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PubMed1979

Effect of 1-methyl-1-nitrosourea on poly(adenosine diphosphate-ribose) polymerase activity at the nucleosomal level.

The stimulation of poly(adenosine diphosphate ribose) [poly(ADP-ribose)] polymerase activity at the nuclear level after damage of HeLa cells by 1-methyl-1-nitrosourea has been previously reported. We have observed a similar activation of the enzyme after treatment of cells with MNU at the nucleosomal level of chromatin (greater than 1N). This stimulation of enzyme activity did not occur through an inhibition of the glycohydrolase enzyme which cleaves poly(ADP-ribose), or elongation of poly(ADP-ribose) chains, or an increased biosynthesis of enzyme protein. The increased activity appears to be a consequence of the generation of more acceptor sites on nuclear proteins for initiation of poly(ADP-ribose) synthesis. The data indicate that MNU increased the accessibility of nucleosome core histones for modification by poly(ADP) ribosylation.

Chromatin↗
PubMed2005

Chromatin in need of a fix: phosphorylation of H2AX connects chromatin to DNA repair.

A bevy of recent reports have firmly established a mechanistic link between a histone posttranslational modification associated with DNA double-strand breaks and recruitment of chromatin-modifying activities. These papers show that in addition to providing signals for transcriptional regulation, specific histone "codes" can coordinate and target multiple activities involved in DNA repair.

Animals↗
PubMedDate not supplied

Lack of correlation between histone H4 acetylation and transcription during the Physarum cell cycle.

The interaction between nucleosomal proteins and DNA is expected to change during DNA replication as well as during transcription. A possible way of achieving the necessary structural changes is the modification of histones and high mobility group (HMG) proteins. The acetylation of core histones has been studied in various systems (for a review see ref. 1) and a correlation between histone acetylation and transcriptional activity of chromatin has frequently been proposed. In particular, Bradbury and co-workers have reported a cell cycle dependence of histone H4 acetylation in Physarum polycephalum which revealed two correlations: (1) tetraacetylated H4 (H4Ac4) correlated with the rate of transcription and (2) H4 acetylation was inversely correlated with H1 phosphorylation in mitosis. We present evidence here that H4 acetylation does not fit these correlations. Our data clearly show that the acetate content of H4 is high during the S phase, but low during later stages of the cell cycle. H4Ac4 remains at a nearly constant level during the whole cycle, with an elevation during the S phase. Furthermore, experiments with the deacetylase inhibitor sodium-n-butyrate do not support the proposed connection between diacetylated H4 (H4Ac2) and DNA replication. Our data imply that a correlation of H4 acetylation and transcription is unlikely during the cell cycle of Physarum. The conclusions of Bradbury and co-workers are therefore invalid.

Acetylation↗
PubMed2003

Insulation of the chicken beta-globin chromosomal domain from a chromatin-condensing protein, MENT.

Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken beta-globin domain. We observed two sharp transitions of MENT concentration coinciding with the beta-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation.

3T3 Cells↗
PubMed2006

Histone acetylation is associated with differential gene expression in the rapid and robust memory CD8(+) T-cell response.

To understand the molecular basis for the rapid and robust memory T-cell responses, we examined gene expression and chromatin modification by histone H3 lysine 9 (H3K9) acetylation in resting and activated human naive and memory CD8(+) T cells. We found that, although overall gene expression patterns were similar, a number of genes are differentially expressed in either memory or naive cells in their resting and activated states. To further elucidate the basis for differential gene expression, we assessed the role of histone H3K9 acetylation in differential gene expression. Strikingly, higher H3K9 acetylation levels were detected in resting memory cells, prior to their activation, for those genes that were differentially expressed following activation, indicating that hyperacetylation of histone H3K9 may play a role in selective and rapid gene expression of memory CD8(+) T cells. Consistent with this model, we showed that inducing high levels of H3K9 acetylation resulted in an increased expression in naive cells of those genes that are normally expressed differentially in memory cells. Together, these findings suggest that differential gene expression mediated at least in part by histone H3K9 hyperacetylation may be responsible for the rapid and robust memory CD8(+) T-cell response.

Acetylation↗
PubMed2001

The archaeal histone-fold protein HMf organizes DNA into bona fide chromatin fibers.

BACKGROUND: The discovery of histone-like proteins in Archaea urged studies into the possible organization of archaeal genomes in chromatin. Despite recent advances, a variety of structural questions remain unanswered. RESULTS: We have used the atomic force microscope (AFM) with traditional nuclease digestion assays to compare the structure of nucleoprotein complexes reconstituted from tandemly repeated eukaryal nucleosome-positioning sequences and histone octamers, H3/H4 tetramers, and the histone-fold archaeal protein HMf. The data unequivocally show that HMf reconstitutes are indeed organized as chromatin fibers, morphologically indistinguishable from their eukaryal counterparts. The nuclease digestion patterns revealed a clear pattern of protection at regular intervals, again similar to the patterns observed with eukaryal chromatin fibers. In addition, we studied HMf reconstitutes on mononucleosome-sized DNA fragments and observed a great degree of similarity in the internal organization of these particles and those organized by H3/H4 tetramers. A difference in stability was observed at the level of mono-, di-, and triparticles between the HMf particles and canonical octamer-containing nucleosomes. CONCLUSIONS: The in vitro reconstituted HMf-nucleoprotein complexes can be considered as bona fide chromatin structures. The differences in stability at the monoparticle level should be due to structural differences between HMf and core histone H3/H4 tetramers, i.e., to the complete absence in HMf of histone tails beyond the histone fold. We speculate that the existence of core histone tails in eukaryotes may provide a greater stability to nucleosomal particles and also provide the additional ability of chromatin structure to regulate DNA function in eukaryotic cells by posttranslational histone tail modifications.

Animals↗
PubMed2004

Functions for S. cerevisiae Swd2p in 3' end formation of specific mRNAs and snoRNAs and global histone 3 lysine 4 methylation.

The Saccharomyces cerevisiae WD-40 repeat protein Swd2p associates with two functionally distinct multiprotein complexes: the cleavage and polyadenylation factor (CPF) that is involved in pre-mRNA and snoRNA 3' end formation and the SET1 complex (SET1C) that methylates histone 3 lysine 4. Based on bioinformatic analysis we predict a seven-bladed beta-propeller structure for Swd2p proteins. Northern, transcriptional run-on and in vitro 3' end cleavage analyses suggest that temperature sensitive swd2 strains were defective in 3' end formation of specific mRNAs and snoRNAs. Protein-protein interaction studies support a role for Swd2p in the assembly of 3' end formation complexes. Furthermore, histone 3 lysine 4 di-and tri-methylation were adversely affected and telomeres were shortened in swd2 mutants. Underaccumulation of the Set1p methyltransferase accounts for the observed loss of SET1C activity and suggests a requirement for Swd2p for the stability or assembly of this complex. We also provide evidence that the roles of Swd2p as component of CPF and SET1C are functionally independent. Taken together, our results establish a dual requirement for Swd2p in 3' end formation and histone tail modification.

Amino Acid Sequence↗
PubMed2005

Avidin plate assay system for enzymatic characterization of a histone lysine methyltransferase.

Modification of proteins by protein methyltransferases has several important biological functions. Here, we study the methylation of histone H3 tail at position Lys9 by the Dim-5 histone lysine methyltransferase, which is involved in epigenetic signaling and gene silencing and which triggers DNA methylation in Neurospora crassa. We have developed a new assay to detect protein methylation using a biotinylated synthetic peptide substrate and a radioactively labeled coenzyme. We show that the assay is linear with respect to time and enzyme concentration (under multiple turnover conditions) and that its background is very low. Data points were reproducible within 3%. At least 200 pmol of biotinylated peptide is bound completely to the microplate. We employed the assay system to determine the K(m) and k(cat) values of the Dim-5 enzyme for the methylation of a 20 mer peptide to be 7.4 microM and 2.3 min(-1), respectively. In addition, we determined the activity of four Dim-5 variants, ranging from full activity to less than 1% of residual activity. The microplate biotin/avidin peptide methylation assay developed here is convenient, very accurate, reproducible, and inexpensive. Because it yields quantitative results, it can be employed for a characterization of the enzymatic properties of histone lysine methyltransferases and other protein methyltransferases. The assay also is well suited for high-throughput applications.

Avidin↗
PubMed1991

Possible role of histone acetylation and histone H1(0) replacement for the initiation of replication in regenerating rat liver.

The role of histone acetylation and DNA synthesis has been investigated extensively in the regenerating rat liver system in the presence and absence of the cyclophosphamide derivative mafosfamide. We demonstrate a mafosfamide-induced inhibition of maximum histone acetyltransferase activity followed by a second elevation of enzyme activity and an accompanying total suppression of DNA synthesis for 7-8 h. The maximum of histone acetyltransferase activity, in parallel with an elevated acetylation in vivo, the consecutive replacement of histone H1(0) amd initiation of replication occur sequentially in the presence and absence of mafosfamide, but with a temporary delay of 7-8 h. Our data indicate that modifications of histone acetyltransferase (EC 2.3.1.48) activity do not significantly influence the acetylation patterns of histones H3 and H4. The mafosfamide-induced change of histone acetyltransferase activity and acetylation in vivo, the shift of histone H1(0) exchange and the consecutive transition of initiation of replication suggest that these three events might be functionally related.

Acetylation↗
PubMed2024

Reversion from basal histone H4 hypoacetylation at the replication fork increases DNA damage in FANCA deficient cells.

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.

Histones↗
PubMed1998

DNA methylation, nucleosomes and the inheritance of chromatin structure and function.

The replication of the genome during S phase is a crucial period for the establishment and maintenance of programmes of differential gene activity. Existing chromosomal structures are disrupted during replication and reassembled on both daughter chromatids. The capacity to reassemble a particular chromatin structure with defined functional properties reflects the commitment of a cell type to a particular state of determination. The core and linker histones and their modifications, enzymes that modify the histones, DNA methylation and proteins that recognize methylated DNA within chromatin may all play independent or interrelated roles in defining the functional properties of chromatin. Pre-existing protein-DNA interactions and DNA methylation in a parental chromosome will influence the structure and function of daughter chromosomes generating an epigenetic imprint. In this chapter we consider the events occurring at the eukaryotic replication fork, their consequences for pre-existing chromosomal structures and how an epigenetic imprint might be maintained.

Animals↗
PubMed2006

Chromatin architectural proteins.

The accessibility of eukaryotic DNA is dependent upon the hierarchical level of chromatin organization. These include (1) intra-nucleosome interactions, (2) inter-nucleosome interactions and (3) the influence of non-histone chromatin architectural proteins. There appears to be interplay between all these levels, in that one level can override another or that two or more can act in concert. In the first level, the stability of the nucleosome itself is dependent on the number and type of contacts between the core histones and the surrounding DNA, as well as protein-protein interactions within the core histone octamer. Core histone variants, post-translational modifications of the histones, and linker histones binding to the DNA all influence the organization and stability of the nucleosome. When nucleosomes are placed end-to-end in linear chromatin arrays, the second level of organization is revealed. The amino terminal tails of the histone proteins make contacts with adjacent and distant nucleosomes, both within the fiber and between different fibers. The third level of organization is imposed upon these 'intrinsic' constraints, and is due to the influence of chromatin binding proteins that alter the architecture of the underlying fiber. These chromatin architectural proteins can, in some cases, bypass intrinsic constraints and impart their own topological affects, resulting in truly unique, supra-molecular assemblages that undoubtedly influence the accessibility of the underlying DNA. In this review we will provide a brief summary of what has been learned about the intrinsic dynamics of chromatin fibers, and survey the biology and architectural affects of the handful of chromatin architectural proteins that have been identified and characterized. These proteins are likely only a small subset of the architectural proteins encoded within the eukaryotic genome. We hope that an increased understanding and appreciation of the contribution of these proteins to genome accessibility will hasten the identification and characterization of more of these important regulatory factors.

Animals↗
PubMed2006

Chromatin modifications in the germinal vesicle (GV) of mammalian oocytes.

The nucleus of eukaryotic cells is organized into functionally specialized compartments that are essential for the control of gene expression, chromosome architecture and cellular differentiation. The mouse oocyte nucleus or germinal vesicle (GV) exhibits a unique chromatin configuration that is subject to dynamic modifications during oogenesis. This process of 'epigenetic maturation' is critical to confer the female gamete with meiotic as well as developmental competence. In spite of its biological significance, little is known concerning the cellular and molecular mechanisms regulating large-scale chromatin structure in mammalian oocytes. Here, recent findings that provide mechanistic insight into the complex relationship between large-scale chromatin structure and global transcriptional repression in pre-ovulatory oocytes will be discussed. Post-translational modifications of histone proteins such as acetylation and methylation are crucial for heterochromatin formation and thus play a key role in remodeling the oocyte genome. This strategy involves multiple and hierarchical chromatin modifications that regulate nuclear dynamics in response to a developmentally programmed signal(s), presumably of paracrine origin, before the resumption of meiosis. Models for the experimental manipulation of large-scale chromatin structure in vivo and in vitro will be instrumental to determine the key cellular pathways and oocyte-derived factors involved in genome-wide chromatin modifications. Importantly, analysis of the functional differentiation of chromatin structure in the oocyte genome with high resolution and in real time will have wide-ranging implications to understand the role of nuclear organization in meiosis, the events of nuclear reprogramming and the spatio-temporal regulation of gene expression during development and differentiation.

Animals↗
PubMed2003

ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation.

Proteins of the ARGONAUTE family are important in diverse posttranscriptional RNA-mediated gene-silencing systems as well as in transcriptional gene silencing in Drosophila and fission yeast and in programmed DNA elimination in Tetrahymena. We cloned ARGONAUTE4 (AGO4) from a screen for mutants that suppress silencing of the Arabidopsis SUPERMAN (SUP) gene. The ago4-1 mutant reactivated silent SUP alleles and decreased CpNpG and asymmetric DNA methylation as well as histone H3 lysine-9 methylation. In addition, ago4-1 blocked histone and DNA methylation and the accumulation of 25-nucleotide small interfering RNAs (siRNAs) that correspond to the retroelement AtSN1. These results suggest that AGO4 and long siRNAs direct chromatin modifications, including histone methylation and non-CpG DNA methylation.

Alleles↗
PubMed1989

Properties of an adenosine triphosphate-stimulated factor that enhances the nuclear binding of activated glucocorticoid-receptor complex: binding to histone-agarose.

Recently we have purified and characterized a factor in rat liver cytosol that enhances nuclear binding of the activated glucocorticoid-receptor complex in the presence of ATP [an ATP-stimulated translocation promoter (ASTP)]. It has a mol wt of 93,000, S value of 6.5, and pI of 4.5. Here we report studies on the binding of ASTP to histones from calf thymus. Higher salt concentrations were required to disrupt the binding of ASTP to histone-agarose than to disrupt its binding to the anion exchanger diethylaminoethyl-cellulose (half-maximal concentration for inhibition: 200 mM vs. 60 mM KCl). ATP decreased the binding of ASTP to histone-agarose in a dose-dependent fashion, but ADP and AMP had no appreciable effect on the binding. Pyridoxal 5'-phosphate (10 mM) inhibited the binding of ASTP to histone-agarose, whereas 10 mM pyridoxal, pyridoxamine 5'-phosphate, pyridoxamine, and pyridoxine were not inhibitory. ASTP did not bind to histone-agarose that had been pretreated with pyridoxal 5'-phosphate and then reduced with sodium borohydride. In contrast, ASTP pretreated in the same way could bind to histone-agarose. Therefore, modification of lysine residues of histones by pyridoxal 5'-phosphate (Schiff base) presumably inhibited the binding of ASTP to the immobilized histone. Competition experiments with various histones (H1, H2A, H2B, H3, and H4) indicated that ASTP bound to the arginine-rich histones H3 and H4, with preference for H4. These observations together suggest that lysine residues in H4 and H3 are essential for the binding of ASTP to histone. The implications of this interaction are discussed.

Adenosine Triphosphate↗
PubMed1980

Cell cycle-specific enhancement of type C virus activation by sodium n-butyrate.

The effect of sodium n-butyrate on chemical induction of xenotropic virus from synchronized Kirsten sarcoma virus-transformed BALB mouse cells was examined. When added during the last part of the G1 phase, n-butyrate produced a large increase in cycloheximide induction during S phase. Under similar conditions, activation by 5-iododeoxyuridine was inhibited. When added with cycloheximide during the S phase, n-butyrate inhibited activation of virus. Studies with synchronized cultures showed that n-butyrate delayed the onset of DNA synthesis, characteristic of the S phase, and inhibited histone deacetylation in log-phase cells. The effects produced by n-butyrate could, therefore, be the result of lengthening the G1 phase of the cell cycle or a modification of histones affecting transcription during virus activation.

Animals↗
PubMed1997

The ability to organize sperm DNA into functional chromatin is acquired during meiotic maturation in murine oocytes.

Following fertilization of meiotically mature eggs, the chromatin of the sperm becomes biochemically and structurally remodeled within the egg cytoplasm. Despite the essential role of the paternal genome during embryogenesis, little is known of when the activities that regulate this chromatin remodeling appear during oogenesis. To determine whether these activities were acquired during meiotic maturation, we inseminated maturing oocytes of mice shortly after germinal vesicle breakdown. As previously shown, insemination at this stage did not activate the maturing oocytes, which became arrested at metaphase II. Immunofluorescent analysis revealed that at 1 hr postinsemination the sperm chromatin was dispersed and contained protamines but was devoid of core histones H2B and H3. At 4 hr postinsemination, both protamine and core histones were detectable on the sperm chromatin. By 8 hr postinsemination protamines were absent, and histones stained maximally. The appearance of immunoreactive histones was correlated with a morphological transition of the sperm chromatin from the dispersed to a condensed state, which suggests that the assembly of the histones reflected modification of the chromatin to a somatic-like state in which it was competent to respond to the metaphase-promoting factor activity of the oocyte. Both the assembly of histones and chromatin condensation were reversibly blocked when protein synthesis was inhibited, indicating that the remodeling process required proteins synthesized during maturation. Injection of core histones into protein synthesis-inhibited oocytes failed to induce condensation of the sperm chromatin, which implies that correct remodeling requires synthesis during maturation of nonhistone proteins. To test the functional capacity of remodeled sperm chromatin, maturing oocytes were inseminated, allowed to continue maturation for 17 hr and then parthenogenetically activated. Following activation, the sperm-derived chromatin as well as that of the oocyte became decondensed within pronuclei and underwent DNA replication, indicating that sperm chromatin remodeled in maturing oocyte cytoplasm was functionally normal. When the postinsemination incubation time was reduced to 11 hr; however, neither the female nor the male pronuclei underwent DNA replication, implying that factors synthesized late during maturation are required for DNA replication after activation. Taken together, these results indicate that the ability to organize sperm DNA into functional somatic-like chromatin develops in oocytes during meiotic maturation, requires proteins synthesized during maturation, and can be expressed independently of activation.

Animals↗
PubMed2003

The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiae.

Genomes are organized into active regions known as euchromatin and inactive regions known as heterochromatin, or silenced chromatin. This review describes contemporary knowledge and models for how silenced chromatin in Saccharomyces cerevisiae forms, functions, and is inherited. In S. cerevisiae, Sir proteins are the key structural components of silenced chromatin. Sir proteins interact first with silencers, which dictate which regions are silenced, and then with histone tails in nucleosomes as the Sir proteins spread from silencers along chromosomes. Importantly, the spreading of silenced chromatin requires the histone deacetylase activity of Sir2p. This requirement leads to a general model for the spreading and inheritance of silenced chromatin or other special chromatin states. Such chromatin domains are marked by modifications of the nucleosomes or DNA, and this mark is able to recruit an enzyme that makes further marks. Thus, among different organisms, multiple forms of repressive chromatin can be formed using similar strategies but completely different proteins. We also describe emerging evidence that mutations that cause global changes in the modification of histones can alter the balance between euchromatin and silenced chromatin within a cell.

Chromatin↗