Acetylation of histone H4 on lysine 16 (H4-K16Ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. To characterize the structural and functional role of this mark, we used a native chemical ligation strategy to generate histone H4 that was homogeneously acetylated at K16. The incorporation of this modified histone into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and impedes the ability of chromatin to form cross-fiber interactions. H4-K16Ac also inhibits the ability of the adenosine triphosphate-utilizing chromatin assembly and remodeling enzyme ACF to mobilize a mononucleosome, indicating that this single histone modification modulates both higher order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber.

This article reviews the effects of the short-chain fatty acid butyrate on histone deacetylase (HDAC) activity. Sodium butyrate has multiple effects on cultured mammalian cells that include inhibition of proliferation, induction of differentiation and induction or repression of gene expression. The observation that butyrate treatment of cells results in histone hyperacetylation initiated a flurry of activity that led to the discovery that butyrate inhibits HDAC activity. Butyrate has been an essential agent for determining the role of histone acetylation in chromatin structure and function. Interestingly, inhibition of HDAC activity affects the expression of only 2% of mammalian genes. Promoters of butyrate-responsive genes have butyrate response elements, and the action of butyrate is often mediated through Sp1/Sp3 binding sites (e.g., p21(Waf1/Cip1)). We demonstrated that Sp1 and Sp3 recruit HDAC1 and HDAC2, with the latter being phosphorylated by protein kinase CK2. A model is proposed in which inhibition of Sp1/Sp3-associated HDAC activity leads to histone hyperacetylation and transcriptional activation of the p21(Waf1/Cip1) gene; p21(Waf1/Cip1) inhibits cyclin-dependent kinase 2 activity and thereby arrests cell cycling. Pending the cell background, the nonproliferating cells may enter differentiation or apoptotic pathways. The potential of butyrate and HDAC inhibitors in the prevention and treatment of cancer is presented.

Transcriptional repression by nuclear receptors has been correlated to binding of the putative co-repressor, N-CoR. A complex has been identified that contains N-CoR, the Mad presumptive co-repressor mSin3, and the histone deacetylase mRPD3, and which is required for both nuclear receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain family. These data predict that the ligand-induced switch of heterodimeric nuclear receptors from repressor to activator functions involves the exchange of complexes containing histone deacetylases with those that have histone acetylase activity.

Transcriptional repression by Mad-Max heterodimers requires interaction of Mad with the corepressors mSin3A/B. Sin3p, the S. cerevisiae homolog of mSin3, functions in the same pathway as Rpd3p, a protein related to two recently identified mammalian histone deacetylases, HDAC1 and HDAC2. Here, we demonstrate that mSin3A and HDAC1/2 are associated in vivo. HDAC2 binding requires a conserved region of mSin3A capable of mediating transcriptional repression. In addition, Mad1 forms a complex with mSin3 and HDAC2 that contains histone deacetylase activity. Trichostatin A, an inhibitor of histone deacetylases, abolishes Mad repression. We propose that Mad-Max functions by recruiting the mSin3-HDAC corepressor complex that deacetylates nucleosomal histones, producing alterations in chromatin structure that block transcription.

The gene for acute myeloid leukemia-1 (AML-1) is one of the most frequently translocated genes in human cancer. It is targeted by t(8;21) and t(3;21) in AML and by t(12;21) in acute lymphocytic leukemia (39). AML-1 is also indirectly targeted by inv(16), which disrupts core binding factor beta, an AML-1-interacting protein. AML-1 binds the enhancer core motif (TGT/cGGT) and regulates a variety of viral and cellular genes in concert with other factors (31). t(8;21) is one of the most frequent translocations found in AML, comprising 10 to 15% of cases with discernible translocations (39). The t(8;21) fusion protein AML-1/ETO acts as a repressor of transcription in transient-transfection assays (10,31,32,43). When expressed during development, the t(8;21) fusion protein yielded the same phenotype as AML-1 deficiency (37, 45).Although eight-twenty-one (ETO; also known as MTG8 [myeloid tumor gene 8] [8,34]) was identified at the breakpoint of t(8;21), little is known about the normal function of the protein. ETO is the human homologue of the Drosophila Nervy protein (9), and it shares four homologous domains with the Nervy protein. These include a region with extensive homology to a Drosophila coactivator, transcription-activating factor 110 (TAF110), a predicted hydrophobic heptad repeat (HHR), a small domain with no other homology, termed the Nervy domain (27), and the MYND (myeloid-Nervy-DEAF-1 [12]) domain. The MYND domain is present in numerous human, murine, Caenorhabditis elegans, and Drosophila proteins and contains two putative zinc finger (ZF) motifs (9,12,31). ETO is expressed in hematopoietic cells and in the brain, but another closely related family member is ubiquitously expressed (19). A third closely related factor, MTG16, is fused to AML-1 by t(16;21) (20).AML-1 is a site-specific DNA binding protein that can both activate and repress transcription (2, 28, 36). The t(8;21) fusion protein AML-1/ETO contains the N-terminal 177 amino acids of AML-1, including the DNA binding domain, fused to nearly all of ETO (7,8,34). The fusion protein inhibits AML-1-dependent transactivation (10, 32). AML-1/ETO also repressed both basal transcription and Ets-1-dependent activation of the multidrug resistance 1 promoter (27). Similarly, AML-1/ETO inhibited both AML-1 and C/EBP␣-dependent transactivation of the neutrophil protein 3 (NP-3) promoter (44). AML-1/ETO-mediated repression is dependent on both the DNA binding domain of AML-1 and ETO sequences (24). AML-1/ETO acts at substoichiometric levels and thus does not compete with AML-1 for DNA binding sites within promoters, nor does it act to "squelch" transcription (24). Thus, we hypothesized that ETO recruits a corepressor or normally functions as a corepressor to inhibit transcription (30,31,33).Several corepressor proteins have been recently described that associate with histone deacetylases (HDACs) to repress transcription (3,5,13,17,38,40,42). The nuclear hormone corepressor N-CoR was identified through interactions with the thyroid hormone receptor and associa...

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

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.

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