Amino-terminal Tails Of Core Histones Research Articles

The DNA Binding and Catalytic Domains of Poly(ADP-Ribose) Polymerase 1 Cooperate in the Regulation of Chromatin Structure and Transcription

We explored the mechanisms of chromatin compaction and transcriptional regulation by poly(ADP-ribose) polymerase 1 (PARP-1), a nucleosome-binding protein with an NAD(+)-dependent enzymatic activity. By using atomic force microscopy and a complementary set of biochemical assays with reconstituted chromatin, we showed that PARP-1 promotes the localized compaction of chromatin into supranucleosomal structures in a manner independent of the amino-terminal tails of core histones. In addition, we defined the domains of PARP-1 required for nucleosome binding, chromatin compaction, and transcriptional repression. Our results indicate that the DNA binding domain (DBD) of PARP-1 is necessary and sufficient for binding to nucleosomes, yet the DBD alone is unable to promote chromatin compaction and only partially represses RNA polymerase II-dependent transcription in an in vitro assay with chromatin templates (approximately 50% of the repression observed with wild-type PARP-1). Furthermore, our results show that the catalytic domain of PARP-1, which does not bind nucleosomes on its own, cooperates with the DBD to promote chromatin compaction and efficient transcriptional repression in a manner independent of its enzymatic activity. Collectively, our results have revealed a novel function for the catalytic domain in chromatin compaction. In addition, they show that the DBD and catalytic domain cooperate to regulate chromatin structure and chromatin-dependent transcription, providing mechanistic insights into how these domains contribute to the chromatin-dependent functions of PARP-1.

Mice Overexpressing Histone Deacetylase 9 Display Abnormal B Cell Development and Proliferation.

Histone acetylation plays a key role in regulating chromatin structure and gene expression. The activities of histone deacetylases (HDAC), enzymes that remove acetyl groups from lysines located at the amino-terminal tails of core histones, have been implicated in the pathogenesis of hematological malignancies. Consequently, inhibitors of these enzymes (HDACi) are considered an important new class of drugs for use in anti-cancer therapy and are now in various stages of development and clinical trials. For example, depsipeptide has now been granted a fast track designation by the FDA for development as a treatment for CTCL. To date, eleven HDACs have been identified and grouped on the basis of sequence homology to class I (HDACs 1–3 and 8), IIa (4, 5, 7 and 9) and IIb (6, 10 and 11). Depsipeptide and other currently used HDACi lack class and enzyme specificities. Additionally, the contribution of each HDAC to the pathogenesis of a given malignant disease is poorly understood. Improvements in HDACi specificities, together with advances in the understanding of the roles that individual HDACs play in normal hematopoiesis and distinct hematological neoplasms, are required for an effective use of these agents in anti-cancer therapies. We have previously identified and characterized HDAC9 as a class IIa enzyme that within the hematopoietic system is preferentially expressed in the lymphoid lineage. HDAC9 appears to be overexpressed in B lymphoid malignancies and associates at physiological levels with the BCL6 oncoprotein (Petrie et al, JBC 278:16059–72, 2003), a transcriptional repressor implicated in the pathogenesis of non-Hodgkin's lymphoma, suggesting a role for this chromatin modifying enzyme in B-cell transformation. In order to examine the function of HDAC9 in lymphoid development and cancer, we have generated transgenic mice that express the protein throughout the B lymphoid compartment under the control of the immunoglobulin heavy chain enhancer (Eμ). Initial data show that at an average of seven months these mice display splenomegaly and alterations to the splenic and bone marrow lymphoid populations. The presence of immature lymphocytes was also detected in peripheral blood. Consistent with these results and the above hypothesis, knockdown of HDAC9 results in strong inhibition of cell growth. These data indicate that aberrant expression of HDAC9 confers a proliferative advantage and leads to a phenotype resembling a pre-malignant condition.

Nuclear Receptor Recruitment of Histone-Modifying Enzymes to Target Gene Promoters

Nuclear receptors (NRs) compose one of the largest known families of eukaryotic transcription factors and, as such, serve as a paradigm for understanding the fundamental molecular mechanisms of eukaryotic transcriptional regulation. The packaging of eukaryotic genomic DNA into a higher ordered chromatin structure, which generally acts as a barrier to transcription by inhibiting transcription factor accessibility, has a major influence on the mechanisms by which NRs activate or repress gene expression. A major breakthrough in the field's understanding of these mechanisms comes from the recent identification of NR-associated coregulatory factors (i.e., coactivators and corepressors). Although several of these NR cofactors are involved in chromatin remodeling and facilitating the recruitment of the basal transcription machinery, the focus of this chapter is on NR coactivators and corepressors that act to covalently modify the amino-terminal tails of core histones. These modifications (acetylation, methylation, and phosphorylation) are thought to directly affect chromatin structure and?or serve as binding surfaces for other coregulatory proteins. This chapter presents the most current models for NR recruitment of histone-modifying enzymes and then summarizes their functional importance in NR-associated gene expression.

Histone Deacetylase 3 Binds to and Regulates the Multifunctional Transcription Factor TFII-I

Histone deacetylase 3 (HDAC3) is one of four members of the human class I histone deacetylases that are implicated in transcriptional repression through deacetylation of acetyllysines in amino-terminal tails of core histones. In an immunoaffinity purification using anti-HDAC3, transcription factor TFII-I copurified with HDAC3. Specificity of the HDAC3-TFII-I interaction was confirmed by coimmunoprecipitation of epitope-tagged proteins, GST pull-down assays, and protein colocalization with indirect immunofluorescence. An anti-TFII-I immunoprecipitate contained histone deacetylase enzymatic activity. Mutational analyses revealed that the carboxyl-terminal of HDAC3 (residues 373-401) and residues 363-606 of TFII-I were required for the HDAC3-TFII-I interaction. Transcriptional activation by TFII-I was severely reduced by overexpression of HDAC3. These results suggest that HDAC3 modulates some of the functions of TFII-I and provides a link between histone deacetylase and a multifunctional transcriptional activator.

Yng2p-dependent NuA4 Histone H4 Acetylation Activity Is Required for Mitotic and Meiotic Progression

In all eukaryotes, multisubunit histone acetyltransferase (HAT) complexes acetylate the highly conserved lysine residues in the amino-terminal tails of core histones to regulate chromatin structure and gene expression. One such complex in yeast, NuA4, specifically acetylates nucleosome-associated histone H4. Recent studies have revealed that NuA4 comprises at least 11 subunits, including Yng2p, a yeast homolog of the candidate human tumor suppressor gene, ING1. Consistent with prior data, we find that cells lacking Yng2p are deficient for NuA4 activity and are temperature-sensitive. Furthermore, we show that the NuA4 complex is present in the absence of Yng2p, suggesting that Yng2p functions to maintain or activate NuA4 HAT activity. Sporulation of diploid yng2 mutant cells reveals a defect in meiotic progression, whereas synchronized yng2 mutant cells display a mitotic delay. Surprisingly, genome-wide expression analysis revealed little change from wild type. Nocodazole arrest and release relieves the mitotic defects, suggesting that Yng2p may have a critical function prior to or during metaphase. Rather than a uniform decrease in acetylated forms of histone H4, we find striking cell-to-cell heterogeneity in the loss of acetylated histone H4 in yng2 mutant cells. Treating yng2 mutants with the histone deacetylase inhibitor trichostatin A suppressed the mitotic delay and restored global histone H4 acetylation, arguing that reduced H4 acetylation may underlie the cell cycle delay.

Phosphorylation of Serine 10 in Histone H3 Is Functionally Linked In Vitro and In Vivo to Gcn5-Mediated Acetylation at Lysine 14

Multiple covalent modifications exist in the amino-terminal tails of core histones, but whether a relationship exists between them is unknown. We examined the relationship between serine 10 phosphorylation and lysine 14 acetylation in histone H3 and have found that, in vitro, several HAT enzymes displayed increased activity on H3 peptides bearing phospho-Ser-10. This augmenting effect of Ser-10 phosphorylation on acetylation by yGcn5 was lost by substitution of alanine for arginine 164 [Gcn5(R164A)], a residue close to Ser-10 in the structure of the ternary tGcn5/CoA/histone H3 complex. Gcn5(R164A) had reduced activity in vivo at a subset of Gcn5-dependent promoters, and, strikingly, transcription of this same subset of genes was also impaired by substitution of serine 10 to alanine in the histone H3 tail. These observations suggest that transcriptional regulation occurs by multiple mechanistically linked covalent modifications of histones.

Structure of Tetrahymena GCN5 bound to coenzyme A and a histone H3 peptide.

Gene activation is a highly regulated process that requires the coordinated action of proteins to relieve chromatin repression and to promote transcriptional activation. Nuclear histone acetyltransferase (HAT) enzymes provide a mechanistic link between chromatin destabilization and gene activation by acetylating the epsilon-amino group of specific lysine residues within the aminoterminal tails of core histones to facilitate access to DNA by transcriptional activators. Here we report the high-resolution crystal structure of the HAT domain of Tetrahymena GCN5 (tGCN5) bound with both its physiologically relevant ligands, coenzyme A (CoA) and a histone H3 peptide, and the structures of nascent tGCN5 and a tGCN5/acetyl-CoA complex. Our structural data reveal histone-binding specificity for a random-coil structure containing a G-K-X-P recognition sequence, and show that CoA is essential for reorienting the enzyme for histone binding. Catalysis appears to involve water-mediated proton extraction from the substrate lysine by a glutamic acid general base and a backbone amide that stabilizes the transition-state reaction intermediate. Comparison with related N-acetyltransferases indicates a conserved structural framework for CoA binding and catalysis, and structural variability in regions associated with substrate-specific binding.

Transcription of mononucleosomal particles acetylated in the presence of n-butyrate

Although a correlation between chemical acetylation of the amino-terminal tails of core histones and stimulation of RNA synthesis has been reported for nucleosomal core particles (Piñeiro et al. (1991) Biochem. Biophys. Res. Commun. 177:370), no differences in transcription are detected between acetylated and nonacetylated mononucleosomal particles obtained from HeLa cells in the presence and absence of n-butyrate. Apparently, the lysine residues modified in the presence of n-butyrate are not the same responsible for the observed acetylation-induced transcription. The acetylation obtained with n-butyrate might be significantly different from that present in transcriptionally active chromatin.

Interaction of RNA polymerase II with acetylated nucleosomal core particles

Chemical acetylation of nucleosomal cores is accompanied by an increase in their efficiency as in vitro transcription templates. Low amounts of acetic anhydride cause preferential modification of the amino-terminal tails of core histones. Modification of these domains, which causes moderate structural effects, is apparently correlated with the observed stimulation of RNA synthesis. In contrast, extensive modification of the globular regions of core histones, which is accompanied by a large structural relaxation of the particle, causes little additional effect on transcription. Acetylation of the amino-terminal domains of histones might stimulate transcription by changing the interaction of the histone tails with components of the transcriptional machinery.