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At least 1,621 records · Page 90Linked to original sources
PubMed2002

Histone H3 lysine 9 methylation occurs rapidly at the onset of random X chromosome inactivation.

In female mammals, a single X chromosome is stably and heritably silenced early in embryogenesis. The inactive X is characterized by asynchronous DNA replication and epigenetic chromatin modifications, including DNA methylation, histone H3/H4 hypoacetylation, and incorporation of a variant histone macroH2A. X inactivation is initiated by a cis-acting RNA molecule, the X-inactive specific transcript (Xist), which coats the chromosome. However, the mechanism by which Xist induces chromosome silencing is poorly understood. An important approach towards answering this question has been to determine the temporal order of epigenetic chromatin modifications in an in vitro model system, differentiating XX embryonic stem (ES) cells, and thereby to identify candidate targets for Xist RNA. To date, these studies have demonstrated that, following accumulation of Xist RNA, the transition to late replication of the X chromosome is the earliest detectable event. H4 hypoacetylation, macroH2A1.2 incorporation, and DNA methylation all occur subsequently. Recently, it has been shown that chromatin of the inactive X is also characterized by methylation of histone H3 at lysine 9 (H3-K9). Here we show that H3-K9 methylation is a very early event in the process of X inactivation, which closely parallels the onset of Xist RNA accumulation.

Cell Differentiation↗
PubMedDate not supplied

Application of high-performance capillary electrophoresis to the analysis of H1 histones.

High-performance capillary electrophoresis for the separation of rat testis H1 histone variants and their phosphorylated modifications is described. The influence of buffer pH, hydroxypropylmethyl cellulose, and buffer concentration has been investigated. Under optimized conditions (500 mM phosphate buffer, pH 2, 0.03% hydroxypropylmethyl cellulose) using an uncoated capillary, eight H1 histone subfractions, including two H1(0) histones and H1t and their phosphorylated modifications, are resolved. Application of capillary electrophoresis to the separation of H1 histones provides an important new alternative to high-performance liquid chromatography (HPLC) and traditional gel electrophoresis.

Buffers↗
PubMed2003

Herpes simplex virus 1 gene expression is accelerated by inhibitors of histone deacetylases in rabbit skin cells infected with a mutant carrying a cDNA copy of the infected-cell protein no. 0.

An earlier report showed that the expression of viral genes by a herpes simplex virus 1 mutant [HSV-1(vCPc0)] in which the wild-type, spliced gene encoding infected-cell protein no. 0 (ICP0) was replaced by a cDNA copy is dependent on both the cell type and multiplicity of infection. At low multiplicities of infection, viral gene expression in rabbit skin cells was delayed by many hours, although ultimately virus yield was comparable to that of the wild-type virus. This defect was rescued by replacement of the cDNA copy with the wild-type gene. To test the hypothesis that the delay reflected a dysfunction of ICP0 in altering the structure of host protein-viral DNA complexes, we examined the state of histone deacetylases (HDACs) (HDAC1, HDAC2, and HDAC3). We report the following. (i) HDAC1 and HDAC2, but not HDAC3, were modified in infected cells. The modification was mediated by the viral protein kinase U(S)3 and occurred between 3 and 6 h after infection with wild-type virus but was delayed in rabbit skin cells infected with HSV-1(vCPc0) mutant, concordant with a delay in the expression of viral genes. (ii) Pretreatment of rabbit skin cells with inhibitors of HDAC activity (e.g., sodium butyrate, Helminthosporium carbonum toxin, or trichostatin A) accelerated the expression of HSV-1(vCPc0) but not that of wild-type virus. We conclude the following. (i) In the interval in which HSV-1(vCPc0) DNA is silent, its DNA is in chromatin-like structures amenable to modification by inhibitors of histone deacetylases. (ii) Expression of wild-type virus genes in these cells precluded the formation of DNA-protein structures that would be affected by either the HDACs or their inhibitors. (iii) Since the defect in HSV-1(vCPc0) maps to ICP0, the results suggest that this protein initiates the process of divestiture of viral DNA from tight chromatin structures but could be replaced by other viral proteins in cells infected with a large number of virions.

Animals↗
PubMed2005

Do protein motifs read the histone code?

The existence of different patterns of chemical modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) of the histone tails led, some years ago, to the histone code hypothesis. According to this hypothesis, these modifications would provide binding sites for proteins that can change the chromatin state to either active or repressed. Interestingly, some protein domains present in histone-modifying enzymes are known to interact with these covalent marks in the histone tails. This was first shown for the bromodomain, which was found to interact selectively with acetylated lysines at the histone tails. More recently, it has been described that the chromodomain can be targeted to methylation marks in histone N-terminal domains. Finally, the interaction between the SANT domain and histones is also well documented. Overall, experimental evidence suggests that these domains could be involved in the recruitment of histone-modifying enzymes to discrete chromosomal locations, and/or in the regulation their enzymatic activity. Within this context, we review the distribution of bromodomains, chromodomains and SANT domains among chromatin-modifying enzymes and discuss how they can contribute to the translation of the histone code.

Adenosine Triphosphate↗
PubMed2005

K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase.

Histones are modified post-translationally, e.g. by methylation of lysine and arginine residues, and by phosphorylation of serine residues. These modifications regulate processes such as gene expression, DNA repair, and mitosis and meiosis. Recently, evidence has been provided that histones are also modified by covalent binding of the vitamin biotin. The aims of this study were to identify biotinylation sites in histone H3, and to investigate the crosstalk among histone biotinylation, methylation and phosphorylation. Synthetic peptides based on the sequence of human histone H3 were used as substrates for enzymatic biotinylation by biotinidase; biotin in peptides was probed using streptavidin peroxidase. These studies provided evidence that K4, K9 and K18 in histone H3 are good targets for biotinylation; K14 and K23 are relatively poor targets. Antibodies were generated to histone H3, biotinylated either at K4, K9 or K18. These antibodies localized to nuclei in human placental cells in immunocytochemistry and immunoblotting experiments, suggesting that lysines in histone H3 are biotinylated in vivo. Dimethylation of R2, R8 and R17 increased biotinylation of K4, K9 and K18, respectively, by biotinidase; phosphorylation of S10 abolished biotinylation of K9. These observations are consistent with crosstalk between biotinylation of histones and other known modifications of histones. We speculate that this crosstalk provides a link to known roles for biotin in gene expression and cell proliferation.

Amino Acid Sequence↗
PubMed2006

The RNA polymerase II kinase Ctk1 regulates positioning of a 5' histone methylation boundary along genes.

In yeast and other eukaryotes, the histone methyltransferase Set1 mediates methylation of lysine 4 on histone H3 (H3K4me). This modification marks the 5' end of transcribed genes in a 5'-to-3' tri- to di- to monomethyl gradient and promotes association of chromatin-remodeling and histone-modifying enzymes. Here we show that Ctk1, the serine 2 C-terminal domain (CTD) kinase for RNA polymerase II (RNAP II), regulates H3K4 methylation. We found that CTK1 deletion nearly abolished H3K4 monomethylation yet caused a significant increase in H3K4 di- and trimethylation. Both in individual genes and genome-wide, loss of CTK1 disrupted the H3K4 methylation patterns normally observed. H3K4me2 and H3K4me3 spread 3' into the bodies of genes, while H3K4 monomethylation was diminished. These effects were dependent on the catalytic activity of Ctk1 but are independent of Set2-mediated H3K36 methylation. Furthermore, these effects are not due to spurious transcription initiation in the bodies of genes, to changes in RNAP II occupancy, to changes in serine 5 CTD phosphorylation patterns, or to "transcriptional stress." These data show that Ctk1 acts to restrict the spread of H3K4 methylation through a mechanism that is independent of a general transcription defect. The evidence presented suggests that Ctk1 controls the maintenance of suppressive chromatin in the coding regions of genes by both promoting H3K36 methylation, which leads to histone deacetylation, and preventing the 3' spread of H3K4 trimethylation, a mark associated with transcriptional initiation.

5' Flanking Region↗
PubMed1981

Histone acetylase from Drosophila melanogaster specific for H4.

Histone acetylation is a rapid and reversible modification which introduces significant changes in histone-DNA interactions. Such changes have been correlated with different states of DNA transcription and replication in the cell. We have purified a histone acetylase about 1200-fold from extracts of Drosophila melanogaster embryos. Major steps in the purification include chromatography on histone-Sepharose and Bio-Rex 70. This enzyme, the only histone acetylase detected in these extracts, acetylates only histone H4. All of the acetate groups are introduced within the NH2-terminal amino acids 4 to 17. This 14-residue peptide contains the four lysines which are acetylated in vivo. The acetylase is inhibited by its substrate, histone H4, and by several highly charged polymers including polylysine, polyarginine, DNA, RNA, and polyglutamic acid. It is not inhibited by polyethyleneimine, spermine, or the other histones H2A, H2B, H3, or H1. The enzyme does not acetylate H4 which is in chromatin. This enzyme is most likely involved in the acetylation of newly synthesized histones in the cytoplasm prior to chromatin assembly.

Acetyltransferases↗
PubMed1981

Multiple structural features are responsible for the nuclease sensitivity of the active ovalbumin gene.

The ovalbumin gene in chick oviduct nuclei or nucleosomes is digested preferentially by either DNase I or staphylococcal nuclease. Staphylococcal nuclease preferentially cuts between and within core particles of the oviduct ovalbumin gene; thus, the ovalbumin gene is more quickly degraded to mononucleosomes and the DNA within these monomers is digested to a nonhybridizable size significantly faster than the chicken globin gene. Mono- and oligonucleosomes generated by partial staphylococcal nuclease digestion at 0 degrees C, but not at 37 degrees C, retain equal sensitivity to DNase I. Most of this sensitivity persists when histone H1 and most of the non-histone chromosomal proteins are removed with 0.6 M NaCl. On the basis of these observations, we propose that nuclease sensitivity of the oviduct ovalbumin gene is due to covalent modifications of the core histones and that this sensitivity is amplified by interaction of other chromosomal proteins with these modified histones.

Animals↗
PubMed2006

Chromatin assembly factor 1 interacts with histone H3 methylated at lysine 79 in the processes of epigenetic silencing and DNA repair.

In eukaryotic cells, chromatin is classified into euchromatin, which is active in transcription, and heterochromatin that silences transcription. Histones in these two domains contain distinct modifications. Chromatin assembly factor 1 (CAF-1) is a highly conserved protein that functions in DNA replication, DNA repair, and heterochromatin silencing. CAF-1 binds histones H3 and H4 and deposits histones onto DNA to form nucleosomes. However, modifications on H3 and H4 associated with CAF-1 are not known. Here, we have purified a complex containing CAF-1 and H3 and H4 from yeast cells and determined the modifications present on these histones using linear ion trap FT-ICR mass spectrometry. H4 that copurified with CAF-1 was a mixture of isoforms acetylated at lysines 5, 8, 12, and 16, whereas an H3 peptide methylated at lysine 79 and an H3 peptide acetylated at lysine 56 were detected. In yeast cell extracts, these two H3 modifications peaked in the late S phase with different kinetics. Moreover, the association of CAF-1 with H3 methylated at lysine 79 appeared to occur in the late S phase. Finally, cells lacking both Dot1p, the methyltransferase that methylates H3 lysine 79, and Cac1p, the large subunit of CAF-1, exhibited a dramatic loss of telomeric silencing and increased sensitivity to DNA damaging agents. Together, these data indicate that CAF-1 interacts with H3 methylated at lysine 79 during the processes of epigenetic silencing and DNA repair.

Amino Acid Sequence↗
PubMed2001

Modification of octamer binding transcriptional factor is related to H2B histone gene repression during dimethyl sulfoxide-dependent differentiation of HL-60 cells.

Transcriptional regulation of H2B histone gene during dimethyl sulfoxide (DMSO)-dependent differentiation of HL-60 cells has been investigated using DNase I footprinting and DNA mobility shift assay. The level of histone H2B mRNA showed a slight decline at 2 days and hardly detectable at 4 days after DMSO treatment. H2B histone mRNA was repressed in proportion to the concentration of DMSO. In DNase I footprinting analysis, one nuclear factor (octamer binding transcription factor, OTF) bound at -42 bp (octamer motif, ATTTGCAT) in undifferentiated HL-60 cells. The binding pattern of OTF was unchanged during DMSO-dependent differentiation. One protein complex (OTF) was detected by DNA mobility shift assay in undifferentiated HL-60 cells. The mobility of OTF was partially retarded during DMSO-dependent differentiation and the retardant OTF was not newly synthesized protein. These results suggest that the posttranslational modification of OTF may be responsible for the repression of H2B histone gene during DMSO-dependent differentiation of HL-60 cells.

Binding Sites↗
PubMed1986

Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction.

Using S1 nuclease protection assays, we have examined the representation of cell cycle-dependent H4 histone RNAs in the nuclear matrix and nonmatrix nuclear fractions of human cells. Cytoplasmic and nuclear fractions were prepared from exponentially growing HeLa S3 cells by double detergent (sodium deoxycholate and NP40) lysis. The nuclear matrix and nonmatrix nuclear fractions were then prepared by digestion of nuclei with RNase-free DNase I and subsequent high-salt [0.4 M (NH4)2SO4] extraction. Subcellular fractions were characterized by 1) DNA, RNA, and protein composition; 2) electrophoretic analysis of the proteins in each fraction; 3) the representation of 45S ribosomal RNA precursors and processed 18S and 28S ribosomal RNAs; and 4) the presence of mitochondrial RNAs. In contrast to ribosomal and messenger RNA precursors, which are largely associated with the nuclear matrix, the human H4 histone RNAs in the nucleus were found predominantly in the nonmatrix nuclear fraction. The presence of H4 histone RNA in the nonmatrix nuclear fraction appeared to be coupled to DNA replication, since inhibition of DNA synthesis by hydroxyurea resulted in a loss of histone RNA from the nucleus. Our results suggest either that the association of histone RNAs with the nuclear matrix is very transient or that posttranscriptional modifications of the rapidly processed histone gene transcripts do not involve the nuclear matrix.

Cell Cycle↗
PubMed2005

Sum1p, the origin recognition complex, and the spreading of a promoter-specific repressor in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Sum1p is a promoter-specific repressor. A single amino acid change generates the mutant Sum1-1p, which causes regional silencing at new loci where wild-type Sum1p does not act. Thus, Sum1-1p is a model for understanding how the spreading of repressive chromatin is regulated. When wild-type Sum1p was targeted to a locus where mutant Sum1-1p spreads, wild-type Sum1p did not spread as efficiently as mutant Sum1-1p did, despite being in the same genomic context. Thus, the SUM1-1 mutation altered the ability of the protein to spread. The spreading of Sum1-1p required both an enzymatically active deacetylase, Hst1p, and the N-terminal tail of histone H4, consistent with the spreading of Sum1-1p involving sequential modification of and binding to histone tails, as observed for other silencing proteins. Furthermore, deletion of the N-terminal tail of H4 caused Sum1-1p to return to loci where wild-type Sum1p acts, consistent with the SUM1-1 mutation increasing the affinity of the protein for H4 tails. These results imply that the spreading of repressive chromatin proteins is regulated by their affinities for histone tails. Finally, this study uncovered a functional connection between wild-type Sum1p and the origin recognition complex, and this relationship also contributes to mutant Sum1-1p localization.

Binding Sites↗
PubMed1989

The assembly of regularly spaced nucleosomes in the Xenopus oocyte S-150 extract is accompanied by deacetylation of histone H4.

Histone proteins, which were assembled into chromatin using the Xenopus oocyte S-150 extract, were analyzed on acid-urea gels and Triton-acid-urea gels to determine their state of modification. We find that histone H4, which is present in a diacetylated form in the oocyte S-150, gradually loses its acetate groups as the DNA is packaged into chromatin. Thus, this process parallels the one observed in vivo during chromatin formation in growing eucaryotic cells. Histone H4 deacetylation in the oocyte S-150 is a DNA-dependent reaction. This reaction is blocked when butyrate (an inhibitor of histone deacetylase) is added at the onset of the chromatin assembly process. When butyrate is added at the end of the assembly process, no de novo acetylation of the nucleosomal histone H4 is observed. Chromatin with regularly spaced nucleosomes, displaying periodicities ranging from 160 to 220 base pairs, can be assembled in vitro with the oocyte S-150 (Rodríguez-Campos, A., Shimamura, A., and Worcel, A. (1989) J. Mol. Biol., in press). This chromatin may contain either deacetylated histone H4 when assembled under standard conditions or diacetylated H4 when assembled in the presence of butyrate. Both types of chromatin display identical structures upon digestion with nucleases. The potential applications of this system toward the study of the naturally occurring diacetylated histone H4 are discussed.

Acetylation↗
PubMed1993

Dynamic methylation of alfalfa histone H3.

Dynamic lysine methylation of histone H3 in alfalfa tissue culture cells was studied by labeling with tritiated methionine, purification of variants H3.1 and H3.2 by reversed-phase high pressure liquid chromatography and amino acid analysis. Mono- and dimethyl-L-lysine were the major labeled amino acids. Within 100 h of continued growth conversion from N epsilon-monomethyl-L-lysine (MML) to N epsilon-dimethyl-L-lysine (DML) and N epsilon-trimethyl-L-lysine (TML) was observed, consistent with steady-state histone methylation. During the same time 20% of the methylation label was lost from major variant H3.1 protein and more than 50% from the more highly labeled minor variant H3.2. A similar pattern of label incorporation and loss was observed during a study of histone synthesis and turnover. This conforms with the general observation in animal cells that lysine methylation is limited to newly synthesized histone. Increased methylation of the more highly acetylated forms of histone H3 protein indicates limited accessibility of chromatin for histone methylation. After loss of the labile fraction of newly synthesized H3 variants, stably methylated proteins with 30% of the label in MML, 40% in DML, and 25% in TML remain. Turnover of methyl modification groups independent of histone turnover was not detected.

Cells, Cultured↗
PubMed2004

A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications.

We have modified the yeast two-hybrid system to enable the detection of protein-protein interactions that require a specific post-translational modification, using the acetylation of histones and the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II as test modifications. In this tethered catalysis assay, constitutive modification of the protein to be screened for interactions is achieved by fusing it to its cognate modifying enzyme, with the physical linkage resulting in efficient catalysis. This catalysis maintains substrate modification even in the presence of antagonizing enzyme activities. A catalytically inactive mutant of the enzyme is fused to the substrate as a control such that the modification does not occur; this construct enables the rapid identification of modification-independent interactions. We identified proteins with links to chromatin functions that interact with acetylated histones, and proteins that participate in RNA polymerase II functions and in CTD phosphorylation regulation that interact preferentially with the phosphorylated CTD.

Acetylation↗
PubMed2000

Phosphorylation of histone H3 correlates with transcriptionally active loci.

Posttranslational modifications of the N-terminal tails of the core histones within the nucleosome particle are thought to act as signals from the chromatin to the cell for various processes. The experiments presented here show that the acetylation of histones H3 and H4 in polytene chromosomes does not change during heat shock. In contrast, the global level of phosphorylated H3 decreased dramatically during a heat shock, with an observed increase in H3 phosphorylation at the heat shock loci. Additional experiments confirm that this change in phosphorylated H3 distribution is dependent on functional heat shock transcription factor activity. These experiments suggest that H3 phosphorylation has an important role in the induction of transcription during the heat shock response.

Acetylation↗
PubMed2002

Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain.

The N-terminal tails of core histones are subjected to multiple covalent modifications, including acetylation, methylation, and phosphorylation. Similar to acetylation, histone methylation has emerged as an important player in regulating chromatin dynamics and gene activity. Histone methylation occurs on arginine and lysine residues and is catalyzed by two families of proteins, the protein arginine methyltransferase family and the SET-domain-containing methyltransferase family. Here, we report that lysine 79 (K79) of H3, located in the globular domain, can be methylated. K79 methylation occurs in a variety of organisms ranging from yeast to human. In budding yeast, K79 methylation is mediated by the silencing protein DOT1. Consistent with conservation of K79 methylation, DOT1 homologs can be found in a variety of eukaryotic organisms. We identified a human DOT1-like (DOT1L) protein and demonstrated that this protein possesses intrinsic H3-K79-specific histone methyltransferase (HMTase) activity in vitro and in vivo. Furthermore, we found that K79 methylation level is regulated throughout the cell cycle. Thus, our studies reveal a new methylation site and define a novel family of histone lysine methyltransferase.

Amino Acid Sequence↗