The zinc-dependent mammalian histone deacetylase (HDAC) family comprises 11 enzymes, which have specific and critical functions in development and tissue homeostasis. Mounting evidence points to a link between misregulated HDAC activity and many oncologic and nononcologic diseases. Thus the development of HDAC inhibitors for therapeutic treatment garners a lot of interest from academic researchers and biotechnology entrepreneurs. Numerous studies of HDAC inhibitor specificities and molecular mechanisms of action are ongoing. In one of these studies, mass spectrometry was used to characterize the affinities and selectivities of HDAC inhibitors toward native HDAC multiprotein complexes in cell extracts. Such a novel approach reproduces in vivo molecular interactions more accurately than standard studies using purified proteins or protein domains as targets and could be very useful in the isolation of inhibitors with superior clinical efficacy and decreased toxicity compared to the ones presently tested or approved. HDAC inhibitor induced-transcriptional reprogramming, believed to contribute largely to their therapeutic benefits, is achieved through various and complex mechanisms not fully understood, including histone deacetylation, transcription factor or regulator (including HDAC1) deacetylation followed by chromatin remodeling and positive or negative outcome regarding transcription initiation. Although only a very low percentage of protein-coding genes are affected by the action of HDAC inhibitors, about 40% of noncoding microRNAs are upregulated or downregulated. Moreover, a whole new world of long noncoding RNAs is emerging, revealing a new class of potential targets for HDAC inhibition. HDAC inhibitors might also regulate transcription elongation and have been shown to impinge on alternative splicing.

Epigenetics refers to mitotically and/or meiotically heritable variations in gene expression that are not caused by changes in DNA sequence. Epigenetic mechanisms regulate all biological processes from conception to death, including genome reprogramming during early embryogenesis and gametogenesis, cell differentiation and maintenance of a committed lineage. Key epigenetic players are DNA methylation and histone post-translational modifications, which interplay with each other, with regulatory proteins and with non-coding RNAs, to remodel chromatin into domains such as euchromatin, constitutive or facultative heterochromatin and to achieve nuclear compartmentalization. Besides epigenetic mechanisms such as imprinting, chromosome X inactivation or mitotic bookmarking which establish heritable states, other rapid and transient mechanisms, such as histone H3 phosphorylation, allow cells to respond and adapt to environmental stimuli. However, these epigenetic marks can also have long-term effects, for example in learning and memory formation or in cancer. Erroneous epigenetic marks are responsible for a whole gamut of diseases including diseases evident at birth or infancy or diseases becoming symptomatic later in life. Moreover, although epigenetic marks are deposited early in development, adaptations occurring through life can lead to diseases and cancer. With epigenetic marks being reversible, research has started to focus on epigenetic therapy which has had encouraging success. As we witness an explosion of knowledge in the field of epigenetics, we are forced to revisit our dogma. For example, recent studies challenge the idea that DNA methylation is irreversible. Further, research on Rett syndrome has revealed an unforeseen role for methyl-CpG-binding protein 2 (MeCP2) in neurons.

Transcriptionally active chromatin is marked by tri-methylation of histone H3 at lysine 4 (H3K4me3) located after first exons and around transcription start sites. This epigenetic mark is typically restricted to narrow regions at the 5`end of the gene body, though a small subset of genes have a broad H3K4me3 domain which extensively covers the coding region. Although most studies focus on the H3K4me3 mark, the broad H3K4me3 domain is associated with a plethora of histone modifications (e.g., H3 acetylated at K27) and is therein termed broad epigenetic domain. Genes marked with the broad epigenetic domain are involved in cell identity and essential cell functions and have clinical potential as biomarkers for patient stratification. Reducing expression of genes with the broad epigenetic domain may increase the metastatic potential of cancer cells. Enhancers and super-enhancers interact with the broad epigenetic domain marked genes forming a hub of interactions involving nucleosome-depleted regions. Together, the regulatory elements coalesce with transcription factors, chromatin modifying/remodeling enzymes, coactivators, and the Mediator and/or Integrator complex into a transcription factory which may be analogous to a liquid–liquid phase-separated condensate. The broad epigenetic domain has a dynamic chromatin structure which supports frequent transcription bursts. In this review, we present the current knowledge of broad epigenetic domains.

The properties of the nucleosomes of a salt-soluble, transcriptionally active gene-enriched fraction of chicken erythrocyte chromatin were evaluated by hydroxyapatite dissociation chromatography. We have demonstrated previously that the salt-soluble, transcriptionally active gene-enriched polynucleosomes are enriched in dynamically acetylated and ubiquitinated histones, and in an atypical U-shaped nucleosome that possessed about 20% less protein than a typical nucleosome. Further, newly synthesized histones H2A and H2B exchange preferentially with the nucleosomal histones H2A and H2B of this salt-soluble chromatin fraction. Analysis of the histones eluting from the hydroxyapatite-bound chromatin demonstrated that hyperacetylated and ubiquitinated (u), including multi-ubiquitinated, H2A-H2B.1 dimers dissociated at lower concentrations of NaCl than unmodified dimers or dimers with histone variants H2A.Z and/or H2B.2. Cross-linking studies revealed that at least 50% of uH2B.1 was paired with uH2A. uH2A-uH2B.1 dimers dissociated at lower NaCl concentrations than H2A-uH2B.1 dimers. Hyperacetylated histone (H3-H4)2 tetramers also eluted at lower concentrations of NaCl than unmodified tetramers. Our results support the idea that acetylation and ubiquitination of histones H2A and H2B.1 increase the lability of H2A-H2B.1 dimers in transcriptionally active nucleosomes. In contrast, our observations suggest that histone variants H2A.Z and H2B.2. stabilize the association of the H2A-H2B dimer in nucleosomes. The elevated lability of the H2A-H2B dimer may facilitate processes such as the exchange of these dimers with newly synthesized histones, the elongation process of transcription and transcription factor binding.

The beta-globin and histone H5 genes are transcriptionally active in immature chicken erythrocytes and potentially active in mature erythrocytes. In both immature and mature erythrocytes, the majority of these erythroid-specific gene sequences are located in two chromatin fractions: the low-salt-insoluble residual nuclear material and the 0.15 M-NaCl-soluble oligo- and poly-nucleosomes. These salt-soluble chromatin fragments are enriched in hyperacetylated species of H4 and H2B, ubiquitinated and polyubiquitinated species of H2A and H2B and are depleted of linker histones H1 and H5. The competent, transcriptionally inactive embryonic epsilon-globin gene, which is part of the DNAase I-sensitive beta-globin domain, is highly enriched in the 0.15 M-NaCl-soluble polynucleosome fraction but not in the insoluble nuclear material. The repressed vitellogenin gene shows no enrichment in either of these fractions. These results suggest that only those genes that are expressed or have the potential for expression are enriched in the low-salt-insoluble nuclear material of immature or mature erythrocytes. The enrichment of active genes in the low-salt-insoluble residual nuclear material of immature erythrocytes is not dependent on on-going transcription, the presence of RNA or changes in the amount of acetylated histone species. Our results are consistent with the hypothesis that active and potentially active genes are insoluble because of the presence of preinitiation transcription complexes.