Site-specific acetylation of ISWI by GCN5

Background  

The tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition.     

Dietary restriction increases site-specific histone H3 acetylation in rat liver: possible modulation by sirtuins

We studied dietary restriction (DR) related changes of site-specific acetylation of histone H3 in rat livers to explore a possible link to histone modifications and sirtuin levels with anti-aging effects of DR. The acetylation at lysine residue 9, 27 and 56 in H3 was 20-30% higher in DR animals compared with ad libitum fed counterparts. SIRT6, one of histone deacetylases, was significantly decreased by DR and thereby may be involved in an increase in the histone acetylation. Our findings suggest that upregulation of chromatin activities through increased histone acetylation is a mechanism of anti-aging effects of DR.     

Mycardial histone acetylation

Myocardial histone acetylation was investigated in an isolated perfused heart preparation. Radioactive acetate rapidly accumulated in the intracellular compartment which preceded the covalent modification of histones. The acetylation of nucleohistones was rapid and reached a maximum during the first 20 min of perfusion. After 60 min of continuous perfusion there was a three-fold decrease in the amount of acetate bound to histone proteins. Histone H3 appeared to be preferred substrate for acetylation and was most responsive to a change in the concentration of radioactive acetate as well as the perfusion time.     

Demethylase activity is directed by histone acetylation

Mammalian genomes are compartmentalized into dense inactive chromatin that is hypermethylated and active open chromatin that is hypomethylated. It is generally accepted that this bimodal pattern of methylation is established during development and is then faithfully inherited through subsequent cell divisions by a maintenance DNA methyltransferase (DNMT1). The pattern of methylation is believed to direct local histone acetylation states. In contrast to this well accepted consensus, we show here using a transient transfection model that an active demethylase is involved in shaping patterns of methylation in somatic cells. Demethylase activity is directed by the state of histone acetylation, and therefore, the resulting methylation pattern is determined by local histone acetylation states contrary to the accepted model. Our data support a new model suggesting that the pattern of methylation is maintained by a dynamic balance of methylation and demethylation activities and the local state of histone acetylation. This provides a simple mechanism for explaining why active genes are not methylated.     

Core histone acetylation is regulated by linker histone stoichiometry in vivo

We investigated the relationship between linker histone stoichiometry and the acetylation of core histones in vivo. Exponentially growing cell lines induced to overproduce either of two H1 variants, H1(0) or H1c, displayed significantly reduced rates of incorporation of [(3)H]acetate into all four core histones. Pulse-chase experiments indicated that the rates of histone deacetylation were similar in all cell lines. These effects were also observed in nuclei isolated from these cells upon labeling with [(3)H]acetyl-CoA. Nuclear extracts prepared from control and H1-overexpressing cell lines displayed similar levels of histone acetylation activity on chromatin templates prepared from control cells. In contrast, extracts prepared from control cells were significantly less active on chromatin templates prepared from H1-overexpressing cells than on templates prepared from control cells. Reduced levels of acetylation in H1-overproducing cell lines do not appear to depend on higher order chromatin structure, because it persists even after digestion of the chromatin with micrococcal nuclease. The results suggest that alterations in chromatin structure, resulting from changes in linker histone stoichiometry may modulate the levels or rates of core histone acetylation in vivo.     

Genomewide histone acetylation microarrays

Histone acetylation and methylation are important regulators of gene activity. Chromatin immunoprecipitation (ChIP or ChrIP) has made it possible to examine not only the state of histone acetylation at a gene but also that of histone methylation and may soon be extended to other histone modifications such as phosphorylation and ubiquitination. In principle such studies are possible as long as an antibody is available to the particular histone modification. Once a target gene is identified it is instructive to see the effect of mutating putative enzymes responsible for the modification to determine how a particular enzyme is responsible for altering chromatin of that gene. Although specific target genes have been studied that contain such modifications recent technical advances have made it possible to study histone modifications genomewide. This not only allows for alternate views of particular paradigms to be investigated, but also uncovers chromosomal patterns of histone modification that would be missed in analyzing individual genes. We describe here an approach to rapidly study histone modifications genomewide by combining chromatin immunoprecipitation and DNA microarrays.     

Control of cytomegalovirus lytic gene expression by histone acetylation

Permissiveness for human cytomegalovirus (HCMV) infection is dependent on the state of cellular differentiation and has been linked to repression of the viral major immediate early promoter (MIEP). We have used conditionally permissive cells to analyze differential regulation of the MIEP and possible mechanisms involved in latency. Our data suggest that histone deacetylases (HDACs) are involved in repression of the MIEP in non-permissive cells as inhibition of HDACs induces viral permissiveness and increases MIEP activity. Non-permissive cells contain the class I HDAC, HDAC3; super-expression of HDAC3 in normally permissive cells reduces infection and MIEP activity. We further show that the MIEP associates with acetylated histones in permissive cells, and that in peripheral blood monocytes the MIEP associates with heterochromatin protein 1 (HP1), a chromosomal protein implicated in gene silencing. As monocytes are believed to be a site of viral latency in HCMV carriers and reactivated virus is only observed upon differentiation into macrophages, we propose that chromatin remodeling of the MIEP following cellular differentiation could potentially play a role in reactivation of latent HCMV.     

Critical role for the histone H4 N terminus in nucleosome remodeling by ISWI

The ATPase ISWI can be considered the catalytic core of several multiprotein nucleosome remodeling machines. Alone or in the context of nucleosome remodeling factor, the chromatin accessibility complex (CHRAC), or ACF, ISWI catalyzes a number of ATP-dependent transitions of chromatin structure that are currently best explained by its ability to induce nucleosome sliding. In addition, ISWI can function as a nucleosome spacing factor during chromatin assembly, where it will trigger the ordering of newly assembled nucleosomes into regular arrays. Both nucleosome remodeling and nucleosome spacing reactions are mechanistically unexplained. As a step toward defining the interaction of ISWI with its substrate during nucleosome remodeling and chromatin assembly we generated a set of nucleosomes lacking individual histone N termini from recombinant histones. We found the conserved N termini (the N-terminal tails) of histone H4 essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, was critical for CHRAC-induced nucleosome sliding and for the generation of regularity in nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a dependence on the H4 tail for ISWI-nucleosome interactions. We conclude that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate.     

Differential protein acetylation induced by novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors induce the hyperacetylation of nucleosomal histones in carcinoma cells resulting in the expression of repressed genes that cause growth arrest, terminal differentiation, and/or apoptosis. In vitro selectivity of several novel hydroxamate HDAC inhibitors including succinimide macrocyclic hydroxamates and the non-hydroxamate alpha-ketoamide inhibitors was investigated using isolated enzyme preparations and cellular assays. In vitro selectivity for the HDAC isozymes (HDAC1/2, 3, 4/3, and 6) was not observed for these HDAC inhibitors or the reference HDAC inhibitors, MS-275 and SAHA. In T24 and HCT116 cells these compounds caused the accumulation of acetylated histones H3 and H4; however, the succinimide macrocyclic hydroxamates and the alpha-ketoamides did not cause the accumulation of acetylated alpha-tubulin. These data suggest "selectivity" can be observed at the cellular level with HDAC inhibitors and that the nature of the zinc-chelating moiety is an important determinant of activity against tubulin deacetylase.     

Enzymic acetylation of nucleosome histone.

Rat liver chromatin prepared from purified nuclei catalyzed the acetylation of histones in nucleosomes at the same level as that of nuclei. The activity of histone acetyltransferase in chromatin was destroyed by heat treatment at 65 degrees C for 5 min. Histones in exogenously added nucleosomes also served as substrate for the enzyme. The sites of acetylation in the nucleosomes appeared to be in the trypsin-digestable N-terminal regions of histones H4, H3, and H2A, as has been reported in an in vivo system.     

The subunit-exchange model of histone acetylation

Increased histone acetylation has long been linked to gene activation, but little is known about how acetylation levels are regulated, largely because the histone acetyltransferase activities (HATs) responsible for this modification have been cloned only recently. Comparison of the biochemical nature of the Tetrahymena HAT A complex with the genetic and biochemical properties of the Saccharomyces Gcn5p-Ado complex leads us to propose that histone acetylase assemblies may be modular in nature and that this modularity may be an intimate part of the association of these enzymes with chromatin. The 'subunit-exchange' model provides a mechanism for the regulation and targeting of both histone acetylases and deacetylases and has implications for the control of cell growth, proliferation and tumorigenesis.