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At least 19 recordsLinked to original sources
PubMed1991

Histone acetylation in Zea mays.I. Activities of histone acetyltransferases and histone deacetylases.

DEAE-Sepharose chromatography of extracts from Zea mays meristematic cells revealed multiple histone acetyltransferase and histone deacetylase enzyme forms. An improved method for nuclear isolation allowed us to discriminate nuclear and cytoplasmic enzymes. Two nuclear histone acetyltransferases, A1 and A2, a cytoplasmic B-enzyme and two nuclear histone deacetylases, HD1 and HD2, have been identified. The histone specificity of the different enzyme forms has been studied in an in vitro system, using chicken erythrocyte histones as substrate. The cytoplasmic histone acetyltransferase B is the predominant enzyme, which acetylates mainly histone H4 and to a lesser extent H2A. The nuclear histone acetyltransferase A1 preferentially acetylates H3 and also H4, whereas enzyme A2 is specific for H3. This substrate specificity was confirmed with homologous Z. mays histones. The two histone deacetylases differ from each other with respect to ionic strength dependence, inhibition by acetate and butyrate, and substrate specificity. The strong inhibitory effect of acetate on histone deacetylases was exploited to distinguish different histone acetyltransferase forms.

Acetylation↗
PubMed2000

A continuous, nonradioactive assay for histone acetyltransferases.

Histone acetyltransferases (HATs) catalyze the acetyl-group transfer from acetyl-CoA to the epsilon-amino group of specific lysine residues within core histone proteins. HATs and other chromatin-remodeling enzymes have been recently shown to regulate gene activation within specific loci. To facilitate mechanistic studies, we have developed two continuous, nonradioactive assays for the prototypical GCN5 HAT. The CoASH generated in the HAT reactions was continuously measured by using a coupled enzyme system with either alpha-ketoglutarate dehydrogenase or pyruvate dehydrogenase. The CoASH-dependent oxidation of alpha-ketoglutarate or pyruvate is accompanied by the reduction of NAD to NADH, which was measured spectrophotometrically at 340 nm. The steady-state rate constants with substrates acetyl-CoA and a synthetic peptide (corresponding to the first 20 amino acids of H3 histone) were determined. The resulting rate constants were not significantly different between the two coupled assays, providing strong validation of these methods. Rate constants were also determined using the commonly employed radioactive filter-binding assay and compared. The 1.5- to 5-fold lower values obtained in the radioactive end-point assay are discussed in terms of the technical problems and limitations of this assay. The coupled assays should be widely applicable since the production of CoASH is common to all HAT enzymes, regardless of protein substrate.

Acetyl Coenzyme A↗
PubMed2004

Molecular requirements for gene expression mediated by targeted histone acetyltransferases.

Histone acetyltransferases (HATs) play fundamental roles in regulating gene expression. HAT complexes with distinct subunit composition and substrate specificity act on chromatin-embedded genes with different promoter architecture and chromosomal locations. Because requirements for HAT complexes vary, a central question in transcriptional regulation is how different HAT complexes function in different chromosomal contexts. Here, we have tested the ability of targeted yeast HATs to regulate gene expression of an epigenetically silenced locus. Of a panel of HAT fusion proteins targeted to a telomeric reporter gene, Sas3p and Gcn5p selectively increased expression of the silenced gene. Reporter gene expression was not solely dependent on acetyltransferase activity of the targeted HAT. Further analysis of Gcn5p-mediated gene expression revealed collateral requirements for HAT complex subunits Spt8p and Spt3p, which interact with TATA-binding protein, and for a gene-specific transcription factor. These data demonstrate plasticity of gene expression mediated by HATs upon encountering novel promoter architecture and chromatin context. The telomeric location of the reporter gene used in these studies also provides insight into the molecular requirements for heterochromatin boundary formation and for overcoming transcriptional silencing.

Acetyltransferases↗
PubMed2003

Expression, purification, and analysis of MOZ and MORF histone acetyltransferases.

Histone acetylation is one major mechanism by which chromatin structure and function are regulated. Besides histones, many nonhistone proteins are also acetylated in vivo. Aberrant acetylation has been linked to the development of various human diseases. Through acetylating histone and nonhistone proteins, histone acetyltransferases (HATs) play fundamental roles in regulating chromatin remodeling, transcription, and other nuclear processes. Known HATs belong to several groups, including the GCN5/PCAF, p300/CBP, and MYST families. ESA1, SAS3, MOF, TIP60, HBO1, MOZ, and MORF are the MYST family members with demonstrated HAT activity. The MOZ and MORF genes are rearranged by chromosome abnormalities associated with several types of leukemia, so these two HATs have been implicated in leukemogenesis. Compared with p300, CBP, and PCAF, much less is known about MOZ and MORF. To elucidate the function and regulation of these two interesting HATs, we have conducted their initial characterization. Here we describe the expression, purification, and activity analysis of MOZ and MORF. For comparison, we also include the procedure for expression and purification of PCAF. These methods are useful not only for functional characterization of MOZ, MORF, PCAF, and other HATs, but also for preparation of HAT proteins to screen compound libraries and obtain inhibitors with potential therapeutic value.

Acetyltransferases↗
PubMed2003

Quantification of histone acetyltransferase and histone deacetylase transcripts during early bovine embryo development.

Mammalian oocytes are very unique cells with an unlimited developmental potential. These totipotent cells are able to remove existing gene-expression patterns and to impose new ones. However, genome reprogramming is still a mystery. Posttranslational modifications by acetylation of the N-termini portion of histones composing the nucleosome are involved in genome reprogramming. These modifications alter the higher-order chromatin structure to render the DNA accessible to the regulatory and transcriptional machinery. In the present study, we have investigated, to our knowledge for the first time, precise expression patterns of seven genes involved in chromatin structure throughout bovine embryo development. Oocytes harvested from bovine ovaries were used for in vitro production of germinal vesicle oocytes, metaphase II oocytes, 2- and 8-cell embryos, and blastocysts. Total RNA was extracted from pools (triplicates) of 20 oocytes or from embryos of each developmental stage. By means of quantitative reverse transcription-polymerase chain reaction using SYBR Green to detect double-stranded DNA, mRNA expression profiles for histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC7), histone acetyltransferases (GCN5 and HAT1), and histone H2A were established. Transcripts for all genes were detected at all stages from the oocyte to the blastocyst. The HDAC1, HDAC2 (class I HDAC), and HAT1 (type B HAT) revealed similar expression profiles. The HDAC3 (class I HDAC) tends to have an expression profile similar to those of HDAC1, HDAC2, and HAT1, whereas the HDAC7 (class II HDAC) and GCN5 (type A HAT) profiles were different from those three. These results indicate variable levels of histone deacetylases and histone acetyltransferases throughout embryonic development and may indicate the ones that are involved in somatic remodeling.

Acetyltransferases↗
PubMed2006

Dynamic changes in chromatin acetylation and the expression of histone acetyltransferases and histone deacetylases regulate the SM22alpha transcription in response to Smad3-mediated TGFbeta1 signaling.

TGFbeta1 plays critical roles in stimulating smooth muscle gene transcription during myofibroblast and smooth muscle cell (SMC) differentiation. Increasing evidence demonstrates that histone modification plays important roles in regulating gene transcription. Here, we investigated the effect of changes in the expression of histone acetyltransferases (HAT) or histone deacetylases (HDAC) on TGFbeta1-induced SM22 promoter activities. We found that overexpressing HAT proteins such as p300 and CBP enhances TGFbeta1-induced SM22 promoter activities; conversely, overexpressing HAT inhibitor such as Twist1 (but not Twist2/Dermo-1) and E1A suppresses this effect of TGFbeta1. We also found that TSA, a HDAC inhibitor that stimulates histone acetylation of the SM22alpha locus, further enhances the transactivational activity of Smad2, Smad3 and Smad4, and relieves the inhibitory effect of Smad6, Smad7, and the dominant negative mutants of Smads. TGFbeta1 also stimulates the association of Smad3 (a potent transactivator for the SM22 promoter) and p300 by co-immunoprecipitation assay. In contrast, overexpressing HDAC 1-6 inhibits TGFbeta1-induced as well as Smad3 and myocardin-activated SM22 promoter. Moreover, chromatin immunoprecipitation (ChIP) assays show that TGFbeta1 induces histone acetylation at the SM22alpha locus. This study demonstrates that the balance of HAT and HDAC expression affects TGFbeta1-induced SM22alpha transcription; TGFbeta1-induced SM22alpha transcription is accompanied by histone hyperacetylation at the SM22alpha locus. This study provides the first evidence showing that histone hyperacetylation of the SM22 promoter is a target of TGFbeta1 signaling, suggesting that modulation of histone acetylation is involved in the molecular mechanisms of TGFbeta1-regulated SMC gene transcription.

Acetylation↗
PubMed1992

Opposite responses of nuclear spermidine N8-acetyltransferase and histone acetyltransferase activities to regenerative stimuli in rat liver.

Experiments performed in different models of hepatic regeneration at the time of maximal DNA synthesis, determined by thymidine kinase activity assay, demonstrated that spermidine N8-acetyltransferase activity increased 48 hr after CCl4 administration (2-fold), 72 hr after CCl4 plus phenobarbital (3-fold) and 24 hr after partial hepatectomy (4.5-fold). On the contrary, at these times histone acetyltransferase activity diminished (approximately twofold) and was unchanged compared with control values in the liver of hepatotoxin-treated and hepatectomized rats, respectively. Histone acetylation was, however, enhanced 1.5-fold before the onset of DNA replication (14 hr), and 3.4-fold after the peak of DNA synthesis (32 hr) in the liver of hepatectomized rats. alpha-Difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase that was administered to hepatectomized rats, blocked polyamine synthesis, thymidine kinase activity and consequently liver regeneration 24 hr after the surgery. In those conditions, spermidine N8-acetyltransferase activity was decreased approximately twofold, whereas histone acetyltransferase activity was elevated approximately twofold. All these effects were reversed by putrescine coadministration. Altogether, these findings showed that nuclear spermidine N8-acetyltransferase and histone acetyltransferase activities were regulated in opposite ways during the processes associated with liver regeneration. Moreover, they suggested that the polyamines themselves might have a direct or indirect role in this regulation.

Acetyltransferases↗
PubMed2003

Measurement of histone acetyltransferase and histone deacetylase activities and kinetics of histone acetylation.

Dynamic histone acetylation has a role in chromatin remodeling and in the regulation of transcription. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) catalyze reversible histone acetylation. HATs and HDACs exist as multiprotein complexes that have coactivator and corepressor activities, respectively. The steady-state level of acetylation at a chromatin site is determined by the local net activities of these enzymes. Here we describe methods to isolate different subcellular fractions (cytosol, nuclei, tightly bound nuclear, loosely bound nuclear, immunoprecipitated multiprotein complexes, and nuclear matrix) to determine the subcellular distribution of HAT and HDAC activities. Procedures to assay the activities of these enzymes and to measure the kinetics of histone acetylation and deacetylation are presented.

Acetylation↗
PubMed2002

Analysis of histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana suggests functional diversification of chromatin modification among multicellular eukaryotes.

Sequence similarity and profile searching tools were used to analyze the genome sequences of Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster for genes encoding three families of histone deacetylase (HDAC) proteins and three families of histone acetyltransferase (HAT) proteins. Plants, animals and fungi were found to have a single member of each of three subfamilies of the GNAT family of HATs, suggesting conservation of these functions. However, major differences were found with respect to sizes of gene families and multi-domain protein structures within other families of HATs and HDACs, indicating substantial evolutionary diversification. Phylogenetic analysis identified a new class of HDACs within the RPD3/HDA1 family that is represented only in plants and animals. A similar analysis of the plant-specific HD2 family of HDACs suggests a duplication event early in dicot evolution, followed by further diversification in the lineage leading to Arabidopsis. Of three major classes of SIR2-type HDACs that are found in animals, fungi have representatives only in one class, whereas plants have representatives only in the other two. Plants possess five CREB-binding protein (CBP)-type HATs compared with one to two in animals and none in fungi. Domain and phylogenetic analyses of the CBP family proteins showed that this family has evolved three distinct types of CBPs in plants. The domain architecture of CBP and TAF(II)250 families of HATs show significant differences between plants and animals, most notably with respect to bromodomain occurrence and their number. Bromodomain-containing proteins in Arabidopsis differ strikingly from animal bromodomain proteins with respect to the numbers of bromodomains and the other types of domains that are present. The substantial diversification of HATs and HDACs that has occurred since the divergence of plants, animals and fungi suggests a surprising degree of evolutionary plasticity and functional diversification in these core chromatin components.

Acetyltransferases↗
PubMed1978

Calf liver nuclear N-acetyltransferases. Purification and properties of two enzymes with both spermidine acetyltransferase and histone acetyltransferase activities.

Calf liver contains two nuclear N-acetyltransferases which are separated by chromatography on hydroxylapatite. Both acetyltransferase A and acetyltransferase B will transfer acetate from acetyl-CoA to either histone or spermidine. The same protein catalyzes the reaction with both substrates; this is shown by a constant ratio of spermidine to histone activity over a 5,000-fold purification and identical heat denaturation kinetics for both spermidine and histone acetyltransferase activity with each enzyme. Histone is preferentially acetylated when both acceptors are present. Both enzymes preferentially acetylate polyamines (spermidine, spermine, and diaminodipropylamine) to diamines. Acetyltransferase A acetylates histones in the order: whole histone greater than H4 greater than H2A greater than H3 greater than H2B greater than H1; acetyltransferase B in the order: whole histone greater than H4 = H3 greater than H2A greater than H2B greater than H1. Michaelis constants are 2 X 10(-4)M for spermidine and 9 X 10(-6)M for acetyl-CoA. Acetyltransferase A has a molecular weight of 150,000; acetyltransferase B 175,000. Both enzymes are strongly inhibited by p-chloromercuribenzoate and weakly inhibited by EDTA.

Acetyltransferases↗
PubMed2001

Structure and function of histone acetyltransferases.

Histone acetyltranferase (HAT) enzymes are the catalytic subunit of large multisubunit HAT complexes that acetylate the epsilon-amino group of specific lysine residues on histone tails to promote transcriptional activation. Recent structural and functional studies on the divergent HAT enzymes Gcn5/PCAF, Esa1 and Hat1 have provided new insights into the underlying mechanism of histone binding and acetylation by HAT proteins. The three HAT enzymes contain a structurally conserved core domain that plays a functionally conserved role in binding the coenzyme A cofactor and in harboring the putative general base for catalysis. Structurally variable N- and C-terminal domains appear to contain a related scaffold that mediates histone substrate binding. These data provide a framework for understanding the structure and function of other more divergent HAT proteins such as TAF(II)250 and CBP/p300, and provides a starting point for understanding how HAT proteins may cooperate with other factors within in vivo HAT complexes to promote transcriptional activation.

Acetyltransferases↗
PubMed2001

Structure of histone acetyltransferases.

Histone acetyltranferase (HAT) enzymes are the catalytic subunits of multisubunit protein complexes that acetylate specific lysine residues on the N-terminal regions of the histone components of chromatin to promote gene activation. These enzymes, which now include more than 20 members, fall into distinct families that generally have high sequence similarity and related substrate specificity within families, but have divergent sequence and substrate specificity between families. Significant insights into the mode of catalysis and histone substrate binding have been provided by the structure determination of the divergent HAT enzymes Hat1, Gcn5/PCAF and Esa1. A comparison of these structures reveals a structurally conserved central core domain that mediates extensive interactions with the acetyl-coenzyme A cofactor, and structurally divergent N and C-terminal domains. A correlation of these structures with other studies reveals that the core domain plays a particularly important role in histone substrate catalysis and that the N and C-terminal domains play important roles in histone substrate binding. These correlations imply a related mode of catalysis and histone substrate binding by a diverse group of HAT enzymes.

Acetyl Coenzyme A↗
PubMed2003

Molecular basis for Gcn5/PCAF histone acetyltransferase selectivity for histone and nonhistone substrates.

Histone acetyltransferase (HAT) proteins often exhibit a high degree of specificity for lysine-bearing protein substrates. We have previously reported on the structure of the Tetrahymena Gcn5 HAT protein (tGcn5) bound to its preferred histone H3 substrate, revealing the mode of substrate binding by the Gcn5/PCAF family of HAT proteins. Interestingly, the Gcn5/PCAF HAT family has a remarkable ability to acetylate lysine residues within diverse cognate sites such as those found around lysines 14, 8, and 320 of histones H3, H4, and p53, respectively. To investigate the molecular basis for this, we now report on the crystal structures of tGcn5 bound to 19-residue histone H4 and p53 peptides. A comparison of these structures with tGcn5 bound to histone H3 reveals that the Gcn5/PCAF HATs can accommodate divergent substrates by utilizing analogous interactions with the lysine target and two C-terminal residues with a related chemical nature, suggesting that these interactions play a general role in Gcn5/PCAF substrate binding selectivity. In contrast, while the histone H3 complex shows extensive interactions with tGcn5 and peptide residues N-terminal to the target lysine, the corresponding residues in histone H4 and p53 are disordered, suggesting that the N-terminal substrate region plays an important role in the enhanced affinity of the Gcn5/PCAF HAT proteins for histone H3. Together, these studies provide a framework for understanding the substrate selectivity of HAT proteins.

Acetyltransferases↗
PubMed2005

Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity.

Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by two classes of enzyme, histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulated HAT and HDAC activity plays a role in the development of a range of cancers. Consequently, inhibitors of these enzymes have potential as anticancer agents. Several HDAC inhibitors have been described; however, few inhibitors of HATs have been disclosed. Following a FlashPlate high-throughput screen, we identified a series of isothiazolone-based HAT inhibitors. Thirty-five N-substituted analogues inhibited both p300/cyclic AMP-responsive element binding protein-binding protein-associated factor (PCAF) and p300 (1 to >50 micromol/L, respectively) and the growth of a panel of human tumor cell lines (50% growth inhibition, 0.8 to >50 micromol/L). CCT077791 and CCT077792 decreased cellular acetylation in a time-dependent manner (2-48 hours of exposure) and a concentration-dependent manner (one to five times, 72 hours, 50% growth inhibition) in HCT116 and HT29 human colon tumor cell lines. CCT077791 reduced total acetylation of histones H3 and H4, levels of specific acetylated lysine marks, and acetylation of alpha-tubulin. Four and 24 hours of exposure to the compounds produced the same extent of growth inhibition as 72 hours of continuous exposure, suggesting that growth arrest was an early event. Chemical reactivity of these compounds, as measured by covalent protein binding and loss of HAT inhibition in the presence of DTT, indicated that reaction with thiol groups might be important in their mechanism of action. As one of the first series of small-molecule inhibitors of HAT activity, further analogue synthesis is being pursued to examine the potential scope for reducing chemical reactivity while maintaining HAT inhibition.

Acetylation↗
PubMed2005

Polyglutamine-expanded spinocerebellar ataxia-7 protein disrupts normal SAGA and SLIK histone acetyltransferase activity.

Histone acetyltransferases have been shown to participate in many essential cellular processes, particularly those associated with activation of transcription. SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) are two highly homologous multisubunit histone acetyltransferase complexes that were originally identified in the yeast Saccharomyces cerevisiae. Here, we identify the protein Sgf73/Sca7 as a component of SAGA and SLIK, and a homologue of the human SCA7-encoded protein ataxin-7, which, in its polyglutamine expanded pathological form, is responsible for the neurodegenerative disease spinocerebellar ataxia 7 (SCA7). Our findings indicate that yeast Sca7 is necessary for the integrity and function of both SAGA and SLIK, and that the human ataxin-7 is able to compliment the loss of Sca7 in yeast. A polyglutamine-expanded version of ataxin-7 assembles a SAGA complex that is depleted of critical proteins that regulate the ability of SAGA to acetylate nucleosomes. These observations have significant implications for the function of the human Sca7 protein in disease pathogenesis.

Acetyltransferases↗
PubMed1999

Structure and ligand of a histone acetyltransferase bromodomain.

Histone acetylation is important in chromatin remodelling and gene activation. Nearly all known histone-acetyltransferase (HAT)-associated transcriptional co-activators contain bromodomains, which are approximately 110-amino-acid modules found in many chromatin-associated proteins. Despite the wide occurrence of these bromodomains, their three-dimensional structure and binding partners remain unknown. Here we report the solution structure of the bromodomain of the HAT co-activator P/CAF (p300/CBP-associated factor). The structure reveals an unusual left-handed up-and-down four-helix bundle. In addition, we show by a combination of structural and site-directed mutagenesis studies that bromodomains can interact specifically with acetylated lysine, making them the first known protein modules to do so. The nature of the recognition of acetyl-lysine by the P/CAF bromodomain is similar to that of acetyl-CoA by histone acetyltransferase. Thus, the bromodomain is functionally linked to the HAT activity of co-activators in the regulation of gene transcription.

Acetyltransferases↗