HDAC Inhibition Rewires Epigenome, Transcriptome, and 3D Chromatin in Mouse Embryonic Stem Cells
HDAC inhibition significantly alters the epigenome and transcriptome in mouse embryonic stem cells (mESCs). This profound re-engineering provides critical insights into the dynamic control of cell identity and memory.
Epigenome and Transcriptome Changes
Acute treatment with Trichostatin A (TSA), an HDAC inhibitor, induces genome-wide H3K27 hyperacetylation. This modification shifts a larger portion of the genome into an active state, promoting developmental processes and suppressing pluripotency.
Specifically, genes associated with stem cell maintenance are downregulated, while those linked to neural lineage maturation are upregulated.
These findings indicate that HDAC inhibition encourages gene expression programs that facilitate the exit from pluripotency.
Global and Local Architectural Changes
TSA treatment results in a marked increase in trans-chromosomal contacts and a decrease in cis-interactions. It also leads to a reduction in A compartment interactions, which are characteristic of mESCs, while B-B interactions become more prominent.
Biophysical modeling suggests that these changes are consistent with an increase in chromatin fiber stiffness, particularly within A domains, and predict a peripheral displacement of A domains.
Furthermore, the treatment alters chromatin looping, weakening CTCF-mediated loops but strengthening non-CTCF loops, which carry either active or repressive chromatin signatures. These effects were corroborated by a different nuclear HDAC inhibitor, romidepsin.
Impact on Gene Expression Regulation
Upregulated genes show enrichment for increased H3K27ac and H3K4me1, along with enhanced chromatin accessibility. Bivalent genes appear susceptible to derepression without losing H3K27me3.
Gene upregulation is linked to ectopic enhancer activation, occurring without changes in enhancer-promoter (E-P) contacts.
Conversely, downregulated genes are associated with a loss of activating histone marks and increased binding of transcriptional regulators like Myc and YY1. In these cases, promoter loops around downregulated transcription start sites (TSSs) become stronger, and de novo loops emerge at H3K9me3 sites, suggesting a role for repressive chromatin contacts in gene downregulation.
Recovery and Cellular Memory
After TSA removal, mESCs exhibit a remarkable capacity for recovery.
Protein acetylation, the histone landscape (including H3K27ac, H3K4me1, and chromatin accessibility), and gene expression are largely restored within 24 hours.
However, a minor fraction of genes (164) shows residual deregulation, indicating that cells retain a partial memory of the TSA pulse.
This partial memory, characterized by incomplete architectural recovery (e.g., persistent cis-contact depletion and B-B interaction prominence), is observed even when gene expression recovers. Repeated exposure to TSA leads to less complete recovery, with a larger number of genes (767) remaining deregulated and associated with developmental processes, suggesting an aggravating effect on cellular identity.
Regulatory 3D Contacts and Lasting Changes
Lasting gene expression changes are linked to specific architectural features. Upregulated genes that do not recover exhibit strong pre-existing E-P contacts and a higher propensity for looping, even though activating chromatin signals are restored. For downregulated genes that do not recover, an abundance of H3K27me3 signal, indicative of Polycomb targeting, is observed.
Critically, Polycomb loops gain substantial and persistent strength at these non-recovering TSSs and genome-wide, often without major changes in H3K27me3 levels at loop anchors.
This suggests that bolstered repressive Polycomb loops may perpetuate altered activity states.
PRC1-Mediated Downregulation Memory
Increased binding of Ring1B, a subunit of Polycomb repressive complex 1 (PRC1), is detected after recovery from TSA, supporting PRC1's role in the memory response. Disrupting PRC1-mediated looping by depleting PCGF2 (another PRC1 subunit) significantly reduces the number of genes exhibiting sustained downregulation following TSA treatment.
This demonstrates that PRC1-mediated spatial clustering is a key mechanism responsible for the TSA-induced, sustained downregulation observed at Polycomb target genes, effectively modulating the cellular memory response.