Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Dec 27;14(1):58.
doi: 10.1186/s13072-021-00432-5.

Changes in chromatin accessibility landscape and histone H3 core acetylation during valproic acid-induced differentiation of embryonic stem cells

Claudia Baumann  1   2 , Xiangyu Zhang  1   2 , Ling Zhu  3 , Yuhong Fan  3   4 , Rabindranath De La Fuente  5   6
Affiliations

Changes in chromatin accessibility landscape and histone H3 core acetylation during valproic acid-induced differentiation of embryonic stem cells

Claudia Baumann et al. Epigenetics Chromatin. .

Abstract

Directed differentiation of mouse embryonic stem cells (mESCs) or induced pluripotent stem cells (iPSCs) provides powerful models to dissect the molecular mechanisms leading to the formation of specific cell lineages. Treatment with histone deacetylase inhibitors can significantly enhance the efficiency of directed differentiation. However, the mechanisms are not well understood. Here, we use CUT&RUN in combination with ATAC-seq to determine changes in both histone modifications and genome-wide chromatin accessibility following valproic acid (VPA) exposure. VPA induced a significant increase in global histone H3 acetylation (H3K56ac), a core histone modification affecting nucleosome stability, as well as enrichment at loci associated with cytoskeletal organization and cellular morphogenesis. In addition, VPA altered the levels of linker histone H1 subtypes and the total histone H1/nucleosome ratio indicative of initial differentiation events. Notably, ATAC-seq analysis revealed changes in chromatin accessibility of genes involved in regulation of CDK serine/threonine kinase activity and DNA duplex unwinding. Importantly, changes in chromatin accessibility were evident at several key genomic loci, such as the pluripotency factor Lefty, cardiac muscle troponin Tnnt2, and the homeodomain factor Hopx, which play critical roles in cardiomyocyte differentiation. Massive parallel transcription factor (TF) footprinting also indicates an increased occupancy of TFs involved in differentiation toward mesoderm and endoderm lineages and a loss of footprints of POU5F1/SOX2 pluripotency factors following VPA treatment. Our results provide the first genome-wide analysis of the chromatin landscape following VPA-induced differentiation in mESCs and provide new mechanistic insight into the intricate molecular processes that govern departure from pluripotency and early lineage commitment.

Keywords: ATAC-seq; Chromatin accessibility; Differentiation; Embryonic stem cells; HDAC; HDACi; Next-generation sequencing; VPA.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
VPA treatment induces global histone H3K56 hyperacetylation. A Treatment of mESCs with 2 mM VPA for 48 h increases the levels of H3K56 acetylation (green) compared to untreated control cultures. Scale bar = 20 μm. B High-resolution confocal imaging of individual control and VPA-treated nuclei stained with H3K56ac (green). DNA is counterstained using DAPI (blue). Fluorescence intensity across nuclei is visualized by line scan (red arrows) graphs. DAPI bright chromocenters are highlighted (shaded blue). C High-content confocal analysis using threshold maps for the quantification of H3K56ac fluorescence intensity and nuclear circumference reveals a higher proportion of nuclei with bright H3K56ac staining (yellow) in VPA-treated cultures compared to controls. Nuclear circumference measurements indicate no significant differences (Cntl: n = 98; VPA: n = 108). Scale bar = 20 μm. Data are expressed as the mean ± SD of three independent replicates. Unpaired t-test, two tailed resulted in P < 0.001. n.s.—not significant
Fig. 2
Fig. 2
HDAC inhibition alters linker histone H1 subtype expression in mESCs. A Reverse-phase HPLC (RP-HPLC) and Mass Spectrometry (inset) analysis of histone H1 subtypes in control and VPA-treated (2 mM, 48 h) mESCs. X axis: elution time [min]; Y axis: absorbency at 214 nm [mAU, milli-absorbency units]. The inset shows the relative signal intensity of H1d and H1e mass spectral peaks in the H1d/H1e fraction collected from HPLC eluates. Prominent changes are indicated in red arrows. B Quantification of H1/nucleosome ratios of individual H1 subtypes as well as of total H1 in control versus VPA-treated mESCs as determined by RP-HPLC and Mass Spectrometry. Data are expressed as the mean ± SD over three independent replicates. Unpaired t-test, two tailed; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001
Fig. 3
Fig. 3
Altered chromatin accessibility following VPA-induced differentiation. (A) ATAC-seq heatmaps of tag distributions across promoters in control and VPA-treated (2 mM, 48 h) samples. B Volcano Plot of differential ATAC-seq peaks. Loci with significant loss (red, n = 3923) and gain (green, n = 1614) of chromatin accessibility in VPA-treated cells are highlighted. C Comparative analysis of changes in the accessibility of peaks overlapping with different types of transposable elements (TEs) as a proportion of the total number of differential TE-overlapping peaks (%). D Top-most regulated loci with increased chromatin accessibility. E Top 10 over-represented GO Terms of biological processes (-log p-value). F Over-represented Kegg pathways identified in VPA-treated cells (-log p-value)
Fig. 4
Fig. 4
VPA induces changes in chromatin accessibility at key genomic loci and alters TF occupancy in mESCs. A UCSC browser views of ATAC-seq peak patterns at the transcription start site of pluripotency genes Oct4 (Pou5f1) and Nanog suggesting reduced chromatin accessibility in response to VPA treatment (shaded boxes). Analysis of protein expression levels of POU5F1 (OCT4) by immunochemistry and western blotting in control and VPA-treated mESCs. Tubulin expression was used as housekeeping control. Immunochemical UCSC browser views of Tnnt2 and Hopx loci in control and VPA-treated mESCs. VPA treatment induces changes in chromatin accessibility (shaded boxes). B Comparison of transcription factor occupancy between control and VPA-treated cells. The Volcano plot shows the differential binding activity against the −log10(p-value). Each dot represents an individual motif. The top 5th percentile of enriched TFs in control samples are labeled blue, while the top 5th percentile of TFs with enriched activity in VPA-treated cells are marked in red. Aggregated footprint plots and binding motifs for representative TFs are shown. The number of TFs motifs with differential occupancy is classified by TF family and shown in the bar graph
Fig. 5
Fig. 5
Cut&Run locus specific and GO Terms. A Heatmaps were generated using the midpoint of the signal block obtained from SEACR analysis. Deeptools were used for heatmap visualization. B Comparative analysis of loci with gained accessibility relative to genebodies and promoter regions in control (Cntl) and VPA-treated (VPA) samples. C Top 15 over-represented GO Terms in biological processes (−log p-value). IGV browser views of differential H3K56ac enrichment at the Pax6 (D) and the Asf1a locus (E) in response to VPA treatment. Coherent broad domains of enrichment were identified by SEACR analysis and are denoted as black bars
Fig. 6
Fig. 6
Integrative ATAC-seq/CUT&RUN analysis. A Venn diagram illustrating correlative changes in chromatin accessibility and H3K56ac enrichment patterns at TSS. B Top 12 over-represented GO Terms in genes with concomitantly increased accessibility and H3K56ac occupancy. C IGV browser alignment of H3K56ac CUT&RUN enrichment and ATAC-seq patterns at the Tnnt3 locus. Coherent broad domains of H3K56ac enrichment were identified by SEACR analysis and are denoted as blue bars. Differential enrichment and correlative accessibility change at the TSS is indicated in shaded box
Fig. 7
Fig. 7
Chromatin landscape at the Lefty locus in mESCs and during induced differentiation. A IGV browser alignment of H3K56ac CUT&RUN enrichment and ATAC-seq patterns at the Lefty locus. Coherent broad domains of H3K56ac enrichment were identified by SEACR analysis and are denoted as blue bars. Differential enrichment and correlative accessibility change at known enhancers (shaded light green) and TSS (shaded gray). B Putative model of key transitions in chromatin accessibility, nucleosome stability and transcription factor binding* at enhancers*# and TSS sites within the Lefty locus in response to HDAC inhibitor treatment (adapted from *[65] and #[64]). C H3K56ac and OCT4 (POU5F1) ChIP-qPCR data following immunoprecipitation and amplification of regulatory sequences of the Lefty1 locus (P < 0.05). IgG was used as negative control
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Luger K, et al. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature. 1997;389(6648):251–60. - PubMed
    1. Woodcock CL, Skoultchi AI, Fan Y. Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length. Chromosome Res. 2006;14(1):17–25. - PubMed
    1. Bian Q, Belmont AS. Revisiting higher-order and large-scale chromatin organization. Curr Opin Cell Biol. 2012;24(3):359–366. - PMC - PubMed
    1. Belmont AS. Large-scale chromatin organization: the good, the surprising, and the still perplexing. Curr Opin Cell Biol. 2014;26:69–78. - PMC - PubMed
    1. Meshorer E, et al. Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells. Dev Cell. 2006;10(1):105–116. - PMC - PubMed

Publication types