Histone deacetylases maintain expression of the pluripotent gene network via recruitment of RNA polymerase II to coding and noncoding loci.

Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Deacetylation of histone tails results in chromatin tightening, and therefore, HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of Hdac1 and Hdac2 in embryonic stem cells (ESCs) reduces expression of the pluripotency-associated transcription factors Pou5f1, Sox2, and Nanog (PSN). By shaping global histone acetylation patterns, HDACs indirectly regulate the activity of acetyl-lysine readers, such as the transcriptional activator BRD4. Here, we use inhibitors of HDACs and BRD4 (LBH589 and JQ1, respectively) in combination with precision nuclear run-on and sequencing (PRO-seq) to examine their roles in defining the ESC transcriptome. Both LBH589 and JQ1 cause a marked reduction in the pluripotent gene network. However, although JQ1 treatment induces widespread transcriptional pausing, HDAC inhibition causes a reduction in both paused and elongating polymerase, suggesting an overall reduction in polymerase recruitment. Using enhancer RNA (eRNA) expression to measure enhancer activity, we find that LBH589-sensitive eRNAs are preferentially associated with superenhancers and PSN binding sites. These findings suggest that HDAC activity is required to maintain pluripotency by regulating the PSN enhancer network via the recruitment of RNA polymerase II.

Co-repressor, co-activator and general transcription factor: the many faces of the Sin3 histone deacetylase (HDAC) complex.

At face value, the Sin3 histone deacetylase (HDAC) complex appears to be a prototypical co-repressor complex, that is, a multi-protein complex recruited to chromatin by DNA bound repressor proteins to facilitate local histone deacetylation and transcriptional repression. While this is almost certainly part of its role, Sin3 stubbornly refuses to be pigeon-holed in quite this way. Genome-wide mapping studies have found that Sin3 localises predominantly to the promoters of actively transcribed genes. While Sin3 knockout studies in various species result in a combination of both up- and down-regulated genes. Furthermore, genes such as the stem cell factor, Nanog, are dependent on the direct association of Sin3 for active transcription to occur. Sin3 appears to have properties of a co-repressor, co-activator and general transcription factor, and has thus been termed a co-regulator complex. Through a series of unique domains, Sin3 is able to assemble HDAC1/2, chromatin adaptors and transcription factors in a series of functionally and compositionally distinct complexes to modify chromatin at both gene-specific and global levels. Unsurprisingly, therefore, Sin3/HDAC1 have been implicated in the regulation of numerous cellular processes, including mammalian development, maintenance of pluripotency, cell cycle regulation and diseases such as cancer.

KAT2A complexes ATAC and SAGA play unique roles in cell maintenance and identity in hematopoiesis and leukemia.

Epigenetic histone modifiers are key regulators of cell fate decisions in normal and malignant hematopoiesis. Their enzymatic activities are of particular significance as putative therapeutic targets in leukemia. In contrast, less is known about the contextual role in which those enzymatic activities are exercised and specifically how different macromolecular complexes configure the same enzymatic activity with distinct molecular and cellular consequences. We focus on KAT2A, a lysine acetyltransferase responsible for histone H3 lysine 9 acetylation, which we recently identified as a dependence in acute myeloid leukemia stem cells and that participates in 2 distinct macromolecular complexes: Ada two-A-containing (ATAC) and Spt-Ada-Gcn5-Acetyltransferase (SAGA). Through analysis of human cord blood hematopoietic stem cells and progenitors, and of myeloid leukemia cells, we identify unique respective contributions of the ATAC complex to regulation of biosynthetic activity in undifferentiated self-renewing cells and of the SAGA complex to stabilization or correct progression of cell type-specific programs with putative preservation of cell identity. Cell type and stage-specific dependencies on ATAC and SAGA-regulated programs explain multilevel KAT2A requirements in leukemia and in erythroid lineage specification and development. Importantly, they set a paradigm against which lineage specification and identity can be explored across developmental stem cell systems.

The MiDAC histone deacetylase complex is essential for embryonic development and has a unique multivalent structure.

MiDAC is one of seven distinct, large multi-protein complexes that recruit class I histone deacetylases to the genome to regulate gene expression. Despite implications of involvement in cell cycle regulation and in several cancers, surprisingly little is known about the function or structure of MiDAC. Here we show that MiDAC is important for chromosome alignment during mitosis in cancer cell lines. Mice lacking the MiDAC proteins, DNTTIP1 or MIDEAS, die with identical phenotypes during late embryogenesis due to perturbations in gene expression that result in heart malformation and haematopoietic failure. This suggests that MiDAC has an essential and unique function that cannot be compensated by other HDAC complexes. Consistent with this, the cryoEM structure of MiDAC reveals a unique and distinctive mode of assembly. Four copies of HDAC1 are positioned at the periphery with outward-facing active sites suggesting that the complex may target multiple nucleosomes implying a processive deacetylase function.

Sin3A recruits Tet1 to the PAH1 domain via a highly conserved Sin3-Interaction Domain.

The Sin3A complex acts as a transcriptional hub, integrating the function of diverse transcription factors with histone modifying enzymes, notably, histone deacetylases (HDAC) 1 and 2. The Sin3A protein sits at the centre of the complex, mediating multiple simultaneous protein-protein interactions via its four paired-amphipathic helix (PAH) domains (PAH1-4). The PAH domains contain a conserved four helical bundle, generating a hydrophobic cleft into which the single-helix of a Sin3-interaction domain (SID) is able to insert and bind with high affinity. Although they share a similar mode of interaction, the SIDs of different repressor proteins bind to only one of four potential PAH domains, due to the specific combination of hydrophobic residues at the interface. Here we report the identification of a highly conserved SID in the 5-methylcytosine dioxygenase, Tet1 (Tet1-SID), which interacts directly with the PAH1 domain of Sin3A. Using a combination of NMR spectroscopy and homology modelling we present a model of the PAH1/Tet1-SID complex, which binds in a Type-II orientation similar to Sap25. Mutagenesis of key residues show that the 11-amino acid Tet1-SID is necessary and sufficient for the interaction with Sin3A and is absolutely required for Tet1 to repress transcription in cells.