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PubMed2002

Interplay between two epigenetic marks. DNA methylation and histone H3 lysine 9 methylation.

BACKGROUND: The heterochromatin of many eukaryotes is marked by both DNA methylation and histone H3 lysine 9 (H3-K9) methylation, though the exact relationship between these epigenetic modifications is unknown. In Neurospora, H3-K9 methylation is required for the maintenance of all known DNA methylation. In Arabidopsis, H3-K9 methylation directs some of the CpNpG and asymmetric methylation. However, it is not known in any organism whether DNA methylation may also direct histone H3 methylation. RESULTS: Using chromatin immunoprecipitation (ChIP) assays, we show that Arabidopsis heterochromatin is associated with H3-K9 methylation. This histone methylation is dependent on the KRYPTONITE and DDM1 genes (SU[VAR]3-9 and SWI2/SNF2 homologs, respectively). We also find that a decrease in DNA methylation does not directly cause a loss of H3-K9 methylation. Instead, a decrease in H3-K9 methylation is only seen at loci where transcription is derepressed. CONCLUSIONS: We conclude that DNA methylation does not control the methylation of histone H3-K9. We propose that loss of H3-K9 methylation is due to transcriptional reactivation, coupled with deposition of unmethylated nucleosomes. These findings are consistent with recent observations of DNA replication-independent deposition of histone H3.3 in Drosophila. Our results also suggest that, in Arabidopsis, DNA methylation is sufficient for gene silencing, but H3-K9 methylation is not.

Arabidopsis↗

PubMed2000

Retrovirus vector silencing is de novo methylase independent and marked by a repressive histone code.

Retrovirus vectors are de novo methylated and transcriptionally silent in mammalian stem cells. Here, we identify epigenetic modifications that mark retrovirus-silenced transgenes. We show that murine stem cell virus (MSCV) and human immunodeficiency virus type 1 (HIV-1) vectors dominantly silence a linked locus control region (LCR) beta-globin reporter gene in transgenic mice. MSCV silencing blocks LCR hypersensitive site formation, and silent transgene chromatin is marked differentially by a histone code composed of abundant linker histone H1, deacetylated H3 and acetylated H4. Retrovirus-transduced embryonic stem (ES) cells are silenced predominantly 3 days post-infection, with a small subset expressing enhanced green fluorescent protein to low levels, and silencing is not relieved in de novo methylase-null [dnmt3a-/-;dnmt3b-/-] ES cells. MSCV and HIV-1 sequences also repress reporter transgene expression in Drosophila, demonstrating establishment of silencing in the absence of de novo and maintenance methylases. These findings provide mechanistic insight into a conserved gene silencing mechanism that is de novo methylase independent and that epigenetically marks retrovirus chromatin with a repressive histone code.

Animals↗

PubMed1997

Different strategies of X-inactivation in germinal and somatic cells: histone H4 underacetylation does not mark the inactive X chromosome in the mouse male germline.

It has previously been shown by immunocytochemistry that the inactive X chromosome (Xi) in somatic cells of human and mouse females is marked by underacetylation of histone H4. It has been suggested that this may be important for transcriptional silencing of genes on Xi. We have now investigated X-inactivation in meiotic cells of the male germline. In these cells the single X chromosome is transcriptionally inactive and expresses XIST, a gene that in somatic cells is transcribed only from Xi. By immunostaining with antibodies to H4 acetylated at lysines 5, 8, 12, or 16, we demonstrate that histone H4 on the male X is not underacetylated. We conclude that there is a differential germline strategy for maintenance of X-inactivation and that H4 underacetylation, though associated with the long-term marking of inactive X chromosomes in the female soma, is not always essential for the transcriptional down-regulation of X-linked genes.

Acetylation↗

PubMed2003

Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa.

Besides serving to package nuclear DNA, histones carry information in the form of a diverse array of post-translational modifications. Methylation of histones H3 and H4 has been implicated in long-term epigenetic 'memory'. Dimethylation or trimethylation of Lys4 of histone H3 (H3 Lys4) has been found in expressible euchromatin of yeasts and mammals. In contrast, methylation of Lys9 of histone H3 (H3 Lys9) has been implicated in establishing and maintaining the largely quiescent heterochromatin of mammals, yeasts, Drosophila melanogaster and plants. We have previously shown that a DNA methylation mutant of Neurospora crassa, dim-5 (defective in methylation), has a nonsense mutation in the SET domain of an H3-specific histone methyltransferase and that substitutions of H3 Lys9 cause gross hypomethylation of DNA. Similarly, the KRYPTONITE histone methyltransferase is required for full DNA methylation in Arabidopsis thaliana. We used biochemical, genetic and immunological methods to investigate the specific mark for DNA methylation in N. crassa. Here we show that trimethylated H3 Lys9, but not dimethylated H3 Lys9, marks chromatin regions for cytosine methylation and that DIM-5 specifically creates this mark.

DNA Methylation↗

PubMed2006

Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer.

Many genes associated with CpG islands undergo de novo methylation in cancer. Studies have suggested that the pattern of this modification may be partially determined by an instructive mechanism that recognizes specifically marked regions of the genome. Using chromatin immunoprecipitation analysis, here we show that genes methylated in cancer cells are specifically packaged with nucleosomes containing histone H3 trimethylated on Lys27. This chromatin mark is established on these unmethylated CpG island genes early in development and then maintained in differentiated cell types by the presence of an EZH2-containing Polycomb complex. In cancer cells, as opposed to normal cells, the presence of this complex brings about the recruitment of DNA methyl transferases, leading to de novo methylation. These results suggest that tumor-specific targeting of de novo methylation is pre-programmed by an established epigenetic system that normally has a role in marking embryonic genes for repression.

Caco-2 Cells↗

PubMedDate not supplied

[Effect of thyroxine on the protein makeup of condensed and diffuse chromatin from the liver of rats of different ages].

Contant composition of histones and marked alterations in the non-histone proteins were found in condensed and diffuse chtomatins of rat liver tissue during postnatal ontogenesis both in normal state and in treatment with thyroxine. Thyroxine affected the ratio histones (non-histone proteins in condensed chromatin of animals of the all age groups studied, except the old rats, in which the ration did not differ from the control values. After thyroxine administration the ratio histones) non-histone proteins was increased in diffuse chromatin of predolescent rats; the ratio was slightly decreased in diffuse chromatin of puberal rats and it was remarkably unaltered in the chromatin of adult and t was remarkably unaltered in the chromatin of adult an old animals. Considering the fact that thyroxine increased in the condensed chromatin the content of non-histone proteins and decreased the content of histones, the data obtained suggest that the hormone caused a shift in the ratio condensed/diffuse chromatin towards the increase in content of the diffuse chromatin.

Aging↗

PubMed2005

Monomethyl histone H3 lysine 4 as an epigenetic mark for silenced euchromatin in Chlamydomonas.

Histone Lys methylation plays an important role in determining chromatin states and is mostly catalyzed by SET domain-containing proteins. The outcome, transcriptional repression or activation, depends on the methylated histone residue, the degree of methylation, and the chromatin context. Dimethylation or trimethylation of histone H3 Lys 4 (H3K4me2 or H3K4me3) has been correlated with transcriptionally competent/active genes. However, H3K4 methylation has also been implicated in gene silencing. This dualistic nature of the H3K4 methyl mark has thus far remained unresolved. In the green alga Chlamydomonas reinhardtii, Mut11p, related to a subunit of trithorax-like methyltransferase complexes, is required for transcriptional silencing. Here, we show that Mut11p interacts with conserved components of H3K4 methyltransferase machineries, and an affinity-purified Mut11p complex(es) methylates histones H3, H2A, and H4. Moreover, a Mut11 mutant showed global loss of monomethylated H3K4 (H3K4me1) and an increase in dimethylated H3K4. By chromatin immunoprecipitation analysis, this strain also displayed substantial reduction in H3K4me1 and enrichment in H3K4me2 associated with transcriptionally derepressed genes, transgenes, and retrotransposons. RNA interference-mediated suppression of Set1, encoding an H3K4 methyltransferase, induced similar phenotypes, but of lower magnitude, and no detectable increase in H3K4me2. Together, our results suggest functional differentiation between dimethyl H3K4 and monomethyl H3K4, with the latter operating as an epigenetic mark for repressed euchromatin.

Algal Proteins↗

PubMedDate not supplied

[DNA and nucleotide triphosphate-activated proteinase from rat liver nuclear matrix specific for H1 histone].

The action of DNA and nucleotide phosphate on histone hydrolysis by nuclear matrix preparations from rat liver has been studied. It is shown that proteinase specific for H1 histone is associated with the nuclear matrix. This proteinase is activated by denatured DNA and by DNA treatment with DNase I or gamma-irradiation, but it is not activated by UV-irradiated DNA. In the presence of nucleotide triphosphates, particularly GTP and ATP, proteolysis of H1 histone is markedly increased. The nuclear matrix proteinase specific for H1 histone and activated by DNA or GTP and ATP appears inhibited by antipain, leupeptin, phenylmethylsulfonyl fluoride (the inhibitors of serine proteinases) as well as by dithiotreitol.

Adenosine Triphosphate↗

PubMed2006

Myc-binding-site recognition in the human genome is determined by chromatin context.

Large-scale chromatin immunoprecipitation (ChIP) studies have been effective in unravelling the distribution of DNA-binding transcription factors along eukaryotic genomes, but specificity determinants remain elusive. Gene-regulatory regions display distinct histone variants and modifications (or marks). An attractive hypothesis is that these marks modulate protein recognition, but whether or not this applies to transcription factors remains unknown. Based on large-scale datasets and quantitative ChIP, we dissect the correlations between 35 histone marks and genomic binding by the transcription factor Myc. Our data reveal a relatively simple combinatorial organization of histone marks in human cells, with a few main groups of marks clustering on distinct promoter populations. A stretch of chromatin bearing high H3 K4/K79 methylation and H3 acetylation (or 'euchromatic island'), which is generally associated with a pre-engaged basal transcription machinery, is a strict pre-requisite for recognition of any target site by Myc (whether the consensus CACGTG or an alternative sequence). These data imply that tethering of a transcription factor to restricted chromatin domains is rate-limiting for sequence-specific DNA binding in vivo.

Acetylation↗

PubMed2005

Cell-type-specific epigenetic marking of the IL2 gene at a distal cis-regulatory region in competent, nontranscribing T-cells.

T-cells retain cell-type-specific programming for IL-2 inducibility through many rounds of division without being stimulated to transcribe the locus. To understand the layering of controls needed to poise this gene heritably for activation, we have used chromatin immunoprecipitation to map histone modifications across the murine IL2 locus, from -10.2 through +0.25 kb, in induction-competent and incompetent cells. In highly inducible EL4 T-lineage cells, stimulation with PMA/A23187 induced strong acetylation of histone H3 and H4, in parallel with transcriptional activation, from -4.6 through +0.25 kb. However, dimethylation of histone H3/K4 was already fully elevated across the same restricted domain before stimulation, with little change after stimulation. RNA polymerase II binding, in contrast, was only found at the known promoter region after stimulation. Similar patterns of histone modifications were seen also in normal IL-2-inducible T-lineage cells. However, neither acetylated histone H3, H4 nor dimethylated histone H3/K4 marking was detected, with or without stimulation, in expression-incompetent cells (NIH/3T3 or Scid.adh). These results identify a discrete new domain of IL2 regulatory sequence marked by dimethylated histone H3/K4 in expression-permissive T-cells even when they are not transcribing IL2, setting boundaries for histone H3 and H4 acetylation when the IL2 gene is transcriptionally activated.

5' Flanking Region↗

PubMed1981

Isolation of actively transcribed nucleosomes using immobilized HMG 14 and 17 and an analysis of alpha-globin chromatin.

Taking advantage of the known specificity of interaction of HMG 14 and 17 with actively transcribed chromatin, we have covalently cross-linked these proteins to agarose and used this HMG 14-17 column to purify active nucleosomes. The column has been used to map the DNA regions surrounding the chicken alpha-globin genes for their capacity to bind HMG 14-17. The results show that at a crude level the HMG binding region correlates with the major stable primary transcript and the region of DNAase I sensitivity. At a finer level of analysis, however, the correspondence between DNAase I sensitivity and HMG binding is preserved, yet this chromosomal domain is shown to extend about 1 kb beyond the known primary transcript for the two chicken alpha genes. Why HMG 14 and 17 bind specifically to active nucleosomes is still not known. Our experiments suggest that at least one additional chemical difference distinguishes them. This difference is not reflected in the DNA:protein ratio; in the electrophoretic mobility of the particles or associated DNA; in the inner histone stoichiometry; in a marked degree of histone modification as assayed on Triton-urea gels; or in the presence of nonhistone proteins. Active nucleosomes can be dissociated and reconstituted and their ability to bind to HMG 14-17 restored. The ability to reconstitute binding should make it possible to determine whether the specificity for the interaction resides in the protein or the DNA.

Animals↗

PubMed1979

Evidences of two different sets of histone genes active during embryogenesis of the sea urchin Paracentrotus lividus.

Histone mRNAs at different stages of development were purified by hybridization with the cloned homologous histone genes. The electrophoretic patterns of oocytes, 2-4 blastomeres, 64 cells and morula histone mRNAs was found to be identical, whereas the electrophoretic pattern of mesenchyme blastula histone mRNA was markedly different. The cloned histone DNA of P.lividus was hybridized with the RNA of each stage. The Tm was 74 degrees C in all cases except for the mesenchyme histone mRNAs whose Tm was 59 degrees C, thus suggesting that at least two different clusters of histone genes are active in the course of the sea urchin development.

Animals↗

PubMed2002

Methylation of alpha-type embryonic globin gene alpha pi represses transcription in primary erythroid cells.

The inverse relationship between expression and methylation of beta-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian alpha-type globin genes. The embryonic alpha(pi)-globin promoter was unmethylated, and alpha(pi)-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the alpha(pi) promoter associated with loss of expression of alpha(pi)-globin gene was seen during development in primary erythroid cells. A 315-bp alpha(pi)-globin promoter region was cloned in an expression construct (alpha(pi)pGL3E) containing a luciferase reporter gene and SV40 enhancer. The alpha(pi)pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of alpha(pi)pGL3E plasmid and alpha(pi)-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp alpha(pi)-globin gene promoter fragment formed a methyl cytosine-binding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the alpha(pi)-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian alpha-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.

Acetylation↗

PubMed1981

Characterisation of a chromatin fraction bearing pulse-labelled RNA. 2. Quantification of histones and high-mobility-group proteins.

The histone variants and high-mobility-group (HMG) proteins of a transcribing fraction of chromatin, described in the preceding paper of this journal, have been analysed qualitatively and quantitatively by a combination of one-dimensional and two-dimensional gel electrophoresis. The stoichiometry of the four core histones (all variants included) in this fraction is equimolar and is not detectably different from that in the nontranscribing fraction or in total chromatin. The molar ratio of histone H1 to the core histones is markedly lower, by approximately 72%, than that in the nontranscribing fraction. A minor histone variant identified as M1 (an H2A variant) is detected only in the transcribing fraction, while variant H3.1 is found only in the non-transcribing fraction. Proteins A24, HMG1 and HMG2 are essentially absent from the transcribing fraction; HMG14 is found uniquely in this fraction, while HMG17 occurs at a relatively lower level.

Animals↗

PubMed2006

Free and chromatin-associated mono-, di-, and trimethylation of histone H4-lysine 20 during development and cell cycle progression.

Methylation of specific amino acids in histone tails is responsible for packaging DNA into condensed, repressed chromatin, and into open chromatin that is accessible to the transcription machinery. Monomethylation and trimethylation of histone H4-lysine 20 (H4-K20) control the formation of repressed chromatin. Using antibodies that specifically recognize the three methyl marks of histone H4-K20, we characterized their regulation during the cell cycle and throughout development. We find free mono- and trimethylated histone H4-K20 in unfertilized Drosophila eggs and in S2 tissue culture cells. Soluble mono-. di-, and trimethylated H4-K20 are also found in HeLa cells. These soluble modified histones may represent a pool of free histones that can rapidly be incorporated into chromatin. The three methyl marks are each regulated differentially during development and their detection on western blots does not overlap with their detection on chromosomes. Monomethylated H4-K20 is detected on condensed chromosomes throughout development, while di- and trimethylated H4-K20 are detected on metaphase chromosomes at specific stages. Our results suggest that the detection of methylated H4-K20 on chromosomes may reveal chromatin packaging rather than the distribution of the methyl marks.

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↗

PubMed1994

Effects of halothane and propofol on purified brain protein kinase C activation.

BACKGROUND: Protein kinase C (PKC) has been implicated as a target for general anesthetic action in the central nervous system. Previous reports have described either stimulation or inhibition of PKC activity by general anesthetics. This study examines the effects of halothane and propofol on the activity of purified rat brain PKC under various assay conditions. METHODS: PKC was assayed in vitro using three previously characterized artificial substrates and three different lipid preparations in the absence or presence of halothane or propofol. RESULTS: Both halothane (50% effective concentration = 2.2 vol%) and propofol (50% effective concentration = 240 microM) markedly stimulated histone H1 phosphorylation by PKC in the presence of a lipid vesicle preparation consisting of phosphatidylcholine, phosphatidylserine, and diacylglycerol. Less marked or no stimulation of PKC by both anesthetics was observed in the presence of a phosphatidylserine/diacylglycerol dispersion or using protamine or poly(lysine, serine) as substrate. Neither anesthetic significantly stimulated PKC activity in the presence of phosphatidylserine/diacylglycerol/Triton X-100 mixed micelles using histone H1, protamine or poly(lysine, serine) as substrate. Slight inhibition of PKC activity by halothane was observed under specific assay conditions with protamine as substrate. The activity of the catalytic fragment of PKC or of two lipid-independent second messenger-regulated protein kinases with conserved catalytic domains was not significantly affected by halothane. CONCLUSIONS: Both halothane and propofol stimulated purified brain PKC activity in vitro assayed with physiologically relevant lipid bilayers in the absence or presence of Ca2+. This effect appears to be mediated through the lipid-binding regulatory domain of PKC. The potencies of halothane and propofol in stimulating PKC in vitro are consistent with submaximal activation of PKC at clinically effective anesthetic concentrations, the pharmacologic significance of this effect requires confirmation in an intact cellular system.

Anesthesia, General↗

PubMed2006

Loss of linker histone H1 in cellular senescence.

Cellular senescence is a tumor-suppressing mechanism that is accompanied by characteristic chromatin condensation called senescence-associated heterochromatic foci (SAHFs). We found that individual SAHFs originate from individual chromosomes. SAHFs do not show alterations of posttranslational modifications of core histones that mark condensed chromatin in mitotic chromosomes, apoptotic chromatin, or transcriptionally inactive heterochromatin. Remarkably, SAHF-positive senescent cells lose linker histone H1 and exhibit increased levels of chromatin-bound high mobility group A2 (HMGA2). The expression of N-terminally enhanced green fluorescent protein (EGFP)-tagged histone H1 induces premature senescence phenotypes, including increased levels of phosphorylated p53, p21, and hypophosphorylated Rb, and a decrease in the chromatin-bound endogenous histone H1 level but not in p16 level accumulation or SAHF formation. However, the simultaneous ectopic expression of hemagglutinin-tagged HMGA2 and N-terminally EGFP-tagged histone H1 leads to significant SAHF formation (P < 0.001). It is known that histone H1 and HMG proteins compete for a common binding site, the linker DNA. These results suggest that SAHFs are a novel type of chromatin condensation involving alterations in linker DNA-binding proteins.

Bromodeoxyuridine↗