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PubMed2005

Histone H4 lysine 91 acetylation a core domain modification associated with chromatin assembly.

The acetylation of the NH2-terminal tail of histone H4 by type B histone acetyltransferases (HATs) is involved in the process of chromatin assembly. Histone H4 associated with a nuclear type B HAT complex contains modifications in its globular core domain as well. In particular, acetylation was found at lysine 91. A mutation that alters this residue, which lies in the interface between histone H3/H4 tetramers and H2A/H2B dimers, confers phenotypes consistent with defects in chromatin assembly such as sensitivity to DNA damaging agents and derepression and alteration of silent chromatin structure. In addition, this mutation destabilizes the histone octamer, leading to defects in chromatin structure. These results indicate an important role for histone modifications outside the NH2-tail domains in the processes of chromatin assembly, DNA repair, and transcriptional silencing.

Acetylation↗
PubMed2001

The biological functions of the methyl-CpG-binding protein MeCP2 and its implication in Rett syndrome.

Methylation of DNA is essential for development in the mouse and plays an important role in inactivation of the X-chromosome, genomic imprinting and gene silencing. The properties of the methyl-CpG binding proteins (MeCPs) are being proved to be the key to interpreting the connection between DNA methylation and transcriptional repression. The founder member of the family, MeCP2, consists of a single polypeptide that contains both a methyl-CpG binding domain (MBD) and transcriptional repression domain (TRD). MBD binds to a single symmetrically methylated CpG site and is responsible for chromatin localization of the protein. NMR studies have revealed that the MBD adopts a wedge-shaped molecular structure. The TRD interacts with Sin3, which is known to form complexes with histone deacetylases. MeCP2-mediated transcriptional repression may involve two distinct mechanisms, one being dependent on chromatin modification by histone deacetylation and the other being chromatin independent. Mutations in MeCP2 gene cause the X-linked neurodevelopmental disease Rett syndrome. The spectrum of mutations reflects the importance of the MBD and TRD domains. We speculate that abnormal gene expression in Rett patients leads to dysfunction of the central nervous system. We propose a genetic therapeutic approach based on activation of the wild type copy of the MeCP2 gene located in the inactive X chromosome.

Animals↗
PubMed2004

Germline cyst development and imprinting in male mealybug Planococcus citri.

In the epigenetic modifications involved in the phenomenon of imprinting, which is thought to take place during gametogenesis, one of the primary roles is exerted by histone tail modifications acting on chromatin structure. What is more, in insects like mealybugs, with a lecanoid chromosome system, imprinting is strictly related to sex determination. In many diverse species gametes originate in specific, highly evolutionarily conserved structures called germline cysts. The use of staining techniques specific for fusomal components like F-actin has allowed us to describe for the first time the morphogenesis of male germline cysts in the mealybug Planococcus citri. Antibodies to anti-methylated lysine 9 of histone H3 (MeLy9-H3) and anti-heterochromatin protein 1 (HP1) were used during cyst formation to investigate the involvement of these epigenetic modifications in the phenomenon of imprinting and their possible concerted action in sex determination in P. citri. These observations indicate: (i) a specific role for F-actin in the segregation, typical of the lecanoid chromosome system, of genomes of paternal origin; (ii) that the two vital gametes originating from a given meiosis, although carrying the same genome, differ in the levels of both MeLy9-H3 and HP1, one of them being more heavily labelled by both antibodies.

Actins↗
PubMed2002

Allele-specific histone lysine methylation marks regulatory regions at imprinted mouse genes.

In different eukaryotic model systems, chromatin and gene expression are modulated by post-translational modification of histone tails. In this in vivo study, histone methylation and acetylation are investigated along the imprinted mouse genes Snrpn, Igf2r and U2af1-rs1. These imprinted genes all have a CpG-rich regulatory element at which methylation is present on the maternal allele, and originates from the female germ line. At these 'differentially methylated regions' (DMRs), histone H3 on the paternal allele has lysine-4 methylation and is acetylated. On the maternally inherited allele, in contrast, chromatin is marked by hypermethylation on lysine-9 of H3. Allele-specific patterns of lysine-4 and lysine-9 methylation are also detected at other regions of the imprinted loci. For the DMR at the U2af1-rs1 gene, we establish that the methyl-CpG-binding-domain (MBD) proteins MeCP2, MBD1 and MBD3 are associated with the maternal allele. These data support the hypothesis that MBD protein-associated histone deacetylase/chromatin-remodelling complexes are recruited to the parental allele that has methylated DNA and H3-K9 methylation, and are prevented from binding to the opposite allele by H3 lysine-4 methylation.

Animals↗
PubMed2005

Maintenance of low histone ubiquitylation by Ubp10 correlates with telomere-proximal Sir2 association and gene silencing.

Low levels of histone covalent modifications are associated with gene silencing at telomeres and other regions in the yeast S. cerevisiae. Although the histone deacetylase Sir2 maintains low acetylation, mechanisms responsible for low H2B ubiquitylation and low H3 methylation are unknown. Here, we show that the ubiquitin protease Ubp10 targets H2B for deubiquitylation, helping to localize Sir2 to the telomere. Ubp10 exhibits reciprocal Sir2-dependent preferential localization proximal to telomeres, where Ubp10 serves to maintain low H2B Lys123 ubiquitylation in this region and, through previously characterized crosstalk, maintains low H3 Lys4 and Lys79 methylation in a slightly broader region. Ubp10 is also localized to the rDNA locus, a second silenced domain, where it similarly maintains low histone methylation. We compare Ubp10 to Ubp8, the SAGA-associated H2B deubiquitylase involved in gene activation, and show that telomeric and gene-silencing functions are specific to Ubp10. Our results suggest that these H2B-deubiquitylating enzymes have distinct genomic functions.

Acetylation↗
PubMed2001

Sp1 and ETS family transcription factors regulate the mouse Mta2 gene expression.

Dynamic changes in chromatin structure through nucleosome remodelling and core histone tail acetylation play important roles in transcriptional regulation. The purification and functional characterization of a nucleosome remodelling and histone deacetylase complex, NuRD, has suggested that nucleosome remodelling and core histone tail modification are potentially linked processes. MTA2, a component of the NuRD complex, plays an important role in regulating histone deacetylase activity of the NuRD complex. Similar to the candidate metastasis associated protein MTA1, an elevated level of MTA2 correlates with cellular proliferation. To understand the regulation of Mta2 transcription, we characterized the mouse Mta2 gene and its transcriptional regulatory elements. We found that MTA2 is encoded by 18 exons that span 10 kb. Primer extension analysis identified a major transcriptional start site locates 259 base pairs upstream of the ATG translational start codon. Transient transfection studies localized its promoter, lacking a canonical TATA box, to within 60 base pairs upstream of the transcriptional start site. Gel-mobility-shift and mutagenesis studies revealed that Sp1 and ETS elements play important roles in regulating Mta2 transcription. Information concerning the regulation of the Mta2 gene expression will be useful in understanding the regulation of NuRD histone deacetylase activity, which in turn will help in our general understanding of the transcriptional repression mechanism.

3T3 Cells↗
PubMed2005

Quantification of acetylation at proximal lysine residues using isotopic labeling and tandem mass spectrometry.

With the emergence of the histone code as a key determinant in the regulation of gene expression, it has been important to develop tools that can not only identify the types and locations of myriad modifications, but also determine how the levels of these modifications change as a result of various processes in a cell. Mass spectrometry has become a method of choice for the investigation of post-translational modifications in histone proteins. Described in this article is a mass spectrometric method that is useful for direct quantification of levels of acetylation at lysines residues in close proximity to one another, as is the case for the amino terminal tail of histone H4. This method involves fragmentation of peptides into b and y ions that contain one or more sites of modification and isotopic labeling which ensures equivalent ionization and fragmentation.

Acetic Acid↗
PubMed2003

Methylation of histone H3 in euchromatin of plant chromosomes depends on basic nuclear DNA content.

Strong methylation of lysine 4 (K4) and low methylation of lysine 9 (K9) have been proposed as modifications of histone H3, typical for transcriptionally active euchromatin and the opposite for inactive heterochromatin. We have analysed the correlation between the global distribution of histone H3, methylated at either lysine 4 or lysine 9, and of microscopically detectable euchromatic or heterochromatic regions in relation to genome size for 24 plant species. Two different distribution patterns of methylated (K9)H3 (Met(K9)H3) were found that depend on genome size. For most species with small genomes (1C <500 Mbp), including Arabidopsis thaliana, strong methylation of (K9)H3 was restricted to constitutive heterochromatin. Species with larger genomes showed a uniform distribution of Met(K9)H3. Contrary to this and regardless of the genome size, methylated (K4)H3 (Met(K4)H3) was found to be enriched within the euchromatin of all species. Transcriptionally less active B chromosomes showed the same patterns as basic A chromosomes. We thus propose that large genomes with high amounts of dispersed repetitive sequences (mainly retroelements) have to silence these sequences and therefore display epigenetic modifications such as methylation of DNA and (K9)H3 also within euchromatic regions.

Cell Nucleus↗
PubMed2006

Histone demethylation by hydroxylation: chemistry in action.

Histone methylation plays an essential role in epigenetic regulation and has been thought to be an irreversible and stable modification of histones. However, several enzymes have recently been discovered to demethylate mono- and dimethylated lysine residues of histone H3 as well as monomethylated arginines via either amine oxidation or deimination, respectively. The JmjC domain-containing histone demethylase 1 (JHDM1), which is conserved from yeast to human, has been demonstrated to demethylate mono- and di- but not trimethylated H3 K36 via hydroxylation of the methyl moiety within the methylated lysine residue. This study broadens our understanding of different types of reaction mechanisms and cofactor requirements for a different category of histone demethylating machinery.

Amino Acid Sequence↗
PubMed2005

Downregulation of lipopolysaccharide response in Drosophila by negative crosstalk between the AP1 and NF-kappaB signaling modules.

IkappaB kinase (IKK) and Jun N-terminal kinase (Jnk) signaling modules are important in the synthesis of immune effector molecules during innate immune responses against lipopolysaccharide and peptidoglycan. However, the regulatory mechanisms required for specificity and termination of these immune responses are unclear. We show here that crosstalk occurred between the drosophila Jnk and IKK pathways, which led to downregulation of each other's activity. The inhibitory action of Jnk was mediated by binding of drosophila activator protein 1 (AP1) to promoters activated by the transcription factor NF-kappaB. This binding led to recruitment of the histone deacetylase dHDAC1 to the promoter of the gene encoding the antibacterial protein Attacin-A and to local modification of histone acetylation content. Thus, AP1 acts as a repressor by recruiting the deacetylase complex to terminate activation of a group of NF-kappaB target genes.

Animals↗
PubMed2000

Histone carbonylation in vivo and in vitro.

Non-enzymic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity. Introduction of carbonyl groups into histones in vivo and in vitro was assessed by Western blot immunoassay and reductive incorporation of tritium from radiolabelled NaBH(4) (sodium borohydride). Histone H1 extracted from bovine thymus, liver and spleen was found to contain significantly elevated amounts of protein-bound carbonyl groups as compared with core histones. The carbonyl content of nuclear proteins of rat pheochromocytoma cells (PC12 cells) was not greatly increased following oxidative stress induced by H(2)O(2), but was significantly increased following alkylating stress induced by N-methyl-N'-nitro-N-nitrosoguanidine or by combined oxidative and alkylating stress. Free ADP-ribose, a reducing sugar generated in the nucleus in proportion to DNA strand breaks, was shown to be a potent histone H1 carbonylating agent in isolated PC12 cell nuclei. Studies of the mechanism of histone H1 modification by ADP-ribose indicate that carbonylation involves formation of a stable acyclic ketoamine. Our results demonstrate preferential histone H1 carbonylation in vivo, with potentially important consequences for chromatin structure and function.

Adenosine Diphosphate Ribose↗
PubMed2001

Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter.

Activation of gene transcription involves chromatin remodeling by coactivator proteins that are recruited by DNA-bound transcription factors. Local modification of chromatin structure at specific gene promoters by ATP-dependent processes and by posttranslational modifications of histone N-terminal tails provides access to RNA polymerase II and its accompanying transcription initiation complex. While the roles of lysine acetylation, serine phosphorylation, and lysine methylation of histones in chromatin remodeling are beginning to emerge, low levels of arginine methylation of histones have only recently been documented, and its physiological role is unknown. The coactivator CARM1 methylates histone H3 at Arg17 and Arg26 in vitro and cooperates synergistically with p160-type coactivators (e.g., GRIP1, SRC-1, ACTR) and coactivators with histone acetyltransferase activity (e.g., p300, CBP) to enhance gene activation by steroid and nuclear hormone receptors (NR) in transient transfection assays. In the current study, CARM1 cooperated with GRIP1 to enhance steroid hormone-dependent activation of stably integrated mouse mammary tumor virus (MMTV) promoters, and this coactivator function required the methyltransferase activity of CARM1. Chromatin immunoprecipitation assays and immunofluorescence studies indicated that CARM1 and the CARM1-methylated form of histone H3 specifically associated with a large tandem array of MMTV promoters in a hormone-dependent manner. Thus, arginine-specific histone methylation by CARM1 is an important part of the transcriptional activation process.

Acetylation↗
PubMed1990

Reactivity and adduct formation of a polyaromatic hydrocarbon, 7-bromomethylbenz[a]anthracene, with chromatin histone proteins.

The alkylation of histones by the direct-acting carcinogen 7-bromomethylbenz[a]anthracene was demonstrated both in vivo and in vitro. The relative molar reactivity for mouse liver histones in vivo was H3 greater than H1 greater than H2b greater than H4 greater than H2a. The in vitro modification of histone H3 was examined in detail. Amino acid adducts stable to acid hydrolysis were separated after acetylation by reversed-phase high-performance liquid chromatography and characterized using ultraviolet absorbance spectra and synthetic amino acid adduct standards. Three major adducts were observed and tentatively identified as cysteinyl, lysyl and histidinyl adducts of histone H3.

Acetylation↗
PubMed2002

Thymocyte apoptosis induced by phosphorylation of histones is associated with the change in chromatin structure to allow easy accessibility of DNase.

The inhibitors of protein phosphatase such as calyculin A and okadaic acid induce the apoptotic cell death in rat thymocytes. To clarify the molecular mechanism of these inhibitor-induced apoptosis, the effect of calyculin A on DNA fragmentation in the isolated nuclei were studied. A significant increase in DNA fragmentation was observed in the nuclei prepared from the cells treated with calyculin A that caused histone hyperphosphorylation. No changes of the activities of caspase-8 and -3 were observed in the extract from the cells treated with calyculin A. The circular dichroism analysis of soluble chromatin from calyculin A-treated thymocyte nuclei indicated that phosphorylation of histones decreased its alpha-helical content. Thus, the change in the chromatin structure may be due to the chemical modification of histones. Moreover, the structural change in chromatin preceded DNA fragmentation in the nuclei. Therefore, these results suggest that the change of chromatin structure allow easy accessibility of nuclear DNase to chromosomal DNA.

Animals↗
PubMed2004

Onset and inheritance of abnormal epigenetic regulation in hematopoietic cells.

Abnormal epigenetic regulation of gene expression contributes significantly to a variety of human pathologies including cancer. Deletion of hypersensitive site 2 (HS2) at the human beta-globin locus control region can lead to abnormal epigenetic regulation of globin genes in transgenic mice. Here, two HS2-deleted transgenic mouse lines were used as model to demonstrate that heritable alteration of chromatin organization at the human beta-globin locus in multipotent hematopoietic progenitors contributes to the abnormal expression of the beta-globin gene in mature erythroid cells. This alteration is characterized by specific patterns of histone covalent modifications that are inherited during erythropoiesis and, moreover, is plastic because it can be reverted by transient treatment with the histone deacetylase inhibitor Trichostatin A. Altogether, our results indicate that aberrant epigenetic regulation can be detected and modified before tissue-specific gene transcription, a finding which may lead to novel strategies for the prevention of chromatin-related pathologies.

Acetylation↗
PubMed2006

Chromatin-modifiying enzymes are essential when the Saccharomyces cerevisiae morphogenesis checkpoint is constitutively activated.

Hsl7p plays a central role in the morphogenesis checkpoint triggered when yeast bud formation is impaired and is proposed to function as an arginine methyltransferase. HSL7 is also essential in the absence of the N-terminal tails of histones H3 or H4. The requirement for H3 and H4 tails may indicate a need for their post-translational modification to bypass the morphogenesis checkpoint. In support of this, the absence of the acetyltransferases Gcn5p or Esa1p, the deacetylase Rpd3p, or the lysine-methyltransferase Set1p resulted in death or extreme sickness in hslDelta mutants. These synthetic interactions involved both the activity of the chromatin-modifying enzymes and the complexes through which they act. Newly reported silencing phenotypes of hsl7Delta mirror those previously reported for gcn5Delta and rpd3Delta, thereby strengthening their functional links. In addition, synthetic interactions and silencing phenotypes were suppressed by inactivation of the morphogenesis checkpoint, either by SWE1 deletion or by preventing Cdc28p phosphorylation. A catalytically dead Hsl7p retained wild-type interactions, implying that modification of histone H3 or H4 N termini by Gcn5p, Esa1p, Rpd3p, and Set1p, but not by Hsl7p, was needed to bypass the morphogenesis checkpoint.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2003

Formation of facultative heterochromatin in the absence of HP1.

Facultative heterochromatin is a cytological manifestation of epigenetic mechanisms that regulate gene expression. Constitutive heterochromatin is marked by distinctive histone H3 methylation and the presence of HP1 proteins, but the chromatin modifications of facultative heterochromatin are less clear. We have examined histone modifications and HP1 in the facultative heterochromatin of nucleated erythrocytes and show that mouse and chicken erythrocytes have different mechanisms of heterochromatin formation. Mouse embryonic erythrocytes have abundant HP1, increased tri-methylation of H3 at K9 and loss of H3 tri-methylation at K27. In contrast, we show that HP1 proteins are lost during the differentiation of chicken erythrocytes, and that H3 tri-methylation at both K9 and K27 is reduced. This coincides with the appearance of the variant linker histone H5. HP1s are also absent from erythrocytes of Xenopus and zebrafish. Our data show that in the same cell lineage there are different mechanisms for forming facultative heterochromatin in vertebrates. To our knowledge, this is the first report of cell types that lack HP1s and that have gross changes in the levels of histone modifications.

3T3 Cells↗
PubMed2003

Transcriptional activation of the enterocyte differentiation marker intestinal alkaline phosphatase is associated with changes in the acetylation state of histone H3 at a specific site within its promoter region in vitro.

Enterocyte differentiation is thought to occur through the transcriptional regulation of a small subset of specific genes. A recent growing body of evidence indicates that post-translational modifications of chromatin proteins (histones) play an important role in the control of gene transcription. Previous work has demonstrated that one such modification, histone acetylation, occurs in an in vitro model of enterocyte differentiation, butyrate-treated HT-29 cells. In the present work, we sought to determine if the epigenetic signal of histone acetylation occurs in an identifiable pattern in association with the transcriptional activation of the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). HT-29 cells were maintained under standard culture conditions and differentiated with sodium butyrate. The chromatin immunoprecipitation (ChIP) assay was used to compare the acetylation state of histones associated with specific regions of the IAP promoter in the two cell populations (undifferentiated vs. differentiated). Chromatin was extracted from cells and cleaved by sonication or enzymatic digestion to obtain fragments of approximately 200 to 600 base-pairs, as confirmed by polymerase chain reaction using primers designed to amplify the IAP segments of interest. The ChIP assay selects DNA sequences that are associated with acetylated histones by immunoprecipitation. Unbound segments represent DNA sequences whose histones are not acetylated. After immunoprecipitation, sequences were detected by radiolabeled polymerase chain reaction, and the relative intensity of the bands was quantified by densitometry. The relative acetylation state of histones at specific sites was determined by comparing the ratios of bound/unbound segments. We determined that in a segment of the IAP promoter between -378 and -303 base-pairs upstream from the transcriptional start site, the acetylation state of histone H3 increased twofold in the differentiated, IAP expressing cells, whereas that of histone H4 remained essentially constant. Additionally, at a distant site, between -1378 and -1303 base-pairs, the acetylation state of H3 and H4 did not change appreciably between the undifferentiated and differentiated cells. We conclude that butyrate-induced differentiation is associated with specific and localized changes in the histone acetylation state within the IAP promoter. These changes within the endogenous IAP gene may underlie its transcriptional activation in the context of the enterocyte differentiation program.

Alkaline Phosphatase↗