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.
Highlights
Chromatin structure and function are largely governed by interactions between DNA and histone proteins that regulate different levels of genome organizationBaumann et al Epigenetics & Chromatin (2021) 14:58 subject to developmental regulation
We show here that mouse embryonic stem cells (mESCs) treated with valproic acid (VPA) undergo striking changes in both nuclear accumulation as well as genome-wide occupancy of histone H3 acetylated at lysine 56 (H3K56ac), an important lateral surface posttranslational modifications (PTMs) that increases the rate of local DNA unwrapping at the DNA entry/exit site of the nucleosome [29,30,31]
To determine the upstream epigenetic changes associated with the onset of directed differentiation of ES cells, we used high-content image analysis [33] to compare the patterns of global histone H3K56 acetylation in control cells with the patterns observed after VPA exposure
Summary
Chromatin structure and function are largely governed by interactions between DNA and histone proteins that regulate different levels of genome organizationBaumann et al Epigenetics & Chromatin (2021) 14:58 subject to developmental regulation. Histone acetylation is of utmost importance for the regulation of chromatin accessibility with far-reaching implications for transcriptional activity, DNA repair, and replication [10,11,12] and requires a dynamic balance between the addition of acetyl groups to lysine residues through the activity of histone acetyltransferases (HATs) and their removal catalyzed by histone deacetylases (HDACs) [13,14,15] Disruptions to these homeostatic mechanisms are commonly associated with pathophysiological changes, in several types of human cancers that show overexpression of HDAC enzymes [16,17,18]. Selective HDAC inhibitors (HDACi), such as valproic acid (VPA), an FDA approved class I deacetylase inhibitor, are important pharmacological compounds for novel epigenetic cancer therapies [19]
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