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PubMed2004

ATRX, a member of the SNF2 family of helicase/ATPases, is required for chromosome alignment and meiotic spindle organization in metaphase II stage mouse oocytes.

ATRX is a centromeric heterochromatin binding protein belonging to the SNF2 family of helicase/ATPases with chromatin remodeling activity. Mutations in the human ATRX gene result in X-linked alpha-thalassaemia with mental retardation (ATRX) syndrome and correlate with changes in methylation of repetitive DNA sequences. We show here that ATRX also functions to regulate key stages of meiosis in mouse oocytes. At the germinal vesicle (GV) stage, ATRX was found associated with the perinucleolar heterochromatin rim in transcriptionally quiescent oocytes. Phosphorylation of ATRX during meiotic maturation is dependent upon calcium calmodulin kinase (CamKII) activity. Meiotic resumption also coincides with deacetylation of histone H4 at lysine 5 (H4K5 Ac) while ATRX and histone H3 methylated on lysine 9 (H3K9) remained bound to the centromeres and interstitial regions of condensing chromosomes, respectively. Inhibition of histone deacetylases (HDACs) with trichostatin A (TSA) disrupted ATRX binding to the centromeres of hyperacetylated chromosomes resulting in abnormal chromosome alignments at metaphase II (MII). Similarly, while selective ablation of ATRX by antibody microinjection and RNA interference (RNAi) had no effect on the progression of meiosis, it had severe consequences for the alignment of chromosomes on the metaphase II spindle. These results suggest that genome-wide epigenetic modifications such as global histone deacetylation are essential for the binding of ATRX to centromeric heterochromatin. Moreover, centromeric ATRX is required for correct chromosome alignment and organization of a bipolar meiotic metaphase II spindle.

Animals↗
PubMed2002

The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation.

DNA methylation plays an important role in mammalian development and correlates with chromatin-associated gene silencing. The recruitment of MeCP2 to methylated CpG dinucleotides represents a major mechanism by which DNA methylation can repress transcription. MeCP2 silences gene expression partly by recruiting histone deacetylase (HDAC) activity, resulting in chromatin remodeling. Here, we show that MeCP2 associates with histone methyltransferase activity in vivo and that this activity is directed against Lys(9) of histone H3. Two characterized repression domains of MeCP2 are involved in tethering the histone methyltransferase to MeCP2. We asked if MeCP2 can deliver Lys(9) H3 methylation to the H19 gene, whose activity it represses. We show that the presence of MeCP2 on nucleosomes within the repressor region of the H19 gene (the differentially methylated domain) coincides with an increase in H3 Lys(9) methylation. Our data provide evidence that MeCP2 reinforces a repressive chromatin state by acting as a bridge between two global epigenetic modifications, DNA methylation and histone methylation.

Animals↗
PubMed2026

A Translocation within the Ogataea Species Complex Alters Local Subtelomeric Chromatin while Maintaining Overall Genome Organization.

Eukaryotic genomic DNA is packaged in the nucleus as chromatin - a DNA-protein aggregate regulating genome function, including transcription. Chromatin is classified as either active euchromatin or silent heterochromatin, with each marked by distinct histone post-translational modifications (PTMs). Chromatin composition also mediates genome organization, including how heterochromatin aggregates at the nuclear periphery while euchromatin localizes to the nucleus center. In fungi, heterochromatic loci cluster, including independent centromere and telomere clusters that form the Rabl chromosome conformation. However, it is unknown if chromatin composition and genome organization are conserved in closely related fungi, and how they are impacted by large-scale chromosomal rearrangements. Here, we examined differences in histone PTM deposition, gene expression, and genome organization in two yeast species from the order Pichiales, which diverged from the common ancestor shared with Saccharomyces cerevisiae more than 200 million years ago. We focused on Ogataea polymorpha, which is used for industrial protein production, and Ogataea haglerorum, an isolate of which harbors a translocation between chromosomes 1 and 6. We show that the enrichment of three activating PTMs - the trimethylation of lysine 4 of histone H3 (H3K4me3) and the acetylation of lysine 9 of histone H3 (H3K9ac) or lysine 16 of histone H4 (H4K16ac) - are similar genome-wide yet individual gene orthologs have distinct chromatin and expression patterns. While both Ogataea genomes organize into a Rabl conformation, the O. haglerorum translocation alters subtelomeric chromatin composition and expression of genes affected by the translocation. Our work highlights the genome function differences that occur on a microevolutionary scale.

Ogataea↗
PubMed1998

Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia.

The transforming proteins of acute promyelocytic leukaemias (APL) are fusions of the promyelocytic leukaemia (PML) and the promyelocytic leukaemia zinc-finger (PLZF) proteins with retinoic acid receptor-alpha (RARalpha). These proteins retain the RARalpha DNA- and retinoic acid (RA)-binding domains, and their ability to block haematopoietic differentiation depends on the RARalpha DNA-binding domain. Thus RA-target genes are downstream effectors. However, treatment with RA induces differentiation of leukaemic blast cells and disease remission in PML-RARalpha APLs, whereas PLZF-RARa APLs are resistant to RA. Transcriptional regulation by RARs involves modifications of chromatin by histone deacetylases, which are recruited to RA-target genes by nuclear co-repressors. Here we show that both PML-RARalpha and PLZF-RARalpha fusion proteins recruit the nuclear co-repressor (N-CoR)-histone deacetylase complex through the RARalpha CoR box. PLZF-RARalpha contains a second, RA-resistant binding site in the PLZF amino-terminal region. High doses of RA release histone deacetylase activity from PML-RARalpha, but not from PLZF-RARalpha. Mutation of the N-CoR binding site abolishes the ability of PML-RARalpha to block differentiation, whereas inhibition of histone deacetylase activity switches the transcriptional and biological effects of PLZF-RARalpha from being an inhibitor to an activator of the RA signalling pathway. Therefore, recruitment of histone deacetylase is crucial to the transforming potential of APL fusion proteins, and the different effects of RA on the stability of the PML-RARalpha and PLZF-RARalpha co-repressor complexes determines the differential response of APLs to RA.

Binding Sites↗
PubMed2026

A translocation within the Ogataea species complex alters local subtelomeric chromatin while maintaining overall genome organization.

Eukaryotic genomic DNA is packaged in the nucleus as chromatin-a DNA-protein aggregate regulating genome function, including transcription. Chromatin is classified as either active euchromatin or silent heterochromatin, with each marked by distinct histone post-translational modifications (PTMs). Chromatin composition also mediates genome organization, including how heterochromatin aggregates at the nuclear periphery while euchromatin localizes to the nucleus center. In fungi, heterochromatic loci cluster, including independent centromere and telomere clusters that form the Rabl chromosome conformation. However, it is unknown if chromatin composition and genome organization are conserved in closely related fungi, and how these features are impacted by large-scale chromosomal rearrangements. Here, we examined differences in histone PTM deposition, gene expression, and genome organization in 2 yeast species from the order Pichiales, which diverged from the common ancestor shared with Saccharomyces cerevisiae more than 200 million years ago. We focused on Ogataea polymorpha, which is used for industrial protein production, and Ogataea haglerorum, an isolate of which harbors a translocation between chromosomes 1 and 6. We show that the enrichment of 3 activating PTMs-the trimethylation of lysine 4 of histone H3 (H3K4me3) and the acetylation of lysine 9 of histone H3 (H3K9ac) or lysine 16 of histone H4 (H4K16ac)-are similar genome-wide, yet gene orthologs have distinct chromatin and expression patterns. While both Ogataea genomes organize into a Rabl conformation, the O. haglerorum translocation alters subtelomeric chromatin composition and expression of genes affected by the translocation. Our work highlights the genome function differences that occur on a microevolutionary scale.

Genome, Fungal↗
PubMed2004

The enhancement of histone H4 and H2A serine 1 phosphorylation during mitosis and S-phase is evolutionarily conserved.

Histone phosphorylation has long been associated with condensed mitotic chromatin; however, the functional roles of these modifications are not yet understood. Histones H1 and H3 are highly phosphorylated from late G2 through telophase in many organisms, and have been implicated in chromatin condensation and sister chromatid segregation. However, mutational analyses in yeast and biochemical experiments with Xenopus extracts have demonstrated that phosphorylation of H1 and H3 is not essential for such processes. In this study, we investigated additional histone phosphorylation events that may have redundant functions to H1 and H3 phosphorylation during mitosis. We developed an antibody to H4 and H2A that are phosphorylated at their respective serine 1 (S1) residues and found that H4S1/H2AS1 are highly phosphorylated in the mitotic chromatin of worm, fly, and mammals. Mitotic H4/H2A phosphorylation has similar timing and localization as H3 phosphorylation, and closely correlates with the chromatin condensation events during mitosis. We also detected a lower level of H4/H2A phosphorylation in 5-bromo-2-deoxyuridine-positive S-phase cells, which corroborates earlier studies that identified H4S1 phosphorylation on newly synthesized histones during S-phase. The evolutionarily conserved phosphorylation of H4/H2A during the cell cycle suggests that they may have a dual purpose in chromatin condensation during mitosis and histone deposition during S-phase.

Amino Acid Sequence↗
PubMed2004

Reproductive epigenetics.

Epigenetics refers to covalent modifications of DNA and core histones that regulate gene activity without altering DNA sequence. To date, the best-characterized DNA modification associated with the modulation of gene activity is methylation of cytosine residues within CpG dinucleotides. Human disorders associated with epigenetic abnormalities include rare imprinting diseases, molar pregnancies, and childhood cancers. Germ cell development and early embryo development are critical times when epigenetic patterns are initiated or maintained. This review focuses on the epigenetic modification DNA methylation and discusses recent progress that has been made in understanding when and how epigenetic patterns are differentially established in the male and female germlines, the mouse, and human disorders associated with abnormalities in epigenetic programming in germ cells and early embryos, as well as genetic and other modulators (e.g. nutrition and drugs) of reproductive epigenetic events.

Animals↗
PubMed1999

Poly-ADP ribose polymerase activates nuclear proteasome to degrade oxidatively damaged histones.

The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the cytoplasm. Nuclear proteins are also subject to oxidation, and the nucleus of mammalian cells contains proteasome. In human beings, tumor cells frequently are subjected to oxidation as a consequence of antitumor chemotherapy, and K562 human myelogenous leukemia cells have a higher nuclear proteasome activity than do nonmalignant cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs and the mechanisms of inducible tumor resistance to oxidation are of obvious importance. After hydrogen peroxide treatment of K562 cells, degradation of the model proteasome peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increases significantly within minutes. Both increased proteolytic susceptibility of the histone substrates (caused by modification by oxidation) and activation of the proteasome enzyme complex occur independently during oxidative stress. This rapid up-regulation of 20S proteasome activity is accompanied by, and depends on, poly-ADP ribosylation of the proteasome, as shown by inhibitor experiments, 14C-ADP ribose incorporation assays, immunoblotting, in vitro reconstitution experiments, and immunoprecipitation of (activated) proteasome with anti-poly-ADP ribose polymerase antibodies. The poly-ADP ribosylation-mediated activated nuclear 20S proteasome is able to remove oxidatively damaged histones more efficiently and therefore is proposed as an oxidant-stimulatable defense or repair system of the nucleus in K562 leukemia cells.

Adenosine Diphosphate Ribose↗
PubMed2023

H3.3 contributes to chromatin accessibility and transcription factor binding at promoter-proximal regulatory elements in embryonic stem cells.

BACKGROUND: The histone variant H3.3 is enriched at active regulatory elements such as promoters and enhancers in mammalian genomes. These regions are highly accessible, creating an environment that is permissive to transcription factor binding and the recruitment of transcriptional coactivators that establish a unique chromatin post-translational landscape. How H3.3 contributes to the establishment and function of chromatin states at these regions is poorly understood. RESULTS: We perform genomic analyses of features associated with active promoter chromatin in mouse embryonic stem cells (ESCs) and find evidence of subtle yet widespread promoter dysregulation in the absence of H3.3. Loss of H3.3 results in reduced chromatin accessibility and transcription factor (TF) binding at promoters of expressed genes in ESCs. Likewise, enrichment of the transcriptional coactivator p300 and downstream histone H3 acetylation at lysine 27 (H3K27ac) is reduced at promoters in the absence of H3.3, along with reduced enrichment of the acetyl lysine reader BRD4. Despite the observed chromatin dysregulation, H3.3 KO ESCs maintain transcription from ESC-specific genes. However, upon undirected differentiation, H3.3 KO cells retain footprinting of ESC-specific TF motifs and fail to generate footprints of lineage-specific TF motifs, in line with their diminished capacity to differentiate. CONCLUSIONS: H3.3 facilitates DNA accessibility, transcription factor binding, and histone post-translational modification at active promoters. While H3.3 is not required for maintaining transcription in ESCs, it does promote de novo transcription factor binding which may contribute to the dysregulation of cellular differentiation in the absence of H3.3.

Animals↗
PubMedDate not supplied

Time-resolved microscopy of chromatin in vitro and in vivo.

In eukaryotic cell nuclei, double-stranded DNA is found in the form of chromatin, a large fiber made up of DNA complexed to histone proteins. In this article, recent studies using fluorescence techniques to look at the dynamics of chromatin, both in vivo and in vitro, are reviewed. Two-photon counterpropagating fluorescence recovery after patterned photobleaching is used to examine chromatin fluctuations on lengthscales ranging from less than 100 nm to microns. By combining in vivo studies with data on isolated nuclei and by measuring how these fluctuations depend on variables like ionic strength and photochemical cross-linking, it is demonstrated that the relatively large-scale motions of chromatin observed in vivo are consistent with smaller scale modifications of the histone-DNA interaction. This connection may provide a means to use conformational dynamics as an in vivo probe of the biochemical events involved in gene expression.

Cell Nucleus↗
PubMed1991

Histones and DNA methylation in mammalian chromatin. II. Presence of non-inhibitory tightly-bound histones.

After removal, by high-salt extraction, of the loosely-bound components present in human placenta chromatin, tightly-bound cationic proteins could be solubilized, by acid extraction, from the 'stripped' chromatin, as well as from the 'stripped' loops or from the 'digested matrix'. These acid-soluble tightly-bound proteins are, in terms of apparent molecular mass and immunoreactivity, quite similar to the 'typical', loosely-bound histones, and, similarly to their 'loosely-bound' counterparts, they can be subdivided in distinct H1-, H2A-, H2B-, H3- and H4-like components, the 'digested matrix' being however characterized by the absence of tightly-bound H1. These tightly-bound histones, at variance from the 'typical' ones, readily find a right-handed helical conformation upon renaturation by progressive dialyses. The H1 components strongly differ also in their effects on enzymic DNA methylation: while 'typical' H1 has a strong inhibitory effect, its tightly-bound counterpart exerts a slight but definite stimulation.

Chromatin↗
PubMed2005

Chromatin, DNA methylation and neuron gene regulation--the purpose of the package.

The accessibility of cognate binding sites within a gene promoter can be modified by the condensation or relaxation of local chromatin structure. Local chromatin structure is in turn programmed by covalent modifications of cytosine bases in DNA and amino acid residues in histone protein tails. These chemical and physical adaptations around gene promoters can significantly change levels of mRNA expression. Furthermore, linear patterns of covalent modification of histone protein tails are emerging as a distinct regulatory code--another form of cellular memory. Because chromatin structure can be modified by conventional pharmacologic therapy, a novel approach to the regulation of neuronal gene expression in clinical populations is possible.

Binding Sites↗
PubMed1992

Multiple mechanisms regulate the proliferation-specific histone gene transcription factor HiNF-D in normal human diploid fibroblasts.

The proliferation-specific transcription factor complex HiNF-D interacts with sequence specificity in a proximal promoter element of the human H4 histone gene FO108, designated Site II. The occupancy of Site II by HiNF-D has been implicated in proper transcription initiation and as a component of the cell cycle regulation of this gene. In the present study we have investigated the role of the HiNF-D/Site II interaction in controlling the level of H4 histone gene transcription during modifications of normal cellular growth. HiNF-D binding activity is present at high levels in rapidly proliferating cultures of human diploid fibroblasts and is reduced to less than 2% upon the cessation of proliferation induced by serum deprivation of sparsely population fibroblast cultures. Density-dependent quiescence also abolishes HiNF-D binding activity. Downregulation of transcription from the H4 gene occurs concomitant with the loss of the HiNF-D/Site II interaction, further suggesting a functional relationship between Site II occupancy and the capacity for transcription. Serum stimulation of quiescent preconfluent cells results in an increase in HiNF-D binding activity as the cells are resuming DNA synthesis and H4 histone gene transcription. Density-inhibited quiescent cells respond to serum stimulation with only a minimal increase in the HiNF-D binding activity, 30% of maximal levels. However, H4 histone gene transcription is stimulated to a level equal to that detected in extracts of the sparsely populated serum-stimulated cultures. These results suggest that there is a threshold level of HiNF-D binding activity necessary for the activation of H4 histone gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood↗
PubMed2003

MAP kinase-mediated phosphoacetylation of histone H3 and inducible gene regulation.

That signalling pathways, particularly the mitogen-activated protein kinase cascades, elicit modification of chromatin proteins such as histone H3 by phosphorylation and/or acetylation concomitant with gene activation is now well established. The picture that is emerging is one of a complex and dynamic pattern of multiple modifications at the H3 tail. Here, we review the inducible gene systems where H3 modifications have been reported and re-evaluate the controversy as to the kinase(s) that phosphorylates it as well as the proposed coupling between H3 phosphorylation and acetylation.

Acetylation↗
PubMed2004

In vivo posttranslational modifications of the high mobility group A1a proteins in breast cancer cells of differing metastatic potential.

The high mobility group (HMG) proteins are important modulators of chromatin structure and gene transcription. Overexpression of HMGA1 proteins in vivo induces neoplastic transformation and promotes a highly metastatic cellular phenotype. This study focuses on characterization of HMGA1a in vivo posttranslational modification (PTM) patterns found in a nonmetastatic and two metastatic lines of MCF-7 human breast cancer cells of differing tumorigenic potential. PTM types and the amino acids on which they occur were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Mass analysis was restricted to MALDI peaks having less than +/-150 parts per million (ppm) error, thereby holding our analysis to a more stringent criterion than previously published work with the HMG proteins. Validation of MALDI-TOF MS analysis was accomplished utilizing electrospray ionization tandem mass spectrometry (ESI MS/MS) and manual analysis of ion fragmentation spectra. Patterns and sites of PTMs identified in this study suggest that HMGA1a proteins, like the histones, exhibit a biochemical modification "code" that relates to cellular function. For example, both increased levels of acetylation and a previously unidentified dimethylation of both lysine and arginine residues were found on HMGA1a proteins from metastatic cells compared to proteins found in their nonmetastatic precursors. Additionally, the types of modification present on lysine-45 (e.g., unmodified, acetylation, or dimethylation) varied, depending on the metastatic potential of cells. These findings suggest that examination of the PTM patterns on HMGA1 proteins may provide valuable information concerning the physiological and phenotypic state of mammalian cells.

Amino Acid Sequence↗
PubMed2004

Microarray profiling of the effects of histone deacetylase inhibitors on gene expression in cancer cell lines.

Chromatin is a highly dynamic environment playing critical roles in the regulation of gene expression. Modifications to the proteins which make up the nucleosome core have been shown to have profound regulatory effects on gene expression. Of these, the best known modification is acetylation of the histone tails. Two enzymes regulate these processes, histone deacetylases and histone acetyltransferases. Both have been shown to have dysregulated functions in certain tumors. Several classes of histone deacetylase inhibitors have been isolated and are currently undergoing evaluation as potential therapeutic modalities in the treatment of cancer. In this study we examined the effects of three such inhibitors on general gene expression in three tumor cell lines derived from three separate tumor types using microarray gene profiling. Our results show that the patterns of alterations which emerge are similar for each cell type.

Carcinoma, Hepatocellular↗
PubMed2003

From discovery to the coming generation of histone deacetylase inhibitors.

Trichostatin A (TSA) is a Streptomyces metabolite that causes differentiation of murine erythroleukemia cells as well as specific inhibition of the cell cycle of some lower eukaryotes and mammalian cells. The targeted molecule of TSA has been shown by genetic and biochemical analyses to be histone deacetylases (HDACs). Histone acetylation is a key modification to control transcription, and HDACs are profoundly involved in pathogenesis of cancer through removing acetyl groups from histones and other transcriptional regulators. Trapoxin (TPX) and FK228 (also known as FR901228 and depsipeptide because FK228 = FR901228 = depsipeptide), structurally unrelated microbial metabolites, were also shown to inhibit HDACs. These HDAC inhibitors cause cell cycle arrest, differentiation and/or apoptosis of many tumors, suggesting their usefulness for chemotherapy and differentiation therapy. In addition, HDAC inhibitors play important roles in identifying the specific function of the enzymes. Indeed, we identified tubulin as one of the substrates of HDAC6 by means of differential sensitivity to HDAC inhibitors. Since recent studies have revealed that HDACs are structurally and functionally diverse, it should be important to develop inhibitors specific to individual enzymes as more promising agents for cancer therapy. We have synthesized novel TSA/TPX hybrids, which will serve as a basis for developing enzyme-specific HDAC inhibitors.

Acetylation↗
PubMed2004

DNA methylation polymorphisms precede any histological sign of atherosclerosis in mice lacking apolipoprotein E.

The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis. To this end, we asked whether the genetically atherosclerosis-prone APOE-null mice show any changes in DNA methylation patterns before the appearance of histologically detectable vascular lesion. We exploited a combination of various techniques: DNA fingerprinting, in vitro methyl-accepting assay, 5-methylcytosine quantitation, histone post-translational modification analysis, Southern blotting, and PCR. Our results show that alterations in DNA methylation profiles, including both hyper- and hypomethylation, were present in aortas and PBMC of 4-week-old mutant mice with no detectable atherosclerotic lesion. Sequencing and expression analysis of 60 leukocytic polymorphisms revealed that epigenetic changes involve transcribed genic sequences, as well as repeated interspersed elements. Furthermore, we showed for the first time that atherogenic lipoproteins promote global DNA hypermethylation in a human monocyte cell line. Taken together, our results unequivocally show that alterations in DNA methylation profiles are early markers of atherosclerosis in a mouse model and may play a causative role in atherogenesis.

Animals↗