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PubMed1980

Fractionation of non-histone chromatin proteins from pig liver and kidney by means of immobilized histone H3.

The non-histone chromatin proteins (NHCp) from pig liver and kidney have been partially fractionated in non-denaturing conditions by the use of histone H3 immobilized on agarose and the fractions obtained have been analysed by SDS-polyacrylamide gel electrophoresis and amino acid analysis. At least six different fractions have been obtained by successive increases of the ionic strength of the medium. Few NHCp have been evidentiated with subunit molecular weights 55,000 and less than 30,000, which diaplay a remarkably high affinity for histone H3, and require 5 M urea to be displaced from the immobilized histone. The elution patterns of the NHCp from liver and kidney, although very similar, reveal some significant differences between the two tissues, which are undetectable by SDS-electrophoresis and which are most likely due to tissue specific proteins. This histone-affinity chromatography appears to be a promising approach for the analysis of specific histone-NHCp interactions, and as a first step for NHCp purification.

Amino Acids↗
PubMed2002

Elongator is a histone H3 and H4 acetyltransferase important for normal histone acetylation levels in vivo.

The elongating, hyperphosphorylated form of RNA polymerase II is associated with the Elongator complex, which has the histone acetyltransferase (HAT) Elp3 as a subunit. Here we show that, in contrast to the isolated Elp3 subunit, the activity of intact Elongator complex is directed specifically toward the amino-terminal tails of histone H3 and H4, and that Elongator can acetylate both core histones and nucleosomal substrates. The predominant acetylation sites are lysine-14 of histone H3 and lysine-8 of histone H4. The three smallest Elongator subunits--Elp4, Elp5, and Elp6--are required for HAT activity, and Elongator binds to both naked and nucleosomal DNA. By using chromatin immunoprecipitation, we show that the levels of multiply acetylated histone H3 and H4 in chromatin are decreased in vivo in yeast cells lacking ELP3.

Acetylation↗
PubMed2002

Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79.

Dot1 is a non-SET domain protein that methylates histone H3 at lysine 79, a surface-exposed residue that lies within the globular domain. In the context of a nucleosome, H3 lysine 79 is located in close proximity with lysine 123 of histone H2B, a major site for ubiquitination by Rad6. Here we show that Rad6-mediated ubiquitination of H2B lysine 123 is important for efficient methylation of lysine 79, but not lysine 36, of histone H3. In contrast, lysine 79 methylation of H3 is not required for ubiquitination of H2B. Our study provides a new example of trans-histone regulation between modifications on different histones. In addition, it suggests that Rad6 affects telomeric silencing, at least in part, by influencing methylation of histone H3.

Histone-Lysine N-Methyltransferase↗
PubMed2003

The Paf1 complex is essential for histone monoubiquitination by the Rad6-Bre1 complex, which signals for histone methylation by COMPASS and Dot1p.

Monoubiquitination of histone H2B, catalyzed by Rad6-Bre1, is required for methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively. The Paf1 protein complex, which associates with RNA polymerase II, is known to be required for these histone H3 methylation events. During the early elongation stage of transcription, the Paf1 complex is required for association of COMPASS with RNA polymerase II, but the role the Paf1 complex plays at the promoter has not been clear. We present evidence that the Paf1 complex is required for monoubiquitination of histone H2B at promoters. Strains deleted for several components of the Paf1 complex are defective in monoubiquitination of histone H2B, which results in the loss of methylation of lysines 4 and 79 of histone H3. We also show that Paf1 complex is required for the interaction of Rad6 and COMPASS with RNA polymerase II. Finally, we show that the Paf1 complex is required for Rad6-Bre1 catalytic activity but not for the recruitment of Rad6-Bre1 to promoters. Thus, in addition to its role during the elongation phase of transcription, the Paf1 complex appears to activate the function but not the placement of the Rad6-Bre1 ubiquitin-protein ligase at the promoters of active genes.

DNA Polymerase II↗
PubMed2006

A highly specific mechanism of histone H3-K4 recognition by histone demethylase LSD1.

Human lysine-specific demethylase (LSD1) is a chromatin-modifying enzyme that specifically removes methyl groups from mono- and dimethylated Lys4 of histone H3 (H3-K4). We used a combination of in vivo and in vitro experiments to characterize the substrate specificity and recognition by LSD1. Biochemical assays on histone peptides show that essentially all epigenetic modifications on the 21 N-terminal amino acids of histone H3 cause a significant reduction in enzymatic activity. Replacement of Lys4 with Arg greatly enhances binding affinity, and a histone peptide incorporating this mutation has a strong inhibitory power. Conversely, a peptide bearing a trimethylated Lys4 is only a weak inhibitor of the enzyme. Rapid kinetics measurements evidence that the enzyme is efficiently reoxidized by molecular oxygen with a second-order rate constant of 9.6x10(3) M-1 s-1, and that the presence of the reaction product does not greatly influence the rate of flavin reoxidation. In vivo experiments provide a correlation between the in vitro inhibitory properties of the tested peptides and their ability of affecting endogenous LSD1 activity. Our results show that epigenetic modifications on histone H3 need to be removed before Lys4 demethylation can efficiently occur. The complex formed by LSD1 with histone deacetylases 1/2 may function as a "double-blade razor" that first eliminates the acetyl groups from acetylated Lys residues and then removes the methyl group from Lys4. We suggest that after H3-K4 demethylation, LSD1 recruits the forthcoming chromatin remodelers leading to the introduction of gene repression marks.

Cell Line↗
PubMed1988

Micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase activity which is highly specific for free histone H4.

Salt extracts prepared from purified micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase (also referred to as histone acetyltransferase) activity which is highly specific for H4 when tested as a free histone. With both extracts, H4 is acetylated first at position 4 (monoacetylated) or positions 4 and 11 (diacetylated), sites diagnostic of deposition-related acetylation of newly synthesized H4 in vivo. As the concentration of cytosolic extract is decreased in the in vitro reactions, acetylation of H3 is also observed. Neither activity acetylates histone in a chromatin form. These activities are distinct from a macronuclear acetylase which acetylates H3 and H4 (macro- or micronuclear) equally well as free histones and which acetylates all four core histones when mononucleosomes are used as substrate. As well, the micronuclear and cytoplasmic activities give similar thermal-inactivation profiles which are different from that of the macronuclear activity. In situ enzyme assays demonstrate a macronuclear-specific activity which acetylates endogenous macronuclear chromatin and an independent micronuclear-cytosolic activity which is able to act upon exogenously added free H4. These results argue strongly that an identical acetylase is responsible for the micronuclear and cytoplasmic activity which is either modified or altogether distinct from that in macronuclei.

Acetylation↗
PubMed2003

Sequence specific transcription factor, JDP2 interacts with histone and inhibits p300-mediated histone acetylation.

Jun dimerization protein 2 (JDP2) is a novel member of AP-1 family and acts as a general repressor of a variety of transcription. JDP2 is able to bind to specific sites in target gene such as c-jun by forming homodimer or heterodimers with a Jun/ATF family member to counteract their transcriptional activity. Previously we showed that JDP2 inhibits the retinoic acid (RA) dependent transcription by recruiting a histone deacetylase 3 (HDAC3) complex to the promoter region of the target genes. We present here that JDP2 has an inhibitory activity of acetylation of all core histones mediated by histone acetyltransferase (HAT) both of p300 and PCAF in vitro. The studies of both histone-binding and HAT-inhibitory activity using a variety of recombinant JDP2(s) indicated that JDP2 might target histone itself through the histone-binding domain of JDP2, which is essential but not sufficient for the inhibition of acetylation. Therefore, our data suggested that HAT-inhibitory activity of JDP2 could in part explain the transcriptional repression of several target genes.

Acetylation↗
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

The Yng1p plant homeodomain finger is a methyl-histone binding module that recognizes lysine 4-methylated histone H3.

The ING (inhibitor of growth) protein family includes a group of homologous nuclear proteins that share a highly conserved plant homeodomain (PHD) finger domain at their carboxyl termini. Members of this family are found in multiprotein complexes that posttranslationally modify histones, suggesting that these proteins serve a general role in permitting various enzymatic activities to interact with nucleosomes. There are three members of the ING family in Saccharomyces cerevisiae: Yng1p, Yng2p, and Pho23p. Yng1p is a component of the NuA3 histone acetyltransferase complex and is required for the interaction of NuA3 with chromatin. To gain insight into the function of the ING proteins, we made use of a genetic strategy to identify genes required for the binding of Yng1p to histones. Using the toxicity of YNG1 overexpression as a tool, we showed that Yng1p interacts with the amino-terminal tail of histone H3 and that this interaction can be disrupted by loss of lysine 4 methylation within this tail. Additionally, we mapped the region of Yng1p required for overexpression of toxicity to the PHD finger, showed that this region capable of binding lysine 4-methylated histone H3 in vitro, and demonstrated that mutations of the PHD finger that abolish binding in vitro are no longer toxic in vivo. These results identify a novel function for the Yng1p PHD finger in promoting stabilization of the NuA3 complex at chromatin through recognition of histone H3 lysine 4 methylation.

Animals↗
PubMed1979

The in vivo distribution of immunoreactive larger than tetrameric polyadenosine diphosphoribose in histone and non-histone protein fractions of rat liver.

The macromolecular association of immunoreactive naturally occurring polyadenosine diphosphoribose n greater than 4 with histones and non-histone proteins was determined with the aid of an improved method of extraction of polyadenosine diphosphoribose and a combination of radioimmunoassay and molecular filtration. More than 99% of the naturally occurring polyadenosine diphosphoribose n greater than 4 was present in rat liver in covalent association with non-histone proteins. The chain length of the polymer varied between n = 4 and n = 34. Less than 1% of naturally occurring polyadenosine diphosphoribose n greater than 4 was almost evenly distributed between histone fractions f1, f2a, f2b, and f3. Adenosine diphosphoribose polymers of relatively long chain length were also detected in the histone fractions. The covalent association of polyadenosine diphosphoribose with non-histone proteins was demonstrated by affinity chromatography.

Animals↗
PubMed2001

Histone methylation versus histone acetylation: new insights into epigenetic regulation.

Post-translational addition of methyl groups to the amino-terminal tails of histone proteins was discovered more than three decades ago. Only now, however, is the biological significance of lysine and arginine methylation of histone tails being elucidated. Recent findings indicate that methylation of certain core histones is catalyzed by a family of conserved proteins known as the histone methyltransferases (HMTs). New evidence suggests that site-specific methylation, catalyzed by HMTs, is associated with various biological processes ranging from transcriptional regulation to epigenetic silencing via heterochromatin assembly. Taken together, these new findings suggest that histone methylation may provide a stable genomic imprint that may serve to regulate gene expression as well as other epigenetic phenomena.

Acetylation↗
PubMed2000

Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation on lysines 8 and 12.

We have investigated the ability of dexamethasone to regulate interleukin-1beta (IL-1beta)-induced gene expression, histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity. Low concentrations of dexamethasone (10(-10) M) repress IL-1beta-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression and fail to stimulate secretory leukocyte proteinase inhibitor expression. Dexamethasone (10(-7) M) and IL-1beta (1 ng/ml) both stimulated HAT activity but showed a different pattern of histone H4 acetylation. Dexamethasone targeted lysines K5 and K16, whereas IL-1beta targeted K8 and K12. Low concentrations of dexamethasone (10(-10) M), which do not transactivate, repressed IL-1beta-stimulated K8 and K12 acetylation. Using chromatin immunoprecipitation assays, we show that dexamethasone inhibits IL-1beta-enhanced acetylated K8-associated GM-CSF promoter enrichment in a concentration-dependent manner. Neither IL-1beta nor dexamethasone elicited any GM-CSF promoter association at acetylated K5 residues. Furthermore, we show that GR acts both as a direct inhibitor of CREB binding protein (CBP)-associated HAT activity and also by recruiting HDAC2 to the p65-CBP HAT complex. This action does not involve de novo synthesis of HDAC protein or altered expression of CBP or p300/CBP-associated factor. This mechanism for glucocorticoid repression is novel and establishes that inhibition of histone acetylation is an additional level of control of inflammatory gene expression. This further suggests that pharmacological manipulation of of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.

Acetylation↗
PubMed2002

Activation of the mouse histone deacetylase 1 gene by cooperative histone phosphorylation and acetylation.

Histone deacetylase 1 (HDAC1) is a major regulator of chromatin structure and gene expression. Tight control of HDAC1 expression is essential for normal cell cycle progression of mammalian cells. HDAC1 mRNA levels are regulated by growth factors and by changes in intracellular deacetylase activity levels. Stimulation of the mitogen-activated protein kinase cascade by anisomycin or growth factors, together with inhibition of deacetylases by trichostatin A (TSA), leads to stable histone H3 phosphoacetylation and strongly induced HDAC1 expression. In contrast, activation of the nucleosomal response by anisomycin alone results only in transient phosphoacetylation of histone H3 without affecting HDAC1 mRNA levels. The transcriptional induction of the HDAC1 gene by anisomycin and TSA is efficiently blocked by H89, an inhibitor of the nucleosomal response. Detailed studies of the kinetics of histone acetylation and phosphorylation show that the two modifications are synergistic and essential for induced HDAC1 transcription. Activation of the HDAC1 gene by anisomycin together with TSA or by growth factors is accompanied by phosphoacetylation of HDAC1 promoter-associated histone H3. Our results present evidence for a precise regulatory mechanism which allows induction of the HDAC1 gene in response to proliferation signals and modulation of HDAC1 expression dependent on intracellular deacetylase levels.

3T3 Cells↗
PubMed1993

Reconstitution of short-spaced chromatin from the histone octamer and either HMG-14,17 or histone H1.

Two new chromatin-assembly reactions are described. The first involves the addition of phosphorylated HMG-14,17 to the histone octamer plus DNA in high concentrations of salt and yields a repeating particle size or spacing of about 165 base-pairs. The second involves the addition of histone H1 to the acetylated histone octamer plus poly(glutamate) in low concentrations of salt, followed by the addition of DNA; and it yields a spacing of about 170 base-pairs. Plots of band size versus band number in gels, often used to determine nucleosome repeat-length, yield slopes of 138 base-pairs for the histone octamer alone, or 155 base-pairs with HMG-14,17 or 160 base-pairs with histone H1, and intercepts of 10,25 and 20 base-pairs, respectively, in the three cases. Attempts were made to combine the spacing activities of HMG-14,17 and histone H1 within a single assembly reaction, to provide an even longer spacing of about 190 base-pairs (as observed in cell extracts to which H1 has been added), but our present methods did not allow this. The two assembly reactions described here will be of use for structural studies of chromatin having defined length and sequence, and potentially of practical use for the regular, ordered condensation of very long DNA.

Animals↗
PubMed1997

An approximately half set of histone genes is enough for cell proliferation and a lack of several histone variants causes protein pattern changes in the DT40 chicken B cell line.

Of the 44 chicken histone genes, 39 are located in a major histone gene cluster of 110 kb, the others residing in four separate regions. The 42 sequenced genes encode six H1 variants, three H2A variants, four H2B variants, two H3 variants, and one histone H4. To clarify the influence on cell functions of simultaneous deletion of an approximately half set of the genes and some of the variants, we generated homozygous chicken DT40 mutants by disruption of two allelic segments of 57 kb, containing the 21 genes, using gene targeting techniques. Analyses with antisense RNA probes common or specific for gene families H1, H2A, H2B, H3 and H4 indicated that the remaining members of each of the gene families were expressed more in the mutants than in DT40 cells, resulting in maintenance of constant steady-state levels of mRNAs. Two-dimensional polyacrylamide gel electrophoresis showed that in the mutants several cellular proteins newly appeared or increased, and some other proteins disappeared or decreased quantitatively. These results demonstrate that all the histone gene families have the inherent ability to compensate for the disruption of a fair number of their own constituents. Furthermore, some of the histone variants are involved in regulation of the expression of putative genes that encode the proteins that varied in mutant DT40 cells, this participation is not compensated for by any residual variant of the same histone subtype(s).

Animals↗
PubMed1984

Isolation of rat testis histone TH2B-x, and interaction of TH2B-x antiserum with histones and mononucleosomes.

A method is reported for the isolation of histone TH2B-x from rat testis by affinity chromatography on an agarose-p-chloromercurianilino column. This purified TH2B-x was used to raise antibodies in the rabbit, and the antiserum was assayed by an enzyme-linked double-antibody procedure. At low concentration the antiserum cross-reacts with histone H2B and with histones TH1-x + H1 to the extent of 11-14% of the interaction with TH2B-x. Antiserum preincubated in three successive H2B-coated tubes still retains 80-89% of the original anti-TH2B-x activity when assayed subsequently in TH2B-x-coated tubes, but cross-reaction with H2B is practically zero. The anti-TH2B-x antibodies also interact with tubes coated with mononucleosomes isolated from nuclei of seminiferous epithelial cells (SEC) of rat testis, but the interaction with mononucleosomes from rat liver nuclei is almost zero. The data suggest that in nucleosomes some of the antigenic determinants which are unique to TH2B-x are accessible, while those determinants which are common to H2B and TH2B-x are not accessible for interaction with antibodies. Competition by mononucleosomes, both from rat testis SEC and rat liver (to a lesser degree), in solution is detected by the reduction of binding of enzyme-labeled IgG to TH2B-x-coated tubes. However, an attempted competition by histones TH2B-x or H2B in solution resulted in an increase in the binding of the enzyme-labeled IgG to the mononucleosome-coated tubes. The interpretation of this type of competition assay is complicated by possible interaction of added histones with the coating mononucleosomes, followed by binding of antibodies to the histones. This TH2B-x antibody should be useful in studying changes in structure and function of chromatin during spermatogenesis and in the isolation of TH2B-x mRNA.

Animals↗
PubMed1989

Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking.

The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA.

Acetylation↗
PubMed1984

Increase in histone acetylation and transitions in histone variants during Friend cell differentiation.

Histone acetylation of Murine Erythroleukemia Cells (MELC) has been re-examined. It is demonstrated that sodium butyrate causes hyperacetylation of core histones in inducible as well as non-inducible MELC strains. This indicates that histone hyperacetylation per se is not sufficient to activate genes. However, [3H]acetate incorporation into core histones of the inducible MELC line F4N increases after induction of differentiation with dimethylsulfoxide (DMSO), in contrast to the non-inducible variant F4+. Thus histone acetylation may play a role as an auxiliary mechanism for gene activation (and inactivation). In addition, the appearance of a histone H3 variant during differentiation of MELC is reported.

Acetylation↗