Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Vrt||IF, WB, ICC||Rb||Serum||Polyclonal Antibody|
|Presentation||Protein A Purified immunoglobulin in 0.1M Tris-glycine, pH 7.4, 0.15M NaCl, 0.05% sodium azide and 30% glycerol.|
|Safety Information according to GHS|
|Material Size||200 µL|
References | 16 Available | See All References
|Reference overview||Application||Pub Med ID|
|Cerebellar oxidative DNA damage and altered DNA methylation in the BTBR T+tf/J mouse model of autism and similarities with human post mortem cerebellum. |
Shpyleva, S; Ivanovsky, S; de Conti, A; Melnyk, S; Tryndyak, V; Beland, FA; James, SJ; Pogribny, IP
PloS one 9 e113712 2014
The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC). The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (Ogg1) and increased expression of de novo DNA methyltransferases 3a and 3b (Dnmt3a and Dnmt3b). Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression 1 (Tet1) and Tet2 genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of OGG1 alterations in pathogenesis of autism.
|Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines. |
Maroschik, B; Gürtler, A; Krämer, A; Rößler, U; Gomolka, M; Hornhardt, S; Mörtl, S; Friedl, AA
Radiation oncology (London, England) 9 15 2014
Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them.A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients.The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h.So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels.
|Caenorhabditis elegans dosage compensation regulates histone H4 chromatin state on X chromosomes. |
Wells, MB; Snyder, MJ; Custer, LM; Csankovszki, G
Molecular and cellular biology 32 1710-9 2012
Dosage compensation equalizes X-linked gene expression between the sexes. This process is achieved in Caenorhabditis elegans by hermaphrodite-specific, dosage compensation complex (DCC)-mediated, 2-fold X chromosome downregulation. How the DCC downregulates gene expression is not known. By analyzing the distribution of histone modifications in nuclei using quantitative fluorescence microscopy, we found that H4K16 acetylation (H4K16ac) is underrepresented and H4K20 monomethylation (H4K20me1) is enriched on hermaphrodite X chromosomes in a DCC-dependent manner. Depletion of H4K16ac also requires the conserved histone deacetylase SIR-2.1, while enrichment of H4K20me1 requires the activities of the histone methyltransferases SET-1 and SET-4. Our data suggest that the mechanism of dosage compensation in C. elegans involves redistribution of chromatin-modifying activities, leading to a depletion of H4K16ac and an enrichment of H4K20me1 on the X chromosomes. These results support conserved roles for histone H4 chromatin modification in worm dosage compensation analogous to those seen in flies, using similar elements and opposing strategies to achieve differential 2-fold changes in X-linked gene expression.
|Drosophila RB proteins repress differentiation-specific genes via two different mechanisms. |
Lee, H; Ohno, K; Voskoboynik, Y; Ragusano, L; Martinez, A; Dimova, DK
Molecular and cellular biology 30 2563-77 2010
The RB and E2F proteins play important roles in the regulation of cell division, cell death, and development by controlling the expression of genes involved in these processes. The mechanisms of repression by the retinoblastoma protein (pRB) have been extensively studied at cell cycle-regulated promoters. However, little is known about developmentally regulated E2F/RB genes. Here, we have taken advantage of the simplicity of the E2F/RB pathway in flies to inspect the regulation of differentiation-specific target genes. These genes are repressed by dE2F2/RBF and a recently identified RB-containing complex, dREAM/MMB, in a cell type- and cell cycle-independent manner. Our studies indicate that the mechanism of repression differs from that of cell cycle-regulated genes. We find that two different activities are involved in their regulation and that in proliferating cells, both are required to maintain repression. First, dE2F2/RBF and dREAM/MMB employ histone deacetylase (HDAC) activities at promoter regions. Remarkably, we have also uncovered an unconventional mechanism of repression by the Polycomb group (PcG) protein Enhancer of zeste [E(Z)], which is involved in silencing of these genes through the dimethylation of histone H3 Lys27 at nucleosomes located downstream of the transcription start sites (TSS).Full Text Article
|Ornithine decarboxylase antizyme induces hypomethylation of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2) in human oral cancer cell line. |
Yamamoto, D; Shima, K; Matsuo, K; Nishioka, T; Chen, CY; Hu, GF; Sasaki, A; Tsuji, T
PloS one 5 e12554 2010
Methylation of CpG islands of genome DNA and lysine residues of histone H3 and H4 tails regulates gene transcription. Inhibition of polyamine synthesis by ornithine decarboxylase antizyme-1 (OAZ) in human oral cancer cell line resulted in accumulation of decarboxylated S-adenosylmethionine (dcSAM), which acts as a competitive inhibitor of methylation reactions. We anticipated that accumulation of dcSAM impaired methylation reactions and resulted in hypomethylation of genome DNA and histone tails.Global methylation state of genome DNA and lysine residues of histone H3 and H4 tails were assayed by Methylation by Isoschizomers (MIAMI) method and western blotting, respectively, in the presence or absence of OAZ expression. Ectopic expression of OAZ mediated hypomethylation of CpG islands of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2). Protein level of DNA methyltransferase 3B (DNMT3B) and histone H3K9me specific methyltransferase G9a were down-regulated in OAZ transfectant.OAZ induced hypomethylation of CpG islands of global genome DNA and H3K9me2 by down-regulating DNMT3B and G9a protein level. Hypomethylation of CpG islands of genome DNA and histone H3K9me2 is a potent mechanism of induction of the genes related to tumor suppression and DNA double strand break repair.Full Text Article
|The target of the NSD family of histone lysine methyltransferases depends on the nature of the substrate. |
Li, Yan, et al.
J. Biol. Chem., 284: 34283-95 (2009) 2009
The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target.
|Global repression of cancer gene expression in a zebrafish model of melanoma is linked to epigenetic regulation. |
Viviana Anelli,Cristina Santoriello,Martin Distel,Reinhard W Köster,Francesca D Ciccarelli,Marina Mione
Zebrafish 6 2009
We have established a model of melanoma progression in zebrafish through the generation of transgenic lines specifically expressing oncogenic human HRAS in the melanocytic lineage. In these tumors we have carried out quantitative expression analysis of several putative cancer genes, from known and predicted cancer gene lists. In particular, we analyzed 39 out of 101 putative cancer genes identified with a bioinformatics approach and selected for the low frequency of duplication and the high connectivity in protein networks. Data obtained by real-time polymerase chain reaction analysis from zebrafish melanoma tissue shows that the expression of many cancer genes is downregulated in zebrafish melanomas, whereas only cell cycle genes are upregulated. To understand whether this trend is due to global repression of gene expression associated to a repressive chromatin state, we investigated whether changes of histone methylation were detectable in our melanoma model. We found substantial differences in the levels of H3K9me3, H4K20me2, H3K27me3, H3K4me3, and H3R2me2a immunostaining in melanoma tissue when compared with normal skin. Thus our analysis suggests that in our model, like in human melanoma, important changes occur to the methylation status of histones. Although the outcome of these changes is still unknown, they could be responsible for the global repression of gene expression through epigenetic regulation shown in this study.
|Ordered histone modifications are associated with transcriptional poising and activation of the phaseolin promoter. |
Ng, DW; Chandrasekharan, MB; Hall, TC
The Plant cell 18 119-32 2006
The phaseolin (phas) promoter drives copious production of transcripts encoding the protein phaseolin during seed embryogenesis but is silent in vegetative tissues, in which a nucleosome is positioned over its three-phased TATA boxes. Transition from the inactive state in transgenic Arabidopsis thaliana leaves was accomplished by ectopic expression of the transcription factor Phaseolus vulgaris ABI3-like factor (ALF) and application of abscisic acid (ABA). Placement of hemagglutinin-tagged ALF expression under the control of an estradiol-inducible promoter permitted chromatin immunoprecipitation analysis of chronological changes in histone modifications, notably increased acetylation of H3-K9 and H4-K12, as phas chromatin was remodeled (potentiated). A different array of changes, including acetylation of H3-K14 and methylation of H3-K4, was found to be associated with ABA-mediated activation. Thus, temporal separation of phas potentiation from activation revealed that histone H3 and H4 Lys residues are not globally hyperacetylated during phas expression. Whereas decreases in histone H3 and H4 levels were detected during ALF-mediated remodeling, slight increases occurred after ABA-mediated activation, suggesting the restoration of histone-phas interactions or the replacement of histones in the phas chromatin. The observed histone modifications provide insight into factors involved in the euchromatinization and activation of a plant gene and expand the evidence for histone code conservation among eukaryotes.Full Text Article
|Partitioning of the maize epigenome by the number of methyl groups on histone H3 lysines 9 and 27. |
Shi, J; Dawe, RK
Genetics 173 1571-83 2006
We report a detailed analysis of maize chromosome structure with respect to seven histone H3 methylation states (dimethylation at lysine 4 and mono-, di-, and trimethylation at lysines 9 and 27). Three-dimensional light microscopy and the fine cytological resolution of maize pachytene chromosomes made it possible to compare the distribution of individual histone methylation events to each other and to DNA staining intensity. Major conclusions are that (1) H3K27me2 marks classical heterochromatin; (2) H3K4me2 is limited to areas between and around H3K27me2-marked chromomeres, clearly demarcating the euchromatic gene space; (3) H3K9me2 is restricted to the euchromatic gene space; (4) H3K27me3 occurs in a few (roughly seven) focused euchromatic domains; (5) centromeres and CENP-C are closely associated with H3K9me2 and H3K9me3; and (6) histone H4K20 di- and trimethylation are nearly or completely absent in maize. Each methylation state identifies different regions of the epigenome. We discuss the evolutionary lability of histone methylation profiles and draw a distinction between H3K9me2-mediated gene silencing and heterochromatin formation.Full Text Article
|Western Blotting, Immunofluorescence||16624902|
|Profile of histone lysine methylation across transcribed mammalian chromatin. |
Vakoc, CR; Sachdeva, MM; Wang, H; Blobel, GA
Molecular and cellular biology 26 9185-95 2006
Complex patterns of histone lysine methylation encode distinct functions within chromatin. We previously reported that trimethylation of lysine 9 of histone H3 (H3K9) occurs at both silent heterochromatin and at the transcribed regions of active mammalian genes, suggesting that the extent of histone lysine methylation involved in mammalian gene activation is not completely defined. To identify additional sites of histone methylation that respond to mammalian gene activity, we describe here a comparative assessment of all six known positions of histone lysine methylation and relate them to gene transcription. Using several model loci, we observed high trimethylation of H3K4, H3K9, H3K36, and H3K79 in the transcribed region, consistent with previous findings. We identify H4K20 monomethylation, a modification previously linked with repression, as a mark of transcription elongation in mammalian cells. In contrast, H3K27 monomethylation, a modification enriched at pericentromeric heterochromatin, was observed broadly distributed throughout all euchromatic sites analyzed, with selective depletion in the vicinity of the transcription start sites at active genes. Together, these results underscore that similar to other described methyl-lysine modifications, H4K20 and H3K27 monomethylation are versatile and dynamic with respect to gene activity, suggesting the existence of novel site-specific methyltransferases and demethylases coupled to the transcription cycle.
|Direct interaction between DNMT1 and G9a coordinates DNA and histone methylation during replication. |
Estève, PO; Chin, HG; Smallwood, A; Feehery, GR; Gangisetty, O; Karpf, AR; Carey, MF; Pradhan, S
Genes & development 20 3089-103 2006
Chromatin methylation is necessary for stable repression of gene expression during mammalian development. During cell division, DNMT1 maintains the DNA methylation pattern of the newly synthesized daughter strand, while G9a methylates H3K9. Here, DNMT1 is shown to directly bind G9a both in vivo and in vitro and to colocalize in the nucleus during DNA replication. The complex of DNMT1 and G9a colocalizes with dimethylated H3K9 (H3K9me2) at replication foci. Similarly, another H3K9 histone methyltransferase, SUV39H1, colocalizes with DNMT1 on heterochromatic regions of the nucleoli exclusively before cell division. Both DNMT1 and G9a are loaded onto the chromatin simultaneously in a ternary complex with loading factor PCNA during chromatin replication. Small interfering RNA (siRNA) knockdown of DNMT1 impairs DNA methylation, G9a loading, and H3K9 methylation on chromatin and rDNA repeats, confirming DNMT1 as the primary loading factor. Additionally, the complex of DNMT1 and G9a led to enhanced DNA and histone methylation of in vitro assembled chromatin substrates. Thus, direct cooperation between DNMT1 and G9a provides a mechanism of coordinated DNA and H3K9 methylation during cell division.
|Monomethyl histone H3 lysine 4 as an epigenetic mark for silenced euchromatin in Chlamydomonas. |
van Dijk, K; Marley, KE; Jeong, BR; Xu, J; Hesson, J; Cerny, RL; Waterborg, JH; Cerutti, H
The Plant cell 17 2439-53 2005
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.
|Histone H4-lysine 20 monomethylation is increased in promoter and coding regions of active genes and correlates with hyperacetylation. |
Talasz, H; Lindner, HH; Sarg, B; Helliger, W
The Journal of biological chemistry 280 38814-22 2005
Methylation and acetylation of position-specific lysine residues in the N-terminal tail of histones H3 and H4 play an important role in regulating chromatin structure and function. In the case of H3-Lys(4), H3-Lys(9), H3-Lys(27), and H4-Lys(20), the degree of methylation was variable from the mono- to the di- or trimethylated state, each of which was presumed to be involved in the organization of chromatin and the activation or repression of genes. Here we investigated the interplay between histone H4-Lys(20) mono- and trim-ethylation and H4 acetylation at induced (beta-major/beta-minor glo-bin), repressed (c-myc), and silent (embryonic beta-globin) genes during in vitro differentiation of mouse erythroleukemia cells. By using chromatin immunoprecipitation, we found that the beta-major and beta-minor promoter and the beta-globin coding regions as well as the promoter and the transcribed exon 2 regions of the highly expressed c-myc gene were hyperacetylated and monomethylated at H4-Lys(20). Although activation of the beta-globin gene resulted in an increase in hyperacetylated, monomethylated H4, down-regulation of the c-myc gene did not cause a decrease in hyperacetylated, monomethylated H4-Lys(20), thus showing a stable pattern of histone modifications. Immunofluorescence microscopy studies revealed that monomethylated H4-Lys(20) mainly overlaps with RNA pol II-stained euchromatic regions, thus indicating an association with transcriptionally engaged chromatin. Our chromatin immunoprecipitation results demonstrated that in contrast to trimethylated H4-Lys(20), which was found to inversely correlate with H4 hyper-acetylation, H4-Lys(20) monomethylation is compatible with histone H4 hyperacetylation and correlates with the transcriptionally active or competent chromatin state.
|A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. |
Schotta, G; Lachner, M; Sarma, K; Ebert, A; Sengupta, R; Reuter, G; Reinberg, D; Jenuwein, T
Genes & development 18 1251-62 2004
Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. Here we show that H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, the Suv4-20 homolog is a novel PEV modifier to regulate position-effect variegation. Together, our data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin.
|A chromosomal memory triggered by Xist regulates histone methylation in X inactivation. |
Kohlmaier, A; Savarese, F; Lachner, M; Martens, J; Jenuwein, T; Wutz, A
PLoS biology 2 E171 2004
We have elucidated the kinetics of histone methylation during X inactivation using an inducible Xist expression system in mouse embryonic stem (ES) cells. Previous reports showed that the ability of Xist to trigger silencing is restricted to an early window in ES cell differentiation. Here we show that this window is also important for establishing methylation patterns on the potential inactive X chromosome. By immunofluorescence and chromatin immunoprecipitation experiments we show that histone H3 lysine 27 trimethylation (H3K27m3) and H4 lysine 20 monomethylation (H4K20m1) are associated with Xist expression in undifferentiated ES cells and mark the initiation of X inactivation. Both marks depend on Xist RNA localisation but are independent of silencing. Induction of Xist expression after the initiation window leads to a markedly reduced ability to induce H3K27m3, whereas expression before the restrictive time point allows efficient H3K27m3 establishment. Our data show that Xist expression early in ES cell differentiation establishes a chromosomal memory, which is maintained in the absence of silencing. One consequence of this memory is the ability to introduce H3K27m3 efficiently after the restrictive time point on the chromosome that has expressed Xist early. Our results suggest that this silencing-independent chromosomal memory has important implications for the maintenance of X inactivation, where previously self-perpetuating heterochromatin structures were viewed as the principal form of memory.Full Text Article
|Purification and functional characterization of SET8, a nucleosomal histone H4-lysine 20-specific methyltransferase. |
Fang, Jia, et al.
Curr. Biol., 12: 1086-99 (2002) 2002
BACKGROUND: Covalent modifications of histone N-terminal tails play fundamental roles in regulating chromatin structure and function. Extensive studies have established that acetylation of specific lysine residues in the histone tails plays an important role in transcriptional regulation. Besides acetylation, recent studies have revealed that histone methylation also has significant effects on heterochromatin formation and transcriptional regulation. Histone methylation occurs on specific arginine and lysine residues of histones H3 and H4. Thus far, only 2 residues on histone H4 are known to be methylated. While H4-arginine 3 (H4-R3) methylation is mediated by PRMT1, the enzyme(s) responsible for H4-lysine 20 (H4-K20) methylation is not known.RESULTS: To gain insight into the function of H4-K20 methylation, we set out to identify the enzyme responsible for this modification. We purified and cloned a novel human SET domain-containing protein, named SET8, which specifically methylates H4 at K20. SET8 is a single subunit enzyme and prefers nucleosomal substrates. We find that H4-K20 methylation occurs in a wide range of higher eukaryotic organisms and that SET8 homologs exist in C. elegans and Drosophila. We demonstrate that the Drosophila SET8 homolog has the same substrate specificity as its human counterpart. Importantly, disruption of SET8 in Drosophila reduces levels of H4-K20 methylation in vivo and results in lethality. Although H4-K20 methylation does not correlate with gene activity, it appears to be regulated during the cell cycle.CONCLUSIONS: We identified and characterized an evolutionarily conserved nucleosomal H4-K20-specific methyltransferase and demonstrated its essential role in Drosophila development.