Dilshad H. Khan

Roles of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitors.

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.

Pre-mRNA splicing: Role of epigenetics and implications in disease

Epigenetics refer to a variety of processes that have long-term effects on gene expression programs without changes in DNA sequence. Key players in epigenetic control are histone modifications and DNA methylation which, in concert with chromatin remodeling complexes, nuclear architecture and microRNAs, define the chromatin structure of a gene and its transcriptional activity. There is a growing awareness that histone modifications and chromatin organization influence pre-mRNA splicing. Further there is emerging evidence that pre-mRNA splicing itself influences chromatin organization. In the mammalian genome around 95% of multi-exon genes generate alternatively spliced transcripts, the products of which create proteins with different functions. It is now established that several human diseases are a direct consequence of aberrant splicing events. In this review we present the interplay between epigenetic mechanisms and splicing regulation, as well as discuss recent studies on the role of histone deacetylases in splicing activities.

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.

Protein Kinase CK2 Regulates the Dimerization of Histone Deacetylase 1 (HDAC1) and HDAC2 during Mitosis

Background: HDAC1 and -2 homo- or heterodimers within corepressor complexes are displaced from chromatin during mitosis. Results: CK2-mediated mitotic phosphorylated HDAC1 and -2 are dissociated from each other but not from corepressor complexes. Conclusion: HDAC1 or HDAC2 homodimers, but not heterodimers, are responsible for the activity of mitotic corepressor complexes. Significance: Mitotically phosphorylated HDAC1 and -2 potentially target different cellular proteins. Histone deacetylase 1 (HDAC1) and HDAC2 are components of corepressor complexes that are involved in chromatin remodeling and regulation of gene expression by regulating dynamic protein acetylation. HDAC1 and -2 form homo- and heterodimers, and their activity is dependent upon dimer formation. Phosphorylation of HDAC1 and/or HDAC2 in interphase cells is required for the formation of HDAC corepressor complexes. In this study, we show that during mitosis, HDAC2 and, to a lesser extent, HDAC1 phosphorylation levels dramatically increase. When HDAC1 and -2 are displaced from the chromosome during metaphase, they dissociate from each other, but each enzyme remains in association with components of the HDAC corepressor complexes Sin3, NuRD, and CoREST as homodimers. Enzyme inhibition studies and mutational analyses demonstrated that protein kinase CK2-catalyzed phosphorylation of HDAC1 and -2 is crucial for the dissociation of these two enzymes. These results suggest that corepressor complexes, including HDAC1 or HDAC2 homodimers, might target different cellular proteins during mitosis.

Dynamic distribution of HDAC1 and HDAC2 during mitosis: Association with F-actin

During mitosis, histone deacetylase 2 (HDAC2) becomes highly phosphorylated through the action of CK2, and HDAC1 and 2 are displaced from mitotic chromosomes. HDAC1 and 2 are components of corepressor complexes, which function with lysine acetyltransferases to catalyze dynamic protein acetylation and regulate gene expression. In this study, we show that HDAC1 and 2 associate with F‐actin in mitotic cells. Inhibition of Aurora B or protein kinase CK2 did not prevent the displacement of HDAC1 and 2 from mitotic chromosomes in HeLa cells. Further, proteins of the HDAC1 and 2 corepressor complexes and transcription factors recruiting these corepressors to chromatin were dissociated from mitotic chromosomes independent of Aurora B activity. HDAC1 and 2 returned to the nuclei of daughter cells during lamin A/C reassembly and before Sp1, Sp3, and RNA polymerase II. Our results show that HDAC1 and 2 corepressor complexes are removed from the mitotic chromosomes and are available early in the events leading to the re‐establishment of the gene expression program in daughter cells. J. Cell. Physiol. 228: 1525–1535, 2013.

Nucleosomal response, immediate-early gene expression and cell transformation.

Multistep tumorigenesis is a progression of events resulting from alterations in the processing of the genetic information. These alterations result from stable genetic changes (mutations) in tumor suppressor genes and oncogenes (e.g. ras) and potentially reversible epigenetic changes (i.e. modifications in gene function without a change in DNA sequence) (Bird, 2007; Egger et al., 2004; Espino et al., 2005; Hake et al., 2004; Vogelstein and Kinzler, 2004). DNA methylation and protein modifications are two epigenetic mechanisms that are altered in cancer cells (Gal-Yam et al., 2008; Gronbaek et al., 2007). Chromatin modifying enzymes, catalyzing DNA methylation and protein modifications have a central role in the genesis of cancer (Ballestar and Esteller, 2008; Esteller, 2008; Gal-Yam et al., 2008; Gronbaek et al., 2007; Hake et al., 2004; Iacobuzio-Donahue, 2009; Medina and Cespedes, 2008; Momparler, 2003). In this review we will discuss how activation of signal transduction pathways, which are often deregulated in cancer cells, results in alterations in gene expression programming through histone modifications, with a focus on histone H3 phosphorylation.

HDAC inhibitors prevent the induction of the immediate-early gene FOSL1, but do not alter the nucleosome response.

Dynamic histone acetylation, catalyzed by lysine acetyltransferases and HDACs, is critical to IEG expression. Expression of IEGs, such as FOSL1, is induced by several signal transduction pathways resulting in activation of the protein kinase MSK and phosphorylation of histone H3 at serine 10 of nucleosomes (the nucleosome response) at the upstream promoter and regulatory region of target genes. HDAC inhibitors prevent FOSL1 gene induction and the association of HDAC1, 2 and 3 with the gene body. However, HDAC inhibitors did not prevent the nucleosome response. Thus HDAC inhibitors perturb events downstream of the nucleosome response required for FOSL1 transcription initiation.

Dynamic Histone Acetylation of H3K4me3 Nucleosome Regulates MCL1 Pre-mRNA Splicing.

Pre‐mRNA splicing is a cotranscriptional process affected by the chromatin architecture along the body of coding genes. Recruited to the pre‐mRNA by splicing factors, histone deacetylases (HDACs) and K‐acetyltransferases (KATs) catalyze dynamic histone acetylation along the gene. In colon carcinoma HCT 116 cells, HDAC inhibition specifically increased KAT2B occupancy as well as H3 and H4 acetylation of the H3K4 trimethylated (H3K4me3) nucleosome positioned over alternative exon 2 of the MCL1 gene, an event paralleled with the exclusion of exon 2. These results were reproduced in MDA‐MB‐231, but not in MCF7 breast adenocarcinoma cells. These later cells have much higher levels of demethylase KDM5B than either HCT 116 or MDA‐MB‐231 cells. We show that H3K4me3 steady‐state levels and H3K4me3 occupancy at the end of exon 1 and over exon 2 of the MCL1 gene were lower in MCF7 than in MDA‐MB‐231 cells. Furthermore, in MCF7 cells, there was minimal effect of HDAC inhibition on H3/H4 acetylation and H3K4me3 levels along the MCL1 gene and no change in pre‐mRNA splicing choice. These results show that, upon HDAC inhibition, the H3K4me3 mark plays a critical role in the exclusion of exon 2 from the MCL1 pre‐mRNA. J. Cell. Physiol. 231: 2196–2204, 2016.

Mitogen-induced distinct epialleles are phosphorylated at either H3S10 or H3S28, depending on H3K27 acetylation

Upon mitogenic induction of immediate-early genes, phosphorylation of histone H3 at S10 or S28 occurs on different alleles. S28ph depends on CBP/p300-mediated K27ac, whereas H3 acetylated on K9 by PCAF is phosphorylated on S10. The redundant roles of S10ph and S28ph and their random targeting on distinct alleles may enable a fast response.

Epigenetics: Chromatin Organization and Function

Epigenetics refer to processes such as histone post-translational modifications (PTMs), DNA methylation and RNA that regulate gene activity and expression but are not dependent on alterations in DNA sequence. Herein, we review histone PTMs, histone variants and DNA modifications in the functioning of the nucleosome as an epigenetic signalling module. The majority of the human genome is transcribed, with most of the genome producing non-coding RNA, some of which is a component of the nuclear matrix, a dynamic RNA protein nuclear sub-structure. Non-coding RNA and coding RNA are associated with epigenetic modifiers, architectural chromatin proteins, coactivators and corepressors. The impact of changes in DNA sequence (single nucleotide polymorphisms) on the epigenome is discussed.