μM); EX-527 (5 nM); CAY10683 (1 μM); MS-275 (1 μM). Below: quantification of the immunofluorescence results. Intensity values represent the mean ± SEM of at least 20 cells for every stage. All treatments (both in HeLa-S3 and MEF cells) beside CAY10863 significantly affect H3K9ac levels at metaphase (P < 10−15, FDR corrected t test). Scale bars: 5 μm. For a boxplot version of the graphs, see Fig S3.
- Figure S2. Box plot representation of the results of Fig 2
(A, B) For HeLa-S3 (A) and for MEF (B).
- Figure S3. Quantification of the effects of key HDACi on H3K9ac levels on metaphase chromosomes.
Quantification of the immunofluorescence results of HeLa-S3 cells enriched for mitotic stages stained for H3K9ac. Intensity values represent the mean ± SEM of 88, 128, 91, and 103 cells for NT, CAY, MS, and RG treatments, respectively. H3K9ac levels at metaphase chromosomes treated with either RG or MS are significantly different from both NT and CAY treated cells (P < 10−26; two side t test).
- Figure 3. Identification of the chromatin localization patterns of key histone acetyl transferases (HATs) and HDACs during mitosis.
(A, B, C) Immunofluorescence of HeLa-S3 cells enriched for mitotic stages, stained for DNA (blue) and for various HDACs (A), sirtuins (B), and HATs (C) (green). Representative pictures from the major mitosis stages and interphase are shown, along with a quantification of at least 20 cells from each stage. Intensity values represent the mean ± SEM. Note that only HDAC3, P300, and HAT-1 are retained on the mitotic chromatin along all mitotic stages, whereas the other proteins show partial retention at prophase. (D) All factors (beside HDAC3, P300, and HAT1) show significant reduction at metaphase (P < 10−16, FDR-corrected t test) (D) Immunoblots showing the abundance of the indicated chromatin modifiers in the chromatin fraction of interphase (L1) and mitotic (L2) HeLa-S3 cells. Total histone H3 serves as loading control. Scale bars: 5 μm.
Source data are available for this figure.
Source Data for Figure 3[LSA-2022-01433_SdataF3.pdf]
- Figure 4. An in vitro assay supports an indirect modulation H3K9ac levels by impacting histone acetyl transferase (HAT) activity.
(A) HAT activity colorimetric assay results (mean ± standard error, N = 2) for HeLa-S3 cells either not treated (NT) or treated with EX-527 (5 nM). (B) Immunoblot analysis showing H3K9 acetylation on His-tagged H3 after HAT activity of HeLa mitotic chromatin either not treated (NT) or treated with EX-527 (5 nM) on His-tagged H3. Total histone H3 serves as loading control. (C) Immunoprecipitation of HDAC3 tested by immunoblotting. HDAC3 antibodies and protein A magnetic beads were used to immunoprecipitate (IP) HDAC3 complexes from 1 mg of HeLa mitotic extract either not treated (NT) or treated with RGFP966 (40 μM) or EX-527 (5 nM). (i) Showing Ponceau S staining of HDAC3 immunocomplex; (ii) showing immunoblot using HDAC3 antibodies developed by ECL method; (iii) showing Ponceau S staining of HDAC3 activity assay reaction subjected to immunoblot; and (iv) showing immunoblot analysis of immunoprecipitated HDAC3 activity on acetylated his-tagged H3 protein.
- Figure 5. Detection of genomic patterns associated with the activity of HDAC2 and HDAC3.
(A) Metagene plots showing H3K9ac promoter occupancy in chicken DT-40 cells before (left) and after (right) normalization. (B) Genomic viewer (IGV) tracks representing the H3K9ac ChIP-seq enrichment for the indicated conditions. (C) Metagene plots showing H3K9ac occupancy around promoters, enhancers and insulators in HeLa-S3 cells. The data were normalized using the DT-40 promoter data. Similar results were obtained in a biological repeat (Fig S5).
- Figure S4. Metagene analyses of chicken enhancers and insulators.
Metagene plots showing H3K9ac enhancer and insulator occupancy in chicken DT-40 cells before (left) and after (right) normalization (according to promoter occupancy [see the Materials and Methods section]).
- Figure S5. HDACs activity at transcriptionally associated regions (replicates).
(A) Left shows UCSC representation of ∼150 kb region (Chr14:24,085,920-24,238,259) of H3K9ac ChIP-seq results under the indicated conditions. Data scaled to show inhibitor-treated peaks. Unsynchronized sample peaks out of range are highlighted in pink. Right shows a portion of the same genomic region but all samples are normalized equally. (B) Metagene plots showing H3K9ac occupancy around promoters, enhancers and insulators in HeLa-S3 cells. The data were normalized using the DT-40 promoter data.
- Figure S6. IGV plots of regions specifically effected by HDAC2 or HDAC3.
(A) IGV representation of two genomic regions with RGFP966 and CAY10683 specific peaks. The locations of the differential peaks are indicated. (B) Scatter plot and Venn diagram depicting the differences between the H3K9c peaks in STC synchronized cells treated with either RGFP966 or CAY10683.
- Figure S7. HDAC2 and SIRT1 are released from the mitotic chromatin.
Immunofluorescence of HeLa-S3 cells cross-linked and fixed with DSG and FA. Arrows point to cells in metaphase. Note that both SIRT1 and HDAC2 are depleted from the mitotic chromosomes.
Tables
Name Specificity Concentration used References TSA Pan-HDACs 150 nM Khan et al (2008) and Lobera et al (2013) CHIDAMIDE HDAC1,2,3,10 10 µM Ning et al (2012) RGFP966 HDAC3 40 µM Malvaez et al (2013) CAY10683 HDAC2 and HDAC6 1 µM Pavlik et al (2013) and Bhattad et al (2020) MS-275 HDAC1,2,3 1 µM Khan et al (2008) NICOTINAMIDE Pan-sirtuins 10 mM Hu et al (2014) EX-527 SIRT1 5 nM Hu et al (2014)
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