Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Vrt||ChIP, WB||Rb||Serum||Polyclonal Antibody|
|Presentation||Rabbit antiserum polyclonal IgG in buffer containing 0.05% sodium azide.
Frozen at -20°C.
|Safety Information according to GHS|
|Material Size||100 µL|
|Anti-dimethyl-Histone H3 (Lys36) - 1966941||1966941|
|Anti-dimethyl-Histone H3 (Lys36) - 22435||22435|
|Anti-dimethyl-Histone H3 (Lys36) - 30257||30257|
|Anti-dimethyl-Histone H3 (Lys36) - DAM1641102||DAM1641102|
|Anti-dimethyl-Histone H3 (Lys36) - DAM1745326||DAM1745326|
|Anti-dimethyl-Histone H3 (Lys36) - DAM1787879||DAM1787879|
|Anti-dimethyl-Histone H3 (Lys36) - JBC1361840||JBC1361840|
|Anti-dimethyl-Histone H3 (Lys36) - JBC1374254||JBC1374254|
|Anti-dimethyl-Histone H3 (Lys36) - JBC1374254||JBC1374254|
|Anti-dimethyl-Histone H3 (Lys36) - JBC1873475||JBC1873475|
References | 46 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae. |
Cucinotta, CE; Young, AN; Klucevsek, KM; Arndt, KM
PLoS genetics 11 e1005420 2015
Eukaryotes regulate gene expression and other nuclear processes through the posttranslational modification of histones. In S. cerevisiae, the mono-ubiquitylation of histone H2B on lysine 123 (H2B K123ub) affects nucleosome stability, broadly influences gene expression and other DNA-templated processes, and is a prerequisite for additional conserved histone modifications that are associated with active transcription, namely the methylation of lysine residues in H3. While the enzymes that promote these chromatin marks are known, regions of the nucleosome required for the recruitment of these enzymes are undefined. To identify histone residues required for H2B K123ub, we exploited a functional interaction between the ubiquitin-protein ligase, Rkr1/Ltn1, and H2B K123ub in S. cerevisiae. Specifically, we performed a synthetic lethal screen with cells lacking RKR1 and a comprehensive library of H2A and H2B residue substitutions, and identified H2A residues that are required for H2B K123ub. Many of these residues map to the nucleosome acidic patch. The substitutions in the acidic patch confer varying histone modification defects downstream of H2B K123ub, indicating that this region contributes differentially to multiple histone modifications. Interestingly, substitutions in the acidic patch result in decreased recruitment of H2B K123ub machinery to active genes and defects in transcription elongation and termination. Together, our findings reveal a role for the nucleosome acidic patch in recruitment of histone modification machinery and maintenance of transcriptional integrity.
|Histone H3.3 and its proteolytically processed form drive a cellular senescence programme. |
Duarte, LF; Young, AR; Wang, Z; Wu, HA; Panda, T; Kou, Y; Kapoor, A; Hasson, D; Mills, NR; Ma'ayan, A; Narita, M; Bernstein, E
Nature communications 5 5210 2014
The process of cellular senescence generates a repressive chromatin environment, however, the role of histone variants and histone proteolytic cleavage in senescence remains unclear. Here, using models of oncogene-induced and replicative senescence, we report novel histone H3 tail cleavage events mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and the histone variant H3.3 is the preferred cleaved form of H3. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first 21 amino acids of the H3 tail, is sufficient to induce senescence. Further, H3.3cs1 chromatin incorporation is mediated by the HUCA histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our study identifies histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence.
|dRYBP counteracts chromatin-dependent activation and repression of transcription. |
Fereres, S; Simón, R; Mohd-Sarip, A; Verrijzer, CP; Busturia, A
PloS one 9 e113255 2014
Chromatin dependent activation and repression of transcription is regulated by the histone modifying enzymatic activities of the trithorax (trxG) and Polycomb (PcG) proteins. To investigate the mechanisms underlying their mutual antagonistic activities we analyzed the function of Drosophila dRYBP, a conserved PcG- and trxG-associated protein. We show that dRYBP is itself ubiquitylated and binds ubiquitylated proteins. Additionally we show that dRYBP maintains H2A monoubiquitylation, H3K4 monomethylation and H3K36 dimethylation levels and does not affect H3K27 trimethylation levels. Further we show that dRYBP interacts with the repressive SCE and dKDM2 proteins as well as the activating dBRE1 protein. Analysis of homeotic phenotypes and post-translationally modified histones levels show that dRYBP antagonizes dKDM2 and dBRE1 functions by respectively preventing H3K36me2 demethylation and H2B monoubiquitylation. Interestingly, our results show that inactivation of dBRE1 produces trithorax-like related homeotic transformations, suggesting that dBRE1 functions in the regulation of homeotic genes expression. Our findings indicate that dRYBP regulates morphogenesis by counteracting transcriptional repression and activation. Thus, they suggest that dRYBP may participate in the epigenetic plasticity important during normal and pathological development.
|The ASH1-RELATED3 SET-domain protein controls cell division competence of the meristem and the quiescent center of the Arabidopsis primary root. |
Kumpf, R; Thorstensen, T; Rahman, MA; Heyman, J; Nenseth, HZ; Lammens, T; Herrmann, U; Swarup, R; Veiseth, SV; Emberland, G; Bennett, MJ; De Veylder, L; Aalen, RB
Plant physiology 166 632-43 2014
The stem cell niche of the Arabidopsis (Arabidopsis thaliana) primary root apical meristem is composed of the quiescent (or organizing) center surrounded by stem (initial) cells for the different tissues. Initial cells generate a population of transit-amplifying cells that undergo a limited number of cell divisions before elongating and differentiating. It is unclear whether these divisions occur stochastically or in an orderly manner. Using the thymidine analog 5-ethynyl-2'-deoxyuridine to monitor DNA replication of cells of Arabidopsis root meristems, we identified a pattern of two, four, and eight neighboring cells with synchronized replication along the cortical, epidermal, and endodermal cell files, suggested to be daughters, granddaughters, and great-granddaughters of the direct progeny of each stem cell. Markers of mitosis and cytokinesis were not present in the region closest to the transition zone where the cells start to elongate, suggesting that great-granddaughter cells switch synchronously from the mitotic cell cycle to endoreduplication. Mutations in the stem cell niche-expressed ASH1-RELATED3 (ASHR3) gene, encoding a SET-domain protein conferring histone H3 lysine-36 methylation, disrupted this pattern of coordinated DNA replication and cell division and increased the cell division rate in the quiescent center. E2Fa/E2Fb transcription factors controlling the G1-to-S-phase transition regulate ASHR3 expression and bind to the ASHR3 promoter, substantiating a role for ASHR3 in cell division control. The reduced length of the root apical meristem and primary root of the mutant ashr3-1 indicate that synchronization of replication and cell divisions is required for normal root growth and development.
|The demethylase JMJD2C localizes to H3K4me3-positive transcription start sites and is dispensable for embryonic development. |
Pedersen, MT; Agger, K; Laugesen, A; Johansen, JV; Cloos, PA; Christensen, J; Helin, K
Molecular and cellular biology 34 1031-45 2014
The histone demethylase JMJD2C, also known as KDM4C/GASC1, has activity against methylated H3K9 and H3K36 and is amplified and/or overexpressed in human cancers. By the generation of Jmjd2c knockout mice, we demonstrate that loss of Jmjd2c is compatible with cellular proliferation, embryonic stem cell (ESC) self-renewal, and embryonic development. Moreover, we report that JMJD2C localizes to H3K4me3-positive transcription start sites in both primary cells and in the human carcinoma KYSE150 cell line containing an amplification of the JMJD2C locus. Binding is dependent on the double Tudor domain of JMJD2C, which recognizes H3K4me3 but not H4K20me2/me3 in vitro, showing a binding specificity different from that of the double Tudor domains of JMJD2A and JMJD2B. Depletion of JMJD2C in KYSE150 cells has a modest effect on H3K9me3 and H3K36me3 levels but impairs proliferation and leads to deregulated expression of a subset of target genes involved in cell cycle progression. Taking these findings together, we show that JMJD2C is targeted to H3K4me3-positive transcription start sites, where it can contribute to transcriptional regulation, and report that the putative oncogene JMJD2C generally is not required for cellular proliferation or embryonic development.
|Histone methyltransferase MMSET/NSD2 alters EZH2 binding and reprograms the myeloma epigenome through global and focal changes in H3K36 and H3K27 methylation. |
Popovic, R; Martinez-Garcia, E; Giannopoulou, EG; Zhang, Q; Zhang, Q; Ezponda, T; Shah, MY; Zheng, Y; Will, CM; Small, EC; Hua, Y; Bulic, M; Jiang, Y; Carrara, M; Calogero, RA; Kath, WL; Kelleher, NL; Wang, JP; Elemento, O; Licht, JD
PLoS genetics 10 e1004566 2014
Overexpression of the histone methyltransferase MMSET in t(4;14)+ multiple myeloma patients is believed to be the driving factor in the pathogenesis of this subtype of myeloma. MMSET catalyzes dimethylation of lysine 36 on histone H3 (H3K36me2), and its overexpression causes a global increase in H3K36me2, redistributing this mark in a broad, elevated level across the genome. Here, we demonstrate that an increased level of MMSET also induces a global reduction of lysine 27 trimethylation on histone H3 (H3K27me3). Despite the net decrease in H3K27 methylation, specific genomic loci exhibit enhanced recruitment of the EZH2 histone methyltransferase and become hypermethylated on this residue. These effects likely contribute to the myeloma phenotype since MMSET-overexpressing cells displayed increased sensitivity to EZH2 inhibition. Furthermore, we demonstrate that such MMSET-mediated epigenetic changes require a number of functional domains within the protein, including PHD domains that mediate MMSET recruitment to chromatin. In vivo, targeting of MMSET by an inducible shRNA reversed histone methylation changes and led to regression of established tumors in athymic mice. Together, our work elucidates previously unrecognized interplay between MMSET and EZH2 in myeloma oncogenesis and identifies domains to be considered when designing inhibitors of MMSET function.
|Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation. |
Le Dily, F; Baù, D; Pohl, A; Vicent, GP; Serra, F; Soronellas, D; Castellano, G; Wright, RH; Ballare, C; Filion, G; Marti-Renom, MA; Beato, M
Genes & development 28 2151-62 2014
The human genome is segmented into topologically associating domains (TADs), but the role of this conserved organization during transient changes in gene expression is not known. Here we describe the distribution of progestin-induced chromatin modifications and changes in transcriptional activity over TADs in T47D breast cancer cells. Using ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing), Hi-C (chromosome capture followed by high-throughput sequencing), and three-dimensional (3D) modeling techniques, we found that the borders of the ∼ 2000 TADs in these cells are largely maintained after hormone treatment and that up to 20% of the TADs could be considered as discrete regulatory units where the majority of the genes are either transcriptionally activated or repressed in a coordinated fashion. The epigenetic signatures of the TADs are homogeneously modified by hormones in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodeling are accompanied by differential structural changes for activated and repressed TADs, as reflected by specific and opposite changes in the strength of intra-TAD interactions within responsive TADs. Indeed, 3D modeling of the Hi-C data suggested that the structure of TADs was modified upon treatment. The differential responses of TADs to progestins and estrogens suggest that TADs could function as "regulons" to enable spatially proximal genes to be coordinately transcribed in response to hormones.
|NuA4 links methylation of histone H3 lysines 4 and 36 to acetylation of histones H4 and H3. |
Ginsburg, DS; Anlembom, TE; Wang, J; Patel, SR; Li, B; Hinnebusch, AG
The Journal of biological chemistry 289 32656-70 2014
Cotranscriptional methylation of histone H3 lysines 4 and 36 by Set1 and Set2, respectively, stimulates interaction between nucleosomes and histone deacetylase complexes to block cryptic transcription in budding yeast. We previously showed that loss of all H3K4 and H3K36 methylation in a set1Δset2Δ mutant reduces interaction between native nucleosomes and the NuA4 lysine acetyltransferase (KAT) complex. We now provide evidence that NuA4 preferentially binds H3 tails mono- and dimethylated on H3K4 and di- and trimethylated on H3K36, an H3 methylation pattern distinct from that recognized by the RPD3C(S) and Hos2/Set3 histone deacetylase complexes (HDACs). Loss of H3K4 or H3K36 methylation in set1Δ or set2Δ mutants reduces NuA4 interaction with bulk nucleosomes in vitro and in vivo, and reduces NuA4 occupancy of transcribed coding sequences at particular genes. We also provide evidence that NuA4 acetylation of lysine residues in the histone H4 tail stimulates SAGA interaction with nucleosomes and its recruitment to coding sequences and attendant acetylation of histone H3 in vivo. Thus, H3 methylation exerts opposing effects of enhancing nucleosome acetylation by both NuA4 and SAGA as well as stimulating nucleosome deacetylation by multiple HDACs to maintain the proper level of histone acetylation in transcribed coding sequences.
|Pharmacological inhibition of polycomb repressive complex-2 activity induces apoptosis in human colon cancer stem cells. |
Benoit, YD; Witherspoon, MS; Laursen, KB; Guezguez, A; Beauséjour, M; Beaulieu, JF; Lipkin, SM; Gudas, LJ
Experimental cell research 319 1463-70 2013
Colorectal cancer is among the leading causes of cancer death in the USA. The polycomb repressive complex 2 (PRC2), including core components SUZ12 and EZH2, represents a key epigenetic regulator of digestive epithelial cell physiology and was previously shown to promote deleterious effects in a number of human cancers, including colon. Using colon cancer stem cells (CCSC) isolated from human primary colorectal tumors, we demonstrate that SUZ12 knockdown and treatment with DZNep, one of the most potent EZH2 inhibitors, increase apoptosis levels, marked by decreased Akt phosphorylation, in CCSCs, while embryonic stem (ES) cell survival is not affected. Moreover, DZNep treatments lead to increased PTEN expression in these highly tumorigenic cells. Taken together, our findings suggest that pharmacological inhibition of PRC2 histone methyltransferase activity may constitute a new, epigenetic therapeutic strategy to target highly tumorigenic and metastatic colon cancer stem cells.
|Inhibition of PRC2 histone methyltransferase activity increases TRAIL-mediated apoptosis sensitivity in human colon cancer cells. |
Benoit, YD; Laursen, KB; Witherspoon, MS; Lipkin, SM; Gudas, LJ
Journal of cellular physiology 228 764-72 2013
Colorectal cancer is ranked among the top leading causes of cancer death in industrialized populations. Polycomb group proteins, including Suz12 and Ezh2, are epigenetic regulatory proteins that act as transcriptional repressors of many differentiation-associated genes and are overexpressed in a large subset of colorectal cancers. Retinoic acid (RA) acts as a negative regulator of PcG actions in stem cells, but has shown limited therapeutic potential in some solid tumors, including colorectal cancer, in part because of retinoic acid receptor β silencing. Through treatment with RA, Suz12 shRNA knockdown, or Ezh2 pharmacological inhibition with 3-deazaneplanocin A (DZNep), we increased TRAIL-mediated apoptosis in human colorectal cancer cell lines. This increased apoptosis in human colon cancer cells after RA or DZNep treatment was associated with a ~2.5-fold increase in TNFRSF10B (DR5) transcript levels and a 42% reduction in the H3K27me3 epigenetic mark at the TNFRSF10B promoter after DZNep addition. Taken together, our findings indicate that pharmacological inhibition of Polycomb repressive complex 2 histone methyltransferase activity may constitute a new epigenetic therapeutic strategy to overcome RA non-responsiveness in a subset of colorectal tumors by increasing TRAIL-mediated apoptosis sensitivity.
|The histone methyltransferase MMSET regulates class switch recombination. |
Pei, H; Wu, X; Liu, T; Yu, K; Jelinek, DF; Lou, Z
Journal of immunology (Baltimore, Md. : 1950) 190 756-63 2013
Wolf-Hirschhorn syndrome (WHS) is a genetic disease with characteristic facial features and developmental disorders. Of interest, loss of the MMSET gene (also known as WHSC1) is considered to be responsible for the core phenotypes of this disease. Patients with WHS also display Ab deficiency, although the underlying cause of this deficiency is unclear. Recent studies suggest that the histone methyltransferase activity of MMSET plays an important role in the DNA damage response by facilitating the recruitment of 53BP1 to sites of DNA damage. We hypothesize that MMSET also regulates class switch recombination (CSR) through its effect on 53BP1. In this study, we show that MMSET indeed plays an important role in CSR through its histone methyltransferase activity. Knocking down MMSET expression impaired 53BP1 recruitment as well as the germline transcription of the Igh switch regions, resulting in defective CSR but no effect on cell growth and viability. These results suggest that defective CSR caused by MMSET deficiency could be a cause of Ab deficiency in WHS patients.
|The histone methyltransferase MMSET/WHSC1 activates TWIST1 to promote an epithelial-mesenchymal transition and invasive properties of prostate cancer. |
Ezponda, T; Popovic, R; Shah, MY; Martinez-Garcia, E; Zheng, Y; Min, DJ; Will, C; Neri, A; Kelleher, NL; Yu, J; Licht, JD
Oncogene 32 2882-90 2013
Epigenetic deregulation of gene expression has a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma SET domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared with immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, whereas depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial-mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus and leads to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET has a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.
|Genomic insights of protein arginine methyltransferase Hmt1 binding reveals novel regulatory functions. |
Milliman, EJ; Hu, Z; Yu, MC
BMC genomics 13 728 2012
Protein arginine methylation is a post-translational modification involved in important biological processes such as transcription and RNA processing. This modification is catalyzed by both type I and II protein arginine methyltransferases (PRMTs). One of the most conserved type I PRMTs is PRMT1, the homolog of which is Hmt1 in Saccharomyces cerevisiae. Hmt1 has been shown to play a role in various gene expression steps, such as promoting the dynamics of messenger ribonucleoprotein particle (mRNP) biogenesis, pre-mRNA splicing, and silencing of chromatin. To determine the full extent of Hmt1's involvement during gene expression, we carried out a genome-wide location analysis for Hmt1.A comprehensive genome-wide binding profile for Hmt1 was obtained by ChIP-chip using NimbleGen high-resolution tiling microarrays. Of the approximately 1000 Hmt1-binding sites found, the majority fall within or proximal to an ORF. Different occupancy patterns of Hmt1 across genes with different transcriptional rates were found. Interestingly, Hmt1 occupancy is found at a number of other genomic features such as tRNA and snoRNA genes, thereby implicating a regulatory role in the biogenesis of these non-coding RNAs. RNA hybridization analysis shows that Hmt1 loss-of-function mutants display higher steady-state tRNA abundance relative to the wild-type. Co-immunoprecipitation studies demonstrate that Hmt1 interacts with the TFIIIB component Bdp1, suggesting a mechanism for Hmt1 in modulating RNA Pol III transcription to regulate tRNA production.The genome-wide binding profile of Hmt1 reveals multiple potential new roles for Hmt1 in the control of eukaryotic gene expression, especially in the realm of non-coding RNAs. The data obtained here will provide an important blueprint for future mechanistic studies on the described occupancy relationship for genomic features bound by Hmt1.
|Rhythmic oscillation of histone acetylation and methylation at the Arabidopsis central clock loci. |
Song, HR; Noh, YS
Molecules and cells 34 279-87 2012
Circadian clock genes are regulated by a transcriptional-translational feedback loop. In Arabidopsis, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) transcripts are highly expressed in the morning. Translated LHY and CCA1 proteins repress the expression of TIMING OF CAB EXPRESSION 1 (TOC1), which peaks in the evening. TOC1 protein induces expression of LHY and CCA1, forming a negative feedback loop which is believed to constitute the oscillatory mechanism of the clock. The rhythmic oscillation of mouse clock genes mPERIOD 1 (mPER1) and mPER2 has been correlated with regular alteration of chromatin structure through histone acetylation/deacetylation. However, little is known about the relationship between the transcriptional activity of Arabidopsis clock genes and their chromatin status. Here, we report that histone H3 acetylation (H3Ac) and H3 lysine 4 tri-methylation (H3K4me3) levels at LHY, CCA1, and TOC1 are positively correlated with the rhythmic transcript levels of these genes, whereas H3K36me2 level shows a negative correlation. Thus, our study suggests rhythmic transcription of Arabidopsis clock genes might be regulated by rhythmic histone modification, and it provides a platform for future identification of clock-controlling histone modifiers.
|JMJD5, a Jumonji C (JmjC) domain-containing protein, negatively regulates osteoclastogenesis by facilitating NFATc1 protein degradation. |
Youn, MY; Yokoyama, A; Fujiyama-Nakamura, S; Ohtake, F; Minehata, K; Yasuda, H; Suzuki, T; Kato, S; Imai, Y
The Journal of biological chemistry 287 12994-3004 2012
Osteoclastogenesis is a highly regulated process governed by diverse classes of regulators. Among them, nuclear factor of activated T-cells calcineurin-dependent 1 (NFATc1) is the primary osteoclastogenic transcription factor, and its expression is transcriptionally induced during early osteoclastogenesis by receptor activation of nuclear factor κB ligand (RANKL), an osteoclastogenic cytokine. Here, we report the novel enzymatic function of JMJD5, which regulates NFATc1 protein stability. Among the tested Jumonji C (JmjC) domain-containing proteins, decreased mRNA expression levels during osteoclastogenesis were found for JMJD5 in RAW264 cells stimulated by RANKL. To examine the functional role of JMJD5 in osteoclast differentiation, we established stable JMJD5 knockdown cells, and osteoclast formation was assessed. Down-regulated expression of JMJD5 led to accelerated osteoclast formation together with induction of several osteoclast-specific genes such as Ctsk and DC-STAMP, suggesting that JMJD5 is a negative regulator in osteoclast differentiation. Although JMJD5 was recently reported as a histone demethylase for histone H3K36me2, no histone demethylase activity was detected in JMJD5 in vitro or in living cells, even for other methylated histone residues. Instead, JMJD5 co-repressed transcriptional activity by destabilizing NFATc1 protein. Protein hydroxylase activity mediated by the JmjC domain in JMJD5 was required for the observed functions of JMJD5. JMJD5 induced the association of hydroxylated NFATc1 with the E3 ubiquitin ligase Von Hippel-Lindau tumor suppressor (VHL), thereby presumably facilitating proteasomal degradation of NFATc1 via ubiquitination. Taken together, the present study demonstrated that JMJD5 is a post-translational co-repressor for NFATc1 that attenuates osteoclastogenesis.
|HDA6 directly interacts with DNA methyltransferase MET1 and maintains transposable element silencing in Arabidopsis. |
Liu, X; Yu, CW; Duan, J; Luo, M; Wang, K; Tian, G; Cui, Y; Wu, K
Plant physiology 158 119-29 2012
The molecular mechanism of how the histone deacetylase HDA6 participates in maintaining transposable element (TE) silencing in Arabidopsis (Arabidopsis thaliana) is not yet defined. In this study, we show that a subset of TEs was transcriptionally reactivated and that TE reactivation was associated with elevated histone H3 and H4 acetylation as well as increased H3K4Me3 and H3K4Me2 in hda6 mutants. Decreased DNA methylation of the TEs was also detected in hda6 mutants, suggesting that HDA6 silences the TEs by regulating histone acetylation and methylation as well as the DNA methylation status of the TEs. Similarly, transcripts of some of these TEs were also increased in the methyltransferase1 (met1) mutant, with decreased DNA methylation. Furthermore, H4 acetylation, H3K4Me3, H3K4Me2, and H3K36Me2 were enriched at the coregulated TEs in the met1 and hda6 met1 mutants. Protein-protein interaction analysis indicated that HDA6 physically interacts with MET1 in vitro and in vivo, and further deletion analysis demonstrated that the carboxyl-terminal region of HDA6 and the bromo-adjacent homology domain of MET1 were responsible for the interaction. These results suggested that HDA6 and MET1 interact directly and act together to silence TEs by modulating DNA methylation, histone acetylation, and histone methylation status.Full Text Article
|The chromatin remodeler ISWI regulates the cellular response to hypoxia: role of FIH. |
Melvin, A; Mudie, S; Rocha, S
Molecular biology of the cell 22 4171-81 2011
The hypoxia-inducible factor (HIF) is a master regulator of the cellular response to hypoxia. Its levels and activity are controlled by dioxygenases called prolyl-hydroxylases and factor inhibiting HIF (FIH). To activate genes, HIF has to access sequences in DNA that are integrated in chromatin. It is known that the chromatin-remodeling complex switch/sucrose nonfermentable (SWI/SNF) is essential for HIF activity. However, no additional information exists about the role of other chromatin-remodeling enzymes in hypoxia. Here we describe the role of imitation switch (ISWI) in the cellular response to hypoxia. We find that unlike SWI/SNF, ISWI depletion enhances HIF activity without altering its levels. Furthermore, ISWI knockdown only alters a subset of HIF target genes. Mechanistically, we find that ISWI is required for full expression of FIH mRNA and protein levels by changing RNA polymerase II loading to the FIH promoter. Of interest, exogenous FIH can rescue the ISWI-mediated upregulation of CA9 but not BNIP3, suggesting that FIH-independent mechanisms are also involved. Of importance, ISWI depletion alters the cellular response to hypoxia by reducing autophagy and increasing apoptosis. These results demonstrate a novel role for ISWI as a survival factor during the cellular response to hypoxia.
|Role of yeast JmjC-domain containing histone demethylases in actively transcribed regions. |
Kwon DW, Ahn SH
Biochemical and biophysical research communications 410 614-9. Epub 2011 Jun 12. 2011
In budding yeast, there are five JmjC domain-containing proteins, Jhd1, Jhd2, Rph1, Ecm5, and Gis1, which have been suggested to directly remove histone lysine methylation via a hydroxylation reaction. Of these demethylases, the ability of Jhd1 or Rph1 to demethylate histone H3 as a substrate has been identified in vivo. However, the overall roles of endogenous JmjC demethylases in the demethylation of histones encompassed by genes that are constitutively transcribed or their specificities towards histone H3 lysine modification at mono-, di-, or trimethylation states are still unclear. Using chromatin immunoprecipitation with nine specific antibodies directed against mono-, di-, or trimethylated histone H3 at lysines 4, 36, or 79, we show the whole patterns of histone H3 lysine methylation and the net changes in methylations that are caused by the deletion of each of the five JmjC demethylases in actively transcribed regions. Our results show that of the JmjC-containing proteins, Rph1 is the demethylase that is specific for histone H3K36 trimethylation during transcription elongation in vivo, and the abilities of other endogenous JmjC demethylasesto demethylate histone H3 are weak toward histone H3in actively transcribed regions.Copyright © 2011 Elsevier Inc. All rights reserved.
|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
|Dual-specificity histone demethylase KIAA1718 (KDM7A) regulates neural differentiation through FGF4. |
Huang, C; Xiang, Y; Wang, Y; Li, X; Xu, L; Zhu, Z; Zhang, T; Zhu, Q; Zhang, K; Jing, N; Chen, CD
Cell research 20 154-65 2010
Dimethylations of histone H3 lysine 9 and lysine 27 are important epigenetic marks associated with transcription repression. Here, we identified KIAA1718 (KDM7A) as a novel histone demethylase specific for these two repressing marks. Using mouse embryonic stem cells, we demonstrated that KIAA1718 expression increased at the early phase of neural differentiation. Knockdown of the gene blocked neural differentiation and the effect was rescued by the wild-type human gene, and not by a catalytically inactive mutant. In addition, overexpression of KIAA1718 accelerated neural differentiation. We provide the evidence that the pro-neural differentiation effect of KDM7A is mediated through direct transcriptional activation of FGF4, a signal molecule implicated in neural differentiation. Thus, our study identified a dual-specificity histone demethylase that regulates neural differentiation through FGF4.
|Recognition of histone H3K4 trimethylation by the plant homeodomain of PHF2 modulates histone demethylation. |
Wen, H; Li, J; Song, T; Lu, M; Kan, PY; Lee, MG; Sha, B; Shi, X
The Journal of biological chemistry 285 9322-6 2010
Distinct lysine methylation marks on histones create dynamic signatures deciphered by the "effector" modules, although the underlying mechanisms remain unclear. We identified the plant homeodomain- and Jumonji C domain-containing protein PHF2 as a novel histone H3K9 demethylase. We show in biochemical and crystallographic analyses that PHF2 recognizes histone H3K4 trimethylation through its plant homeodomain finger and that this interaction is essential for PHF2 occupancy and H3K9 demethylation at rDNA promoters. Our study provides molecular insights into the mechanism by which distinct effector domains within a protein cooperatively modulate the "cross-talk" of histone modifications.Full Text Article
|Coordinated regulation of active and repressive histone methylations by a dual-specificity histone demethylase ceKDM7A from Caenorhabditis elegans. |
Lin, H; Wang, Y; Wang, Y; Tian, F; Pu, P; Yu, Y; Mao, H; Yang, Y; Wang, P; Hu, L; Lin, Y; Liu, Y; Xu, Y; Chen, CD
Cell research 20 899-907 2010
H3K9me2 and H3K27me2 are important epigenetic marks associated with transcription repression, while H3K4me3 is associated with transcription activation. It has been shown that active and repressive histone methylations distribute in a mutually exclusive manner, but the underlying mechanism was poorly understood. Here we identified ceKDM7A, a PHD (plant homeodomain)- and JmjC domain-containing protein, as a histone demethylase specific for H3K9me2 and H3K27me2. We further demonstrated that the PHD domain of ceKDM7A bound H3K4me3 and H3K4me3 co-localized with ceKDM7A at the genome-wide level. Disruption of the PHD domain binding to H3K4me3 reduced the demethylase activity in vivo, and loss of ceKDM7A reduced the expression of its associated target genes. These results indicate that ceKDM7A is recruited to the promoter to demethylate H3K9me2 and H3K27me2 and activate gene expression through the binding of the PHD domain to H3K4me3. Thus, our study identifies a dual-specificity histone demethylase and provides novel insights into the regulation of histone methylation.
|Chromatin states of core pluripotency-associated genes in pluripotent, multipotent and differentiated cells. |
S Barrand, P Collas
Biochemical and biophysical research communications 391 762-7 2010
Oct4, Nanog and Sox2 constitute a core of transcription factors controlling pluripotency. Differentiation and reprogramming studies have unraveled a few epigenetic modifications associated in relation to the expression state of OCT4, NANOG and SOX2. There is, however, no comprehensive map of chromatin states on these genes in human primary cells at different stages of differentiation. We report here a profile of DNA methylation and of 10 histone modifications on regulatory regions of OCT4, NANOG and SOX2 in embryonal carcinoma cells, mesenchymal stem cells and fibroblasts. Bisulfite sequencing reveals correlation between promoter CpG methylation and repression of OCT4, but not NANOG or SOX2, suggesting distinct repression mechanisms. Whereas none of these genes, even when inactive, harbor repressive trimethylated H3K9, CpG hypomethylated NANOG and SOX2, but not CpG methylated OCT4, are enriched in repressive H3K27me3. H3K79me1 and H3K79me3 tend to parallel each other and are linked to repression. Moreover, we highlight an inverse relationship between H3K27me3 occupancy on promoters and H3K36me3 occupancy on coding regions of OCT4, NANOG and SOX2, suggesting a cross-talk between K27 and K36 methylation. Establishment of distinct repression mechanisms for pluripotency-associated genes may constitute a safeguard system to prevent promiscuous reactivation during development or differentiation.
|PHF8 activates transcription of rRNA genes through H3K4me3 binding and H3K9me12 demethylation. |
Feng W, Yonezawa M, Ye J, Jenuwein T, Grummt I
Nature structural & molecular biology 17 445-50 Epub 2010 Mar 7 2010
Histone lysine methylation is dynamically regulated by lysine methyltransferases and lysine demethylases. Here we show that PHD finger protein 8 (PHF8), a protein containing a PHD finger and a Jumonji C (JmjC) domain, is associated with hypomethylated rRNA genes (rDNA). PHF8 interacts with the RNA polymerase I transcription machinery and with WD repeat-containing protein 5 (WDR5)-containing H3K4 methyltransferase complexes. PHF8 exerts a positive effect on rDNA transcription, with transcriptional activation requiring both the JmjC domain and the PHD finger. PHF8 demethylates H3K9me1/2, and its catalytic activity is stimulated by adjacent H3K4me3. A point mutation within the JmjC domain that is linked to mental retardation with cleft lip and palate (XLMR-CL/P) abolishes demethylase activity and transcriptional activation. Though further work is needed to unravel the contribution of PHF8 activity to mental retardation and cleft lip/palate, our results reveal a functional interplay between H3K4 methylation and H3K9me1/2 demethylation, linking dynamic histone methylation to rDNA transcription and neural disease.
|Histone methyltransferase gene SETD2 is a novel tumor suppressor gene in clear cell renal cell carcinoma. |
Gerben Duns,Eva van den Berg,Inge van Duivenbode,Jan Osinga,Harry Hollema,Robert M W Hofstra,Klaas Kok
Cancer research 70 2010
Sporadic clear cell renal cell carcinoma (cRCC) is genetically characterized by the recurrent loss of the short arm of chromosome 3, with a hotspot for copy number loss in the 3p21 region. We applied a method called gene identification by nonsense-mediated mRNA decay inhibition to a panel of 10 cRCC cell lines with 3p21 copy number loss to identify biallelic inactivated genes located at 3p21. This revealed inactivation of the histone methyltransferase gene SETD2, located on 3p21.31, as a common event in cRCC cells. SETD2 is nonredundantly responsible for trimethylation of the histone mark H3K36. Consistent with this function, we observed loss or a decrease of H3K36me3 in 7 out of the 10 cRCC cell lines. Identification of missense mutations in 2 out of 10 primary cRCC tumor samples added support to the involvement of loss of SETD2 function in the development of cRCC tumors.
|Asf1 can promote trimethylation of H3 K36 by Set2. |
Lin, LJ; Minard, LV; Johnston, GC; Singer, RA; Schultz, MC
Molecular and cellular biology 30 1116-29 2010
Asf1 is a conserved histone H3/H4 chaperone that can assemble and disassemble nucleosomes and promote histone acetylation. Set2 is an H3 K36 methyltransferase. The functions of these proteins intersect in the context of transcription elongation by RNA polymerase II: both contribute to the establishment of repressive chromatin structures that inhibit spurious intragenic transcription. Here we characterize further interactions between budding yeast (Saccharomyces cerevisiae) Asf1 and Set2 using assays of intragenic transcription, H3/H4 posttranslational modification, coding region cross-linking of Asf1 and Set2, and cooccurrence of Asf1 and Set2 in protein complexes. We find that at some genes Asf1 and Set2 control chromatin metabolism as components of separate pathways. However, the existence of a low-abundance complex containing both proteins suggests that Asf1 and Set2 can more directly collaborate in chromatin regulation. Consistent with this possibility, we show that Asf1 stimulates Set2 occupancy of the coding region of a highly transcribed gene by a mechanism that depends on Asf1 binding to H3/H4. This function of Asf1 promotes the switch from di- to trimethylation of H3 K36 at that gene. These results support the view that Set2 function in chromatin metabolism can intimately involve histone chaperone Asf1.Full Text Article
|PHF8 is a histone H3K9me2 demethylase regulating rRNA synthesis. |
Zhu, Z; Wang, Y; Li, X; Wang, Y; Xu, L; Wang, X; Sun, T; Dong, X; Chen, L; Mao, H; Yu, Y; Li, J; Li, J; Chen, PA; Chen, CD
Cell research 20 794-801 2010
Dimethylation of histone H3 lysine 9 (H3K9me2) is an important epigenetic mark associated with transcription repression. Here, we identified PHF8, a JmjC-domain-containing protein, as a histone demethylase specific for this repressing mark. Recombinant full-length wild type protein could remove methylation from H3K9me2, but mutation of a conserved histidine to alanine H247A abolished the demethylase activity. Overexpressed exogenous PHF8 was colocalized with B23 staining. Endogenous PHF8 was also colocalized with B23 and fibrillarin, two well-established nucleolus proteins, suggesting that PHF8 is localized in the nucleolus and may regulate rRNA transcription. Indeed, PHF8 bound to the promoter region of the rDNA gene. Knockdown of PHF8 reduced the expression of rRNA, and overexpression of the gene resulted in upregulation of rRNA transcript. Concomitantly, H3K9me2 level was elevated in the promoter region of the rDNA gene in PHF8 knockdown cells and reduced significantly when the wild type but not the catalytically inactive H247A mutant PHF8 was overexpressed. Thus, our study identified a histone demethylase for H3K9me2 that regulates rRNA transcription.
|The Mi-2/NuRD complex associates with pericentromeric heterochromatin during S phase in rapidly proliferating lymphoid cells. |
Helbling Chadwick, L; Chadwick, BP; Jaye, DL; Wade, PA
Chromosoma 118 445-57 2009
Chromosomal replication results in the duplication not only of DNA sequence but also of the patterns of histone modification, DNA methylation, and nucleoprotein structure that constitute epigenetic information. Pericentromeric heterochromatin in human cells is characterized by unique patterns of histone and DNA modification. Here, we describe association of the Mi-2/NuRD complex with specific segments of pericentromeric heterochromatin consisting of Satellite II/III DNA located on human chromosomes 1, 9, and 16 in some but not all cell types. This association is linked in part to DNA replication and chromatin assembly and may suggest a role in these processes. Mi-2/NuRD accumulation is independent of Polycomb association and is characterized by a unique pattern of histone modification. We propose that Mi-2/NuRD constitutes an enzymatic component of a pathway for assembly and maturation of chromatin utilized by rapidly proliferating lymphoid cells for replication of constitutive heterochromatin.
|The ASH1 HOMOLOG 2 (ASHH2) histone H3 methyltransferase is required for ovule and anther development in Arabidopsis. |
Grini, PE; Thorstensen, T; Alm, V; Vizcay-Barrena, G; Windju, SS; Jørstad, TS; Wilson, ZA; Aalen, RB
PloS one 4 e7817 2009
SET-domain proteins are histone lysine (K) methyltransferases (HMTase) implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2) protein (also called SDG8, EFS and CCR1) has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction.A slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to approximately 90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99) we observed a reduction of H3K36 trimethylation (me3), but not H3K4me3 or H3K36me2.The severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to the observed defects, implicates ASHH2 in regulation of gene expression via H3K36 trimethylation in chromatin of Arabidopsis inflorescences.
|Set2-dependent K36 methylation is regulated by novel intratail interactions within H3. |
James N Psathas,Suting Zheng,Song Tan,Joseph C Reese
Molecular and cellular biology 29 2009
Posttranslational modifications to histones have been studied extensively, but the requirement for the residues within the tails for different stages of transcription is less clear. Using RNR3 as a model, we found that the residues within the N terminus of H3 are predominantly required for steps after transcription initiation and chromatin remodeling. Specifically, deleting as few as 20 amino acids, or substituting glutamines for lysines in the tail, greatly impaired K36 methylation by Set2. The mutations to the tail described here preserve the residues predicted to fill the active site of Set2, and the deletion mimics the recently described cleavage of the H3 tail that occurs during gene activation. Importantly, maintaining the charge of the unmodified tail by arginine substitutions preserves Set2 function in vivo. The H3 tail is dispensable for Set2 recruitment to genes but is required for the catalytic activity of Set2 in vitro. We propose that Set2 activity is controlled by novel intratail interactions which can be influenced by modifications and changes to the structure of the H3 tail to control the dynamics and localization of methylation during elongation.Full Text Article
|Repression of FLOWERING LOCUS T chromatin by functionally redundant histone H3 lysine 4 demethylases in Arabidopsis. |
Jeong, JH; Song, HR; Ko, JH; Jeong, YM; Kwon, YE; Seol, JH; Amasino, RM; Noh, B; Noh, YS
PloS one 4 e8033 2009
FLOWERING LOCUS T (FT) plays a key role as a mobile floral induction signal that initiates the floral transition. Therefore, precise control of FT expression is critical for the reproductive success of flowering plants. Coexistence of bivalent histone H3 lysine 27 trimethylation (H3K27me3) and H3K4me3 marks at the FT locus and the role of H3K27me3 as a strong FT repression mechanism in Arabidopsis have been reported. However, the role of an active mark, H3K4me3, in FT regulation has not been addressed, nor have the components affecting this mark been identified. Mutations in Arabidopsis thaliana Jumonji4 (AtJmj4) and EARLY FLOWERING6 (ELF6), two Arabidopsis genes encoding Jumonji (Jmj) family proteins, caused FT-dependent, additive early flowering correlated with increased expression of FT mRNA and increased H3K4me3 levels within FT chromatin. Purified recombinant AtJmj4 protein possesses specific demethylase activity for mono-, di-, and trimethylated H3K4. Tagged AtJmj4 and ELF6 proteins associate directly with the FT transcription initiation region, a region where the H3K4me3 levels were increased most significantly in the mutants. Thus, our study demonstrates the roles of AtJmj4 and ELF6 as H3K4 demethylases directly repressing FT chromatin and preventing precocious flowering in Arabidopsis.Full Text Article
|Di- and tri- but not monomethylation on histone H3 lysine 36 marks active transcription of genes involved in flowering time regulation and other processes in Arabidopsis thaliana. |
Xu, L; Zhao, Z; Dong, A; Soubigou-Taconnat, L; Renou, JP; Steinmetz, A; Shen, WH
Molecular and cellular biology 28 1348-60 2008
Histone lysines can be mono-, di-, or trimethylated, providing an ample magnitude of epigenetic information for transcription regulation. In fungi, SET2 is the sole methyltransferase responsible for mono-, di-, and trimethylation of H3K36. Here we show that in Arabidopsis thaliana, the degree of H3K36 methylation is regulated by distinct methyltransferases. The SET2 homologs SDG8 and SDG26 each can methylate oligonucleosomes in vitro, and both proteins are localized in the nucleus. While the previously reported loss-of-function sdg8 mutants have an early-flowering phenotype, the loss-of-function sdg26 mutants show a late-flowering phenotype. Consistently, several MADS-box flowering repressors are down-regulated by sdg8 but up-regulated by sdg26. The sdg8 but not the sdg26 mutant plants show a dramatically reduced level of both di- and trimethyl-H3K36 and an increased level of monomethyl-H3K36. SDG8 is thus specifically required for di- and trimethylation of H3K36. Our results further establish that H3K36 di- and tri- but not monomethylation correlates with transcription activation. Finally, we show that SDG8 and VIP4, which encodes a component of the PAF1 complex, act independently and synergistically in transcription regulation. Together our results reveal that the deposition of H3K36 methylation is finely regulated, possibly to cope with the complex regulation of growth and development in higher eukaryotes.Full Text Article
|The Iws1:Spt6:CTD complex controls cotranscriptional mRNA biosynthesis and HYPB/Setd2-mediated histone H3K36 methylation. |
Yoh, SM; Lucas, JS; Jones, KA
Genes & development 22 3422-34 2008
Many steps in gene expression and mRNA biosynthesis are coupled to transcription elongation and organized through the C-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAPII). We showed recently that Spt6, a transcription elongation factor and histone H3 chaperone, binds to the Ser2P CTD and recruits Iws1 and the REF1/Aly mRNA export adaptor to facilitate mRNA export. Here we show that Iws1 also recruits the HYPB/Setd2 histone methyltransferase to the RNAPII elongation complex and is required for H3K36 trimethylation (H3K36me3) across the transcribed region of the c-Myc, HIV-1, and PABPC1 genes in vivo. Interestingly, knockdown of either Iws1 or HYPB/Setd2 also enhanced H3K27me3 at the 5' end of the PABPC1 gene, and depletion of Iws1, but not HYPB/Setd2, increased histone acetylation across the coding regions at the HIV-1 and PABPC1 genes in vivo. Knockdown of HYPB/Setd2, like Iws1, induced bulk HeLa poly(A)+ mRNAs to accumulate in the nucleus. In vitro, recombinant Spt6 binds selectively to a stretch of uninterrupted consensus repeats located in the N-terminal half of the CTD and recruits Iws1. Thus Iws1 connects two distinct CTD-binding proteins, Spt6 and HYPB/Setd2, in a megacomplex that affects mRNA export as well as the histone modification state of active genes.
|Transcription-coupled methylation of histone H3 at lysine 36 regulates dosage compensation by enhancing recruitment of the MSL complex in Drosophila melanogaster. |
Bell, O; Conrad, T; Kind, J; Wirbelauer, C; Akhtar, A; Schübeler, D
Molecular and cellular biology 28 3401-9 2008
In Drosophila melanogaster, dosage compensation relies on the targeting of the male-specific lethal (MSL) complex to hundreds of sites along the male X chromosome. Transcription-coupled methylation of histone H3 lysine 36 is enriched toward the 3' end of active genes, similar to the MSL proteins. Here, we have studied the link between histone H3 methylation and MSL complex targeting using RNA interference and chromatin immunoprecipitation. We show that trimethylation of histone H3 at lysine 36 (H3K36me3) relies on the histone methyltransferase Hypb and is localized promoter distal at dosage-compensated genes, similar to active genes on autosomes. However, H3K36me3 has an X-specific function, as reduction specifically decreases acetylation of histone H4 lysine 16 on the male X chromosome. This hypoacetylation is caused by compromised MSL binding and results in a failure to increase expression twofold. Thus, H3K36me3 marks the body of all active genes yet is utilized in a chromosome-specific manner to enhance histone acetylation at sites of dosage compensation.Full Text Article
|Reciprocal patterns of methylation of H3K36 and H3K27 on proximal vs. distal IgVH genes are modulated by IL-7 and Pax5. |
Xu, CR; Schaffer, L; Head, SR; Feeney, AJ
Proceedings of the National Academy of Sciences of the United States of America 105 8685-90 2008
The usage of greater than 100 functional murine Ig heavy chain V(H) genes, when rearranged to D(H)J(H) genes, generates a diverse antibody repertoire. The V(H) locus encompasses 2.5 Mb, and rearrangement of V(H) genes in the D(H)-distal half of the locus are controlled very differently from the V(H) genes in the proximal end of the locus. The rearrangement of distal but not proximal V(H) genes is impaired in mice deficient in the cytokine IL-7 or its receptor, in the transcription factor Pax5, or in Ezh2, a histone methyltransferase for Lys-27 of histone H3 (H3K27). The relative role of IL-7, Pax5, and Ezh2 in regulating distal vs. proximal V(H) rearrangement is not clear. Here, we show by ChIP and ChIP-on-chip that the active histone modification H3K36me2 is most highly associated with distal V(H) segments and the repressive histone modification H3K27me3 is exclusively present on proximal V(H) segments. We observed an absence of H3K27me3 in fetal pro-B cells, which predominantly rearrange proximal V(H) genes. Absence of IL-7 signaling reduces H3K36me2, and overexpression of IL-7 increases H3K36me2. In contrast, the major effect of the absence of Pax5 is the reduction in H3K27me3. Our data indicate that the cytokine IL-7 and the transcription factor Pax5 influence the rearrangement of the two regions of the V(H) locus by differentially modulating two reciprocal histone modifications during B lymphocyte development.
|dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. |
Lagarou, A; Mohd-Sarip, A; Moshkin, YM; Chalkley, GE; Bezstarosti, K; Demmers, JA; Verrijzer, CP
Genes & development 22 2799-810 2008
Transcription regulation involves enzyme-mediated changes in chromatin structure. Here, we describe a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase dKDM2. In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression.Full Text Article
|Genic and global functions for Paf1C in chromatin modification and gene expression in Arabidopsis. |
Oh, S; Park, S; van Nocker, S
PLoS genetics 4 e1000077 2008
In budding yeast, intragenic histone modification is linked with transcriptional elongation through the conserved regulator Paf1C. To investigate Paf1C-related function in higher eukaryotes, we analyzed the effects of loss of Paf1C on histone H3 density and patterns of H3 methylated at K4, K27, and K36 in Arabidopsis genes, and integrated this with existing gene expression data. Loss of Paf1C did not change global abundance of H3K4me3 or H3K36me2 within chromatin, but instead led to a 3' shift in the distribution of H3K4me3 and a 5' shift in the distribution of H3K36me2 within genes. We found that genes regulated by plant Paf1C showed strong enrichment for both H3K4me3 and H3K27me3 and also showed a high degree of tissue-specific expression. At the Paf1C- and PcG-regulated gene FLC, transcriptional silencing and loss of H3K4me3 and H3K36me2 were accompanied by expansion of H3K27me3 into the promoter and transcriptional start regions and further enrichment of H3K27me3 within the transcribed region. These results highlight both genic and global functions for plant Paf1C in histone modification and gene expression, and link transcriptional activity with cellular memory.Full Text Article
|Two Saccharomyces cerevisiae JmjC domain proteins demethylate histone H3 Lys36 in transcribed regions to promote elongation. |
Kim, T; Buratowski, S
The Journal of biological chemistry 282 20827-35 2007
Histone methylation is a reversible modification regulated by the antagonistic functions of residue-specific histone methyltransferases and demethylases. Although methylation of histone H3 at lysines 4 and 36 is linked to transcription, the roles of histone demethylases in transcription regulation are not understood. Here we show that overexpression of either Jhd1 or Rph1, two JmjC-domain proteins, bypasses the requirement for the positive elongation factor gene BUR1. Biochemical analysis and chromatin immunoprecipitation experiments indicate that Rph1 functions as a specific demethylase for H3 K36me3 and K36me2, directly regulating Lys(36) methylation in transcribed regions. Both Jhd1 and Rph1 are required for normal levels of RNA polymerase II cross-linking to genes. Taken together, these findings indicate that a general function of histone demethylases for H3 Lys(36) is to promote transcription elongation by antagonizing repressive Lys(36) methylation by Set2.
|Identification of JmjC domain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases. |
Hong, S; Cho, YW; Yu, LR; Yu, H; Veenstra, TD; Ge, K
Proceedings of the National Academy of Sciences of the United States of America 104 18439-44 2007
Covalent modifications of histones, such as acetylation and methylation, play important roles in the regulation of gene expression. Histone lysine methylation has been implicated in both gene activation and repression, depending on the specific lysine (K) residue that becomes methylated and the state of methylation (mono-, di-, or trimethylation). Methylation on K4, K9, and K36 of histone H3 has been shown to be reversible and can be removed by site-specific demethylases. However, the enzymes that antagonize methylation on K27 of histone H3 (H3K27), an epigenetic mark important for embryonic stem cell maintenance, Polycomb-mediated gene silencing, and X chromosome inactivation have been elusive. Here we show the JmjC domain-containing protein UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome), as well as the related JMJD3 (jumonji domain containing 3), specifically removes methyl marks on H3K27 in vitro. Further, the demethylase activity of UTX requires a catalytically active JmjC domain. Finally, overexpression of UTX and JMJD3 leads to reduced di- and trimethylation on H3K27 in cells, suggesting that UTX and JMJD3 may function as H3K27 demethylases in vivo. The identification of UTX and JMJD3 as H3K27-specific demethylases provides direct evidence to indicate that similar to methylation on K4, K9, and K36 of histone H3, methylation on H3K27 is also reversible and can be dynamically regulated by site-specific histone methyltransferases and demethylases.
|A plasmid model system shows that Drosophila dosage compensation depends on the global acetylation of histone H4 at lysine 16 and is not affected by depletion of common transcription elongation chromatin marks. |
Yokoyama, R; Pannuti, A; Ling, H; Smith, ER; Lucchesi, JC
Molecular and cellular biology 27 7865-70 2007
Dosage compensation refers to the equalization of most X-linked gene products between males, which have one X chromosome and a single dose of X-linked genes, and females, which have two X's and two doses of such genes. We developed a plasmid-based model of dosage compensation that allows new experimental approaches for the study of this regulatory mechanism. In Drosophila melanogaster, an enhanced rate of transcription of the X chromosome in males is dependent upon the presence of histone H4 acetylated at lysine 16. This chromatin mark occurs throughout active transcriptional units, leading us to the conclusion that the enhanced level of transcription is achieved through an enhanced rate of RNA polymerase elongation. We used the plasmid model to demonstrate that enhancement in the level of transcription does not depend on other histone marks and factors that have been associated with the process of elongation, thereby highlighting the special role played by histone H4 acetylated at lysine 16 in this process.
|Mammalian ASH1L is a histone methyltransferase that occupies the transcribed region of active genes. |
Gregory, GD; Vakoc, CR; Rozovskaia, T; Zheng, X; Patel, S; Nakamura, T; Canaani, E; Blobel, GA
Molecular and cellular biology 27 8466-79 2007
Histone lysine methylation regulates genomic functions, including gene transcription. Previous reports found various degrees of methylation at H3K4, H3K9, and H4K20 within the transcribed region of active mammalian genes. To identify the enzymes responsible for placing these modifications, we examined ASH1L, the mammalian homolog of the Drosophila melanogaster Trithorax group (TrxG) protein Ash1. Drosophila Ash1 has been reported to methylate H3K4, H3K9, and H4K20 at its target sites. Here we demonstrate that mammalian ASH1L associates with the transcribed region of all active genes examined, including Hox genes. The distribution of ASH1L in transcribed chromatin strongly resembles that of methylated H3K4 but not that of H3K9 or H4K20. Accordingly, the SET domain of ASH1L methylates H3K4 in vitro, and knockdown of ASH1L expression reduced H3K4 trimethylation at HoxA10 in vivo. Notably, prior methylation at H3K9 reduced ASH1L-mediated methylation at H3K4, suggesting cross-regulation among these marks. Drosophila ash1 and trithorax interact genetically, and the mammalian TrxG protein MLL1 and ASH1L display highly similar distributions and substrate specificities. However, by using MLL null cell lines we found that their recruitments occur independently of each other. Collectively, our data suggest that ASH1L occupies most, if not all, active genes and methylates histone H3 in a nonredundant fashion at a subset of genes.
|C2H2 zinc finger-SET histone methyltransferase is a plant-specific chromatin modifier. |
Krichevsky, A; Gutgarts, H; Kozlovsky, SV; Tzfira, T; Sutton, A; Sternglanz, R; Mandel, G; Citovsky, V
Developmental biology 303 259-69 2007
Histone modification represents a universal mechanism for regulation of eukaryotic gene expression underlying diverse biological processes from neuronal gene expression in mammals to control of flowering in plants. In animal cells, these chromatin modifications are effected by well-defined multiprotein complexes containing specific histone-modifying activities. In plants, information about the composition of such co-repressor complexes is just beginning to emerge. Here, we report that two Arabidopsis thaliana factors, a SWIRM domain polyamine oxidase protein, AtSWP1, and a plant-specific C2H2 zinc finger-SET domain protein, AtCZS, interact with each other in plant cells and repress expression of a negative regulator of flowering, FLOWERING LOCUS C (FLC) via an autonomous, vernalization-independent pathway. Loss-of-function of either AtSWP1 or AtCZS results in reduced dimethylation of lysine 9 and lysine 27 of histone H3 and hyperacetylation of histone H4 within the FLC locus, in elevated FLC mRNA levels, and in moderately delayed flowering. Thus, AtSWP1 and AtCZS represent two main components of a co-repressor complex that fine tunes flowering and is unique to plants.
|Regulation of histone H3K4 tri-methylation and PAF complex recruitment by the Ccr4-Not complex. |
Mulder, KW; Brenkman, AB; Inagaki, A; van den Broek, NJ; Timmers, HT
Nucleic acids research 35 2428-39 2007
Efficient transcription is linked to modification of chromatin. For instance, tri-methylation of lysine 4 on histone H3 (H3K4) strongly correlates with transcriptional activity and is regulated by the Bur1/2 kinase complex. We found that the evolutionarily conserved Ccr4-Not complex is involved in establishing H3K4 tri-methylation in Saccharomyces cerevisiae. We observed synthetic lethal interactions of Ccr4-Not components with BUR1 and BUR2. Further analysis indicated that the genes encoding the Not-proteins are essential for efficient regulation of H3K4me3, but not H3K4me1/2, H3K36me2 or H3K79me2/3 levels. Moreover, regulation of H3K4me3 levels by NOT4 is independent of defects in RNA polymerase II loading. We found NOT4 to be important for ubiquitylation of histone H2B via recruitment of the PAF complex, but not for recruitment or activation of the Bur1/2 complex. These results suggest a mechanism in which the Ccr4-Not complex functions parallel to or downstream of the Bur1/2 kinase to facilitate H3K4me3 via PAF complex recruitment.Full Text Article
|Regulation of histone modification and cryptic transcription by the Bur1 and Paf1 complexes. |
Yaya Chu, Rajna Simic, Marcie H Warner, Karen M Arndt, Gregory Prelich
The EMBO journal 26 4646-56 2007
The Bur1-Bur2 and Paf1 complexes function during transcription elongation and affect histone modifications. Here we describe new roles for Bur1-Bur2 and the Paf1 complex. We find that histone H3 K36 tri-methylation requires specific components of the Paf1 complex and that K36 tri-methylation is more strongly affected at the 5' ends of genes in paf1delta and bur2delta strains in parallel with increased acetylation of histones H3 and H4. Interestingly, the 5' increase in histone acetylation is independent of K36 methylation, and therefore is mechanistically distinct from the methylation-driven deacetylation that occurs at the 3' ends of genes. Finally, Bur1-Bur2 and the Paf1 complex have a second methylation-independent function, since bur2delta set2delta and paf1delta set2delta double mutants display enhanced histone acetylation at the 3' ends of genes and increased cryptic transcription initiation. These findings identify new functions for the Paf1 and Bur1-Bur2 complexes, provide evidence that histone modifications at the 5' and 3' ends of coding regions are regulated by distinct mechanisms, and reveal that the Bur1-Bur2 and Paf1 complexes repress cryptic transcription through a Set2-independent pathway.Full Text Article
|The BUR1 cyclin-dependent protein kinase is required for the normal pattern of histone methylation by SET2. |
Chu, Y; Sutton, A; Sternglanz, R; Prelich, G
Molecular and cellular biology 26 3029-38 2006
BUR1 and BUR2 encode the catalytic and regulatory subunits of a cyclin-dependent protein kinase complex that is essential for normal growth and has a general role in transcription elongation. To gain insight into its specific role in vivo, we identified mutations that reverse the severe growth defect of bur1Delta cells. This selection identified mutations in SET2, which encodes a histone methylase that targets lysine 36 of histone H3 and, like BUR1, has a poorly characterized role during transcription elongation. This genetic relationship indicates that SET2 activity is required for the growth defect observed in bur1Delta strains. This SET2-dependent growth inhibition occurs via methylation of histone H3 on lysine 36, since a methylation-defective allele of SET2 or a histone H3 K36R mutation also suppressed bur1Delta. We have explored the relationship between BUR1 and SET2 at the biochemical level and find that histone H3 is monomethylated, dimethylated, and trimethylated on lysine 36 in wild-type cells, but trimethylation is significantly reduced in bur1 and bur2 mutant strains. A similar methylation pattern is observed in RNA polymerase II C-terminal domain truncation mutants and in an spt16 mutant strain. Chromatin immunoprecipitation assays reveal that the transcription-dependent increase in trimethylated K36 over open reading frames is significantly reduced in bur2Delta strains. These results establish links between a regulatory protein kinase and histone methylation and lead to a model in which the Bur1-Bur2 complex counteracts an inhibitory effect of Set2-dependent histone methylation.
|Dimethylation of histone H3 at lysine 36 demarcates regulatory and nonregulatory chromatin genome-wide. |
Rao, B; Shibata, Y; Strahl, BD; Lieb, JD
Molecular and cellular biology 25 9447-59 2005
Set2p, which mediates histone H3 lysine 36 dimethylation (H3K36me2) in Saccharomyces cerevisiae, has been shown to associate with RNA polymerase II (RNAP II) at individual loci. Here, chromatin immunoprecipitation-microarray experiments normalized to general nucleosome occupancy reveal that nucleosomes within open reading frames (ORFs) and downstream noncoding chromatin were highly dimethylated at H3K36 and that Set2p activity begins at a stereotypic distance from the initiation of transcription genome-wide. H3K36me2 is scarce in regions upstream of divergently transcribed genes, telomeres, silenced mating loci, and regions transcribed by RNA polymerase III, providing evidence that the enzymatic activity of Set2p is restricted to its association with RNAP II. The presence of H3K36me2 within ORFs correlated with the "on" or "off" state of transcription, but the degree of H3K36 dimethylation within ORFs did not correlate with transcription frequency. This provides evidence that H3K36me2 is established during the initial instances of gene transcription, with subsequent transcription having at most a maintenance role. Accordingly, newly activated genes acquire H3K36me2 in a manner that does not correlate with gene transcript levels. Finally, nucleosomes dimethylated at H3K36 appear to be refractory to loss from highly transcribed chromatin. Thus, H3K36me2, which is highly conserved throughout eukaryotic evolution, provides a stable molecular mechanism for establishing chromatin context throughout the genome by distinguishing potential regulatory regions from transcribed chromatin.Full Text Article