RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

PubMed Central

Zoabi, Muhammad; Nadar-Ponniah, Prathamesh T.; Khoury-Haddad, Hanan; Usaj, Marko; Budowski-Tal, Inbal; Haran, Tali; Henn, Arnon; Mandel-Gutfreund, Yael; Ayoub, Nabieh

2014-01-01

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin. PMID:25378304

H3K27me3 and H3K4me3 chromatin environment at super-induced dehydration stress memory genes of Arabidopsis thaliana.

PubMed

Liu, Ning; Fromm, Michael; Avramova, Zoya

2014-03-01

Pre-exposure to a stress may alter the plant's cellular, biochemical, and/or transcriptional responses during future encounters as a 'memory' from the previous stress. Genes increasing transcription in response to a first dehydration stress, but producing much higher transcript levels in a subsequent stress, represent the super-induced 'transcription memory' genes in Arabidopsis thaliana. The chromatin environment (histone H3 tri-methylations of Lys 4 and Lys 27, H3K4me3, and H3K27me3) studied at five dehydration stress memory genes revealed existence of distinct memory-response subclasses that responded differently to CLF deficiency and displayed different transcriptional activities during the watered recovery periods. Among the most important findings is the novel aspect of the H3K27me3 function observed at specific dehydration stress memory genes. In contrast to its well-known role as a chromatin repressive mechanism at developmentally regulated genes, H3K27me3 did not prevent transcription from the dehydration stress-responding genes. The high H3K27me3 levels present during transcriptionally inactive states did not interfere with the transition to active transcription and with H3K4me3 accumulation. H3K4me3 and H3K27me3 marks function independently and are not mutually exclusive at the dehydration stress-responding memory genes.

UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity.

PubMed

Wang, Chaochen; Lee, Ji-Eun; Cho, Young-Wook; Xiao, Ying; Jin, Qihuang; Liu, Chengyu; Ge, Kai

2012-09-18

To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/β-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.

Association of H3K9me3 and H3K27me3 repressive histone marks with breast cancer subtypes in the Nurses' Health Study.

PubMed

Healey, Megan A; Hu, Rong; Beck, Andrew H; Collins, Laura C; Schnitt, Stuart J; Tamimi, Rulla M; Hazra, Aditi

2014-10-01

Repressive histone tail modifications have been associated with molecular breast cancer subtypes. We investigated whether histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) were associated with tumor features and subtypes while adjusting for prospectively collected reproductive and lifestyle breast cancer risk factors. We have tissue microarray data with immunohistochemical marker information on 804 incident cases of invasive breast cancer diagnosed from 1976-2000 in the Nurses' Health Study. Tissue microarray sections were stained for global H3K9me3 and H3K27me3, and scored into four categories. Multivariate odds ratios (OR) and 95 % confidence intervals (CI) were calculated using logistic regression models for tumor features and subtypes, adjusting for breast cancer risk factors. While there were no significant associations between H3K9me3 and tumor features, H3K27me3 was significantly associated with lower grade tumors compared to high grade tumors in the multivariate model (OR = 1.95, 95 % CI 1.35-2.81, p = 0.0004). H3K27me3 was suggestively associated with estrogen receptor-positive (ER+) tumors (OR = 1.47, 95 % CI 0.97-2.23, p = 0.07). In subtype analyses, H3K27me3 was positively associated with the luminal A subtype compared to all other subtypes (OR = 1.42, 95 % CI 1.14-1.77, p = 0.002), and was inversely associated with HER2-type (OR = 0.58, 95 % CI 0.37-0.91, p = 0.02) and basal-like breast cancer (OR = 0.52, 95 % CI 0.36-0.76, p = 0.0006). In the largest immunohistochemical examination of H3K9me3 and H3K27me3 in breast cancer, we found that H3K27me3 positivity, but not H3K9me3, was associated with lower grade tumors and the luminal A subtype after adjusting for reproductive and lifestyle breast cancer risk factors.

Cis-existence of H3K27me3 and H3K36me2 in mouse embryonic stem cells revealed by specific ions of isobaric modification chromatogram.

PubMed

Mao, Hailei; Han, Gang; Xu, Longyong; Zhu, Duming; Lin, Hanqing; Cao, Xiongwen; Yu, Yi; Chen, Charlie Degui

2015-07-21

Histone H3 lysine 27 trimethylation (H3K27me3) and H3 lysine 36 trimethylation (H3K36me3) are important epigenetic modifications correlated with transcription repression and activation, respectively. These two opposing modifications rarely co-exist in the same H3 polypeptide. However, a small but significant amount of H3 tails are modified with 5 methyl groups on K27 and K36 in mouse embryonic stem cells (mESCs) and it is unclear how the trimethylation is distributed on K27 or K36. A label-free, bottom-up mass spectrum method, named specific ions of isobaric modification chromatogram (SIMC), was established to quantify the relative abundance of K27me2-K36me3 and K27me3-K36me2 in the same histone H3 tail. By using this method, we demonstrated that the H3K27me3-K36me2 comprises about 85 % of the penta-methylated H3 tails at K27 and K36 in mESCs. Upon mESC differentiation, the abundance of H3K27me3-K36me2 significantly decreased, while the level of H3K27me2-K36me3 remains unchanged. Our study not only revealed the cis-existence of H3K27me3-K36me2 in mESCs, but also suggested that this combinatorial histone modification may assume a specific regulatory function during differentiation.

The histone demethylase Jarid1b ensures faithful mouse development by protecting developmental genes from aberrant H3K4me3.

PubMed

Albert, Mareike; Schmitz, Sandra U; Kooistra, Susanne M; Malatesta, Martina; Morales Torres, Cristina; Rekling, Jens C; Johansen, Jens V; Abarrategui, Iratxe; Helin, Kristian

2013-04-01

Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like Pax6 and Otx2 in Jarid1b knockout brains. Taken together, these results suggest that Jarid1b regulates mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications.

The Histone Demethylase Jarid1b Ensures Faithful Mouse Development by Protecting Developmental Genes from Aberrant H3K4me3

PubMed Central

Kooistra, Susanne M.; Malatesta, Martina; Morales Torres, Cristina; Rekling, Jens C.; Johansen, Jens V.; Abarrategui, Iratxe; Helin, Kristian

2013-01-01

Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like Pax6 and Otx2 in Jarid1b knockout brains. Taken together, these results suggest that Jarid1b regulates mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications. PMID:23637629

Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

PubMed Central

Hosogane, Masaki; Funayama, Ryo; Nishida, Yuichiro; Nagashima, Takeshi; Nakayama, Keiko

2013-01-01

Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity. PMID:24009517

JMJD3 Is Crucial for the Female AVPV RIP-Cre Neuron-Controlled Kisspeptin-Estrogen Feedback Loop and Reproductive Function.

PubMed

Song, Anying; Jiang, Shujun; Wang, Qinghua; Zou, Jianghuan; Lin, Zhaoyu; Gao, Xiang

2017-06-01

The hypothalamic-pituitary-gonadal axis controls development, reproduction, and metabolism. Although most studies have focused on the hierarchy from the brain to the gonad, many questions remain unresolved concerning the feedback from the gonad to the central nervous system, especially regarding the potential epigenetic modifications in hypothalamic neurons. In the present report, we generated genetically modified mice lacking histone H3 lysine 27 (H3K27) demethylase Jumonji domain-containing 3 (JMJD3) in hypothalamic rat-insulin-promoter-expressing neurons (RIP-Cre neurons). The female mutant mice displayed late-onset obesity owing to reduced locomotor activity and decreased energy expenditure. JMJD3 deficiency in RIP-Cre neurons also results in delayed pubertal onset, an irregular estrous cycle, impaired fertility, and accelerated ovarian failure in female mice owing to the dysregulation of the hypothalamic-ovarian axis. We found that JMJD3 directly regulates Kiss1 gene expression by binding to the Kiss1 promoter and triggering H3K27me3 demethylation in the anteroventral periventricular (AVPV) nucleus. Further study confirmed that the aberrations arose from impaired kisspeptin signaling in the hypothalamic AVPV nucleus and subsequent estrogen deficiency. Estrogen replacement therapy can reverse obesity in mutant mice. Moreover, we demonstrated that Jmjd3 is an estrogen target gene in the hypothalamus. These results provide direct genetic and molecular evidence that JMJD3 is a key mediator for the kisspeptin-estrogen feedback loop. Copyright © 2017 Endocrine Society.

Reduced H3K27me3 expression in radiation-associated angiosarcoma of the breast.

PubMed

Mentzel, Thomas; Kiss, Katalin

2018-03-01

The diagnosis of radiation-associated angiosarcoma is challenging and there are overlapping clinicopathological features between radiation-associated benign, atypical and malignant vascular lesions. It has been shown convincingly, that the majority of radiation-associated angiosarcomas are characterised by amplification and subsequent overexpression of MYC in contrast to benign and atypical vascular lesions. Given the fact that epigenetic changes play an important role in carcinogenesis and loss of histone H3K27 trimethylation (H3K27me3) has been found in a number of malignant neoplasms including malignant peripheral nerve sheath tumours, especially when associated with previous radiotherapy, we evaluated the immunohistochemical reaction pattern for H3K27me3 in 49 vascular lesions and control cases: normal skin and benign vascular lesions not associated with previous radiotherapy, radiation-associated benign, atypical and malignant vascular lesions and angiosarcomas not associated with previous radiotherapy. We found loss of H3K27me3 expression in most cases of radiation-associated angiosarcomas, whereas endothelial cells in benign and atypical vascular lesions arising after previous radiotherapy stained positively for H3K27me3. The sporadic angiosarcomas stained inconsistently for H3K27me3. Loss of H3K27me3 is typically seen in radiation-associated angiosarcomas, representing an additional diagnostic tool and raises questions in regard to the carcinogenesis of malignant vascular neoplasms.

Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia.

PubMed

Adriaens, Michiel E; Prickaerts, Peggy; Chan-Seng-Yue, Michelle; van den Beucken, Twan; Dahlmans, Vivian E H; Eijssen, Lars M; Beck, Timothy; Wouters, Bradly G; Voncken, Jan Willem; Evelo, Chris T A

2016-01-01

A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. In silico -identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.

Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing

PubMed Central

Tie, Feng; Banerjee, Rakhee; Saiakhova, Alina R.; Howard, Benny; Monteith, Kelsey E.; Scacheri, Peter C.; Cosgrove, Michael S.; Harte, Peter J.

2014-01-01

Trithorax (TRX) antagonizes epigenetic silencing by Polycomb group (PcG) proteins, stimulates enhancer-dependent transcription, and establishes a ‘cellular memory’ of active transcription of PcG-regulated genes. The mechanisms underlying these TRX functions remain largely unknown, but are presumed to involve its histone H3K4 methyltransferase activity. We report that the SET domains of TRX and TRX-related (TRR) have robust histone H3K4 monomethyltransferase activity in vitro and that Tyr3701 of TRX and Tyr2404 of TRR prevent them from being trimethyltransferases. The trxZ11 missense mutation (G3601S), which abolishes H3K4 methyltransferase activity in vitro, reduces the H3K4me1 but not the H3K4me3 level in vivo. trxZ11 also suppresses the impaired silencing phenotypes of the Pc3 mutant, suggesting that H3K4me1 is involved in antagonizing Polycomb silencing. Polycomb silencing is also antagonized by TRX-dependent H3K27 acetylation by CREB-binding protein (CBP). We show that perturbation of Polycomb silencing by TRX overexpression requires CBP. We also show that TRX and TRR are each physically associated with CBP in vivo, that TRX binds directly to the CBP KIX domain, and that the chromatin binding patterns of TRX and TRR are highly correlated with CBP and H3K4me1 genome-wide. In vitro acetylation of H3K27 by CBP is enhanced on K4me1-containing H3 substrates, and independently altering the H3K4me1 level in vivo, via the H3K4 demethylase LSD1, produces concordant changes in H3K27ac. These data indicate that the catalytic activities of TRX and CBP are physically coupled and suggest that both activities play roles in antagonizing Polycomb silencing, stimulating enhancer activity and cellular memory. PMID:24550119

Global analysis of H3K27me3 as an epigenetic marker in prostate cancer progression.

PubMed

Ngollo, Marjolaine; Lebert, Andre; Daures, Marine; Judes, Gaelle; Rifai, Khaldoun; Dubois, Lucas; Kemeny, Jean-Louis; Penault-Llorca, Frederique; Bignon, Yves-Jean; Guy, Laurent; Bernard-Gallon, Dominique

2017-04-12

H3K27me3 histone marks shape the inhibition of gene transcription. In prostate cancer, the deregulation of H3K27me3 marks might play a role in prostate tumor progression. We investigated genome-wide H3K27me3 histone methylation profile using chromatin immunoprecipitation (ChIP) and 2X400K promoter microarrays to identify differentially-enriched regions in biopsy samples from prostate cancer patients. H3K27me3 marks were assessed in 34 prostate tumors: 11 with Gleason score > 7 (GS > 7), 10 with Gleason score ≤ 7 (GS ≤ 7), and 13 morphologically normal prostate samples. Here, H3K27me3 profiling identified an average of 386 enriched-genes on promoter regions in healthy control group versus 545 genes in GS ≤ 7 and 748 genes in GS > 7 group. We then ran a factorial discriminant analysis (FDA) and compared the enriched genes in prostate-tumor biopsies and normal biopsies using ANOVA to identify significantly differentially-enriched genes. The analysis identified ALG5, EXOSC8, CBX1, GRID2, GRIN3B, ING3, MYO1D, NPHP3-AS1, MSH6, FBXO11, SND1, SPATS2, TENM4 and TRA2A genes. These genes are possibly associated with prostate cancer. Notably, the H3K27me3 histone mark emerged as a novel regulatory mechanism in poor-prognosis prostate cancer. Our findings point to epigenetic mark H3K27me3 as an important event in prostate carcinogenesis and progression. The results reported here provide new molecular insights into the pathogenesis of prostate cancer.

Regions of very low H3K27me3 partition the Drosophila genome into topological domains

PubMed Central

Flower, Rosalyn; Choo, Siew Woh

2017-01-01

It is now well established that eukaryote genomes have a common architectural organization into topologically associated domains (TADs) and evidence is accumulating that this organization plays an important role in gene regulation. However, the mechanisms that partition the genome into TADs and the nature of domain boundaries are still poorly understood. We have investigated boundary regions in the Drosophila genome and find that they can be identified as domains of very low H3K27me3. The genome-wide H3K27me3 profile partitions into two states; very low H3K27me3 identifies Depleted (D) domains that contain housekeeping genes and their regulators such as the histone acetyltransferase-containing NSL complex, whereas domains containing moderate-to-high levels of H3K27me3 (Enriched or E domains) are associated with regulated genes, irrespective of whether they are active or inactive. The D domains correlate with the boundaries of TADs and are enriched in a subset of architectural proteins, particularly Chromator, BEAF-32, and Z4/Putzig. However, rather than being clustered at the borders of these domains, these proteins bind throughout the H3K27me3-depleted regions and are much more strongly associated with the transcription start sites of housekeeping genes than with the H3K27me3 domain boundaries. While we have not demonstrated causality, we suggest that the D domain chromatin state, characterised by very low or absent H3K27me3 and established by housekeeping gene regulators, acts to separate topological domains thereby setting up the domain architecture of the genome. PMID:28282436

Natural variation of H3K27me3 distribution between two Arabidopsis accessions and its association with flanking transposable elements

PubMed Central

2012-01-01

Background Histone H3 lysine 27 tri-methylation and lysine 9 di-methylation are independent repressive chromatin modifications in Arabidopsis thaliana. H3K27me3 is established and maintained by Polycomb repressive complexes whereas H3K9me2 is catalyzed by SUVH histone methyltransferases. Both modifications can spread to flanking regions after initialization and were shown to be mutually exclusive in Arabidopsis. Results We analyzed the extent of natural variation of H3K27me3 in the two accessions Landsberg erecta (Ler) and Columbia (Col) and their F1 hybrids. The majority of H3K27me3 target genes in Col were unchanged in Ler and F1 hybrids. A small number of Ler-specific targets were detected and confirmed. Consistent with a cis-regulatory mechanism for establishing H3K27me3, differential targets showed allele-specific H3K27me3 in hybrids. Five Ler-specific targets showed the active mark H3K4me3 in Col and for this group, differential H3K27me3 enrichment accorded to expression variation. On the other hand, the majority of Ler-specific targets were not expressed in Col, Ler or 17 other accessions. Instead of H3K27me3, the antagonistic mark H3K9me2 and other heterochromatic features were observed at these loci in Col. These loci were frequently flanked by transposable elements, which were often missing in the Ler genome assembly. Conclusion There is little variation in H3K27me3 occupancy within the species, although H3K27me3 targets were previously shown as overrepresented among differentially expressed genes. The existing variation in H3K27me3 seems mostly explained by flanking polymorphic transposable elements. These could nucleate heterochromatin, which then spreads into neighboring H3K27me3 genes, thus converting them to H3K9me2 targets. PMID:23253144

Reduced H3K27me3 expression in Merkel cell polyoma virus-positive tumors.

PubMed

Busam, Klaus J; Pulitzer, Melissa P; Coit, Daniel C; Arcila, Maria; Leng, Danielle; Jungbluth, Achim A; Wiesner, Thomas

2017-06-01

Merkel cell carcinoma is a primary cutaneous neuroendocrine carcinoma, which once metastatic is difficult to treat. Recent mutation analyses of Merkel cell carcinoma revealed a low number of mutations in Merkel cell polyomavirus-associated tumors, and a high number of mutations in virus-negative combined squamous cell and neuroendocrine carcinomas of chronically sun-damaged skin. We speculated that the paucity of mutations in virus-positive Merkel cell carcinoma may reflect a pathomechanism that depends on derangements of chromatin without alterations in the DNA sequence (epigenetic dysregulation). One central epigenetic regulator is the Polycomb repressive complex 2 (PRC2), which silences genomic regions by trimethylating (me3) lysine (K) 27 of histone H3, and thereby establishes the histone mark H3K27me3. Recent experimental research data demonstrated that PRC2 loss in mice skin results in the formation of Merkel cells. Prompted by these findings, we explored a possible contribution of PRC2 loss in human Merkel cell carcinoma. We examined the immunohistochemical expression of H3K27me3 in 35 Merkel cell carcinomas with pure histological features (22 primary and 13 metastatic lesions) and in 5 combined squamous and neuroendocrine carcinomas of the skin. We found a strong reduction of H3K27me3 staining in tumors with pure histologic features and virus-positive Merkel cell carcinomas. Combined neuroendocrine carcinomas had no or only minimal loss of H3K27me3 labeling. Our findings suggest that a PRC2-mediated epigenetic deregulation may play a role in the pathogenesis of virus-positive Merkel cell carcinomas and in tumors with pure histologic features.

Targeted inhibition of histone H3K27 demethylation is effective in high-risk neuroblastoma.

PubMed

Lochmann, Timothy L; Powell, Krista M; Ham, Jungoh; Floros, Konstantinos V; Heisey, Daniel A R; Kurupi, Richard I J; Calbert, Marissa L; Ghotra, Maninderjit S; Greninger, Patricia; Dozmorov, Mikhail; Gowda, Madhu; Souers, Andrew J; Reynolds, C Patrick; Benes, Cyril H; Faber, Anthony C

2018-05-16

High-risk neuroblastoma is often distinguished by amplification of MYCN and loss of differentiation potential. We performed high-throughput drug screening of epigenetic-targeted therapies across a large and diverse tumor cell line panel and uncovered the hypersensitivity of neuroblastoma cells to GSK-J4, a small-molecule dual inhibitor of lysine 27 of histone 3 (H3K27) demethylases ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), and histone demethylase Jumonji D3 (JMJD3). Mechanistically, GSK-J4 induced neuroblastoma differentiation and endoplasmic reticulum (ER) stress, with accompanying up-regulation of p53 up-regulated modulator of apoptosis (PUMA) and induction of cell death. Retinoic acid (RA)-resistant neuroblastoma cells were sensitive to GSK-J4. In addition, GSK-J4 was effective at blocking the growth of chemorefractory and patient-derived xenograft models of high-risk neuroblastoma in vivo. Furthermore, GSK-J4 and RA combination increased differentiation and ER stress over GSK-J4 effects and limited the growth of neuroblastomas resistant to either drug alone. In MYCN -amplified neuroblastoma, PUMA induction by GSK-J4 sensitized tumors to the B cell lymphoma 2 (BCL-2) inhibitor venetoclax, demonstrating that epigenetic-targeted therapies and BCL-2 homology domain 3 mimetics can be rationally combined to treat this high-risk subset of neuroblastoma. Therefore, H3K27 demethylation inhibition is a promising therapeutic target to treat high-risk neuroblastoma, and H3K27 demethylation can be part of rational combination therapies to induce robust antineuroblastoma activity. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Jumonji domain-containing protein 3 regulates the early inflammatory response epigenetically in human periodontal ligament cells.

PubMed

Wang, Puyu; Yue, Junli; Xu, Weizhe; Chen, Xi; Yi, Xiaowei; Ye, Ling; Zhang, Lan; Huang, Dingming

2018-05-30

To investigate the role of the histone 3 lysine 27 trimethylation (H3K27me3) demethylase Jumonji domain-containing protein 3 (Jmjd3) in the epigenetic regulation of the inflammatory response in human periodontal ligament cells (HPDLs). HPDLs were stimulated with lipopolysaccharide from E. coli. The expression of Jmjd3 in HPDLs was examined by quantitative real-time polymerase chain reaction (Q-PCR), Western Blot and immunofluorescent staining. Potential target genes were selected by silencing Jmjd3 and were confirmed by Chromatin Immunoprecipitation (ChIP). Q-PCR, Western Blot and immunofluorescent staining revealed that the expression of Jmjd3 was increased in inflamed HPDLs. Knockdown of Jmjd3 led to the suppression of inflammation-induced up-regulation of interleukin-6 and interleukin-12. Moreover, ChIP assays demonstrated that Jmjd3 was recruited to the promoters of interleukin-6 and interleukin-12b and this recruitment was associated with decreased levels of trimethylated histone 3 lysine 27 (H3K27). It was concluded that Jmjd3 regulated the activation of interleukin-6 and interleukin-12b in the early inflammatory response of HPDLs via demethylation of H3K27me3 at promoters. This molecular event may play an important role in the regulation of the inflammatory response in HPDLs. Copyright © 2018. Published by Elsevier Ltd.

Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification

PubMed Central

Xu, Cheng-Ran; Li, Lin-Chen; Donahue, Greg; Ying, Lei; Zhang, Yu-Wei; Gadue, Paul; Zaret, Kenneth S

2014-01-01

Endoderm cells undergo sequential fate choices to generate insulin-secreting beta cells. Ezh2 of the PRC2 complex, which generates H3K27me3, modulates the transition from endoderm to pancreas progenitors, but the role of Ezh2 and H3K27me3 in the next transition to endocrine progenitors is unknown. We isolated endoderm cells, pancreas progenitors, and endocrine progenitors from different staged mouse embryos and analyzed H3K27me3 genome-wide. Unlike the decline in H3K27me3 domains reported during embryonic stem cell differentiation in vitro, we find that H3K27me3 domains increase in number during endocrine progenitor development in vivo. Genes that lose the H3K27me3 mark typically encode transcriptional regulators, including those for pro-endocrine fates, whereas genes that acquire the mark typically are involved in cell biology and morphogenesis. Deletion of Ezh2 at the pancreas progenitor stage enhanced the production of endocrine progenitors and beta cells. Inhibition of EZH2 in embryonic pancreas explants and in human embryonic stem cell cultures increased endocrine progenitors in vitro. Our studies reveal distinct dynamics in H3K27me3 targets in vivo and a means to modulate beta cell development from stem cells. PMID:25107471

JMJ27, an Arabidopsis H3K9 histone demethylase, modulates defense against Pseudomonas syringae and flowering time.

PubMed

Dutta, Aditya; Choudhary, Pratibha; Caruana, Julie; Raina, Ramesh

2017-09-01

Histone methylation is known to dynamically regulate diverse developmental and physiological processes. Histone methyl marks are written by methyltransferases and erased by demethylases, and result in modification of chromatin structure to repress or activate transcription. However, little is known about how histone methylation may regulate defense mechanisms and flowering time in plants. Here we report characterization of JmjC DOMAIN-CONTAINING PROTEIN 27 (JMJ27), an Arabidopsis JHDM2 (JmjC domain-containing histone demethylase 2) family protein, which modulates defense against pathogens and flowering time. JMJ27 is a nuclear protein containing a zinc-finger motif and a catalytic JmjC domain with conserved Fe(II) and α-ketoglutarate binding sites, and displays H3K9me1/2 demethylase activity both in vitro and in vivo. JMJ27 is induced in response to virulent Pseudomonas syringae pathogens and is required for resistance against these pathogens. JMJ27 is a negative modulator of WRKY25 (a repressor of defense) and a positive modulator of several pathogenesis-related (PR) proteins. Additionally, loss of JMJ27 function leads to early flowering. JMJ27 negatively modulates the major flowering regulator CONSTANS (CO) and positively modulates FLOWERING LOCUS C (FLC). Taken together, our results indicate that JMJ27 functions as a histone demethylase to modulate both physiological (defense) and developmental (flowering time) processes in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hypertrimethylation.

PubMed

Ott, Heidi M; Graves, Alan P; Pappalardi, Melissa B; Huddleston, Michael; Halsey, Wendy S; Hughes, Ashley M; Groy, Arthur; Dul, Edward; Jiang, Yong; Bai, Yuchen; Annan, Roland; Verma, Sharad K; Knight, Steven D; Kruger, Ryan G; Dhanak, Dashyant; Schwartz, Benjamin; Tummino, Peter J; Creasy, Caretha L; McCabe, Michael T

2014-12-01

The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell-derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival. ©2014 American Association for Cancer Research.

Comparative Analyses of H3K4 and H3K27 Trimethylations Between the Mouse Cerebrum and Testis

PubMed Central

Cui, Peng; Liu, Wanfei; Zhao, Yuhui; Lin, Qiang; Zhang, Daoyong; Ding, Feng; Xin, Chengqi; Zhang, Zhang; Song, Shuhui; Sun, Fanglin; Yu, Jun; Hu, Songnian

2012-01-01

The global features of H3K4 and H3K27 trimethylations (H3K4me3 and H3K27me3) have been well studied in recent years, but most of these studies were performed in mammalian cell lines. In this work, we generated the genome-wide maps of H3K4me3 and H3K27me3 of mouse cerebrum and testis using ChIP-seq and their high-coverage transcriptomes using ribominus RNA-seq with SOLiD technology. We examined the global patterns of H3K4me3 and H3K27me3 in both tissues and found that modifications are closely-associated with tissue-specific expression, function and development. Moreover, we revealed that H3K4me3 and H3K27me3 rarely occur in silent genes, which contradicts the findings in previous studies. Finally, we observed that bivalent domains, with both H3K4me3 and H3K27me3, existed ubiquitously in both tissues and demonstrated an invariable preference for the regulation of developmentally-related genes. However, the bivalent domains tend towards a “winner-takes-all” approach to regulate the expression of associated genes. We also verified the above results in mouse ES cells. As expected, the results in ES cells are consistent with those in cerebrum and testis. In conclusion, we present two very important findings. One is that H3K4me3 and H3K27me3 rarely occur in silent genes. The other is that bivalent domains may adopt a “winner-takes-all” principle to regulate gene expression. PMID:22768982

Enhancer-associated H3K4 monomethylation by Trithorax-related, the Drosophila homolog of mammalian Mll3/Mll4.

PubMed

Herz, Hans-Martin; Mohan, Man; Garruss, Alexander S; Liang, Kaiwei; Takahashi, Yoh-Hei; Mickey, Kristen; Voets, Olaf; Verrijzer, C Peter; Shilatifard, Ali

2012-12-01

Monomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are implemented by Set1/COMPASS (complex of proteins associated with Set1), there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax, and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of the mammalian Mll3/4 COMPASS-like complexes, can function as a major H3K4 monomethyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac levels in various tissues. Assays with the cut wing margin enhancer implied a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrated that Trr is required to maintain the H3K4me1 and H3K27ac chromatin signature that resembles the histone modification patterns described for enhancers. Furthermore, studies in the mammalian system suggested a role for the Trr homolog Mll3 in similar processes. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 monomethyltransferases Trr/Mll3/Mll4 and the H3K27 demethylase UTX cooperate to regulate the transition from inactive/poised to active enhancers.

Enhancer-associated H3K4 monomethylation by Trithorax-related, the Drosophila homolog of mammalian Mll3/Mll4

PubMed Central

Herz, Hans-Martin; Mohan, Man; Garruss, Alexander S.; Liang, Kaiwei; Takahashi, Yoh-hei; Mickey, Kristen; Voets, Olaf; Verrijzer, C. Peter; Shilatifard, Ali

2012-01-01

Monomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are implemented by Set1/COMPASS (complex of proteins associated with Set1), there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax, and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of the mammalian Mll3/4 COMPASS-like complexes, can function as a major H3K4 monomethyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac levels in various tissues. Assays with the cut wing margin enhancer implied a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrated that Trr is required to maintain the H3K4me1 and H3K27ac chromatin signature that resembles the histone modification patterns described for enhancers. Furthermore, studies in the mammalian system suggested a role for the Trr homolog Mll3 in similar processes. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 monomethyltransferases Trr/Mll3/Mll4 and the H3K27 demethylase UTX cooperate to regulate the transition from inactive/poised to active enhancers. PMID:23166019

H3K27me3 dynamics dictate evolving uterine states in pregnancy and parturition

PubMed Central

Nancy, Patrice; Siewiera, Johan; Tagliani, Elisa; Osokine, Ivan; Manandhar, Priyanka; Clementi, Caterina

2017-01-01

Uncovering the causes of pregnancy complications such as preterm labor requires greater insight into how the uterus remains in a noncontractile state until term and then surmounts this state to enter labor. Here, we show that dynamic generation and erasure of the repressive histone modification tri-methyl histone H3 lysine 27 (H3K27me3) in decidual stromal cells dictate both elements of pregnancy success in mice. In early gestation, H3K27me3-induced transcriptional silencing of select gene targets ensured uterine quiescence by preventing the decidua from expressing parturition-inducing hormone receptors, manifesting type 1 immunity, and most unexpectedly, generating myofibroblasts and associated wound-healing responses. In late gestation, genome-wide H3K27 demethylation allowed for target gene upregulation, decidual activation, and labor entry. Pharmacological inhibition of H3K27 demethylation in late gestation not only prevented term parturition, but also inhibited delivery while maintaining pup viability in a noninflammatory model of preterm parturition. Immunofluorescence analysis of human specimens suggested that similar regulatory events might occur in the human decidua. Together, these results reveal the centrality of regulated gene silencing in the uterine adaptation to pregnancy and suggest new areas in the study and treatment of pregnancy disorders. PMID:29202469

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication

PubMed Central

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-01

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. PMID:27679476

The Histone Modification H3K27me3 Is Retained after Gene Duplication and Correlates with Conserved Noncoding Sequences in Arabidopsis

PubMed Central

Berke, Lidija; Snel, Berend

2014-01-01

The histone modification H3K27me3 is involved in repression of transcription and plays a crucial role in developmental transitions in both animals and plants. It is deposited by PRC2 (Polycomb repressive complex 2), a conserved protein complex. In Arabidopsis thaliana, H3K27me3 is found at 15% of all genes. These tend to encode transcription factors and other regulators important for development. However, it is not known how PRC2 is recruited to target loci nor how this set of target genes arose during Arabidopsis evolution. To resolve the latter, we integrated A. thaliana gene families with five independent genome-wide H3K27me3 data sets. Gene families were either significantly enriched or depleted of H3K27me3, showing a strong impact of shared ancestry to H3K27me3 distribution. To quantify this, we performed ancestral state reconstruction of H3K27me3 on phylogenetic trees of gene families. The set of H3K27me3-marked genes changed less than expected by chance, suggesting that H3K27me3 was retained after gene duplication. This retention suggests that the PRC2-recruiting signal could be encoded in the DNA and also conserved among certain duplicated genes. Indeed, H3K27me3-marked genes were overrepresented among paralogs sharing conserved noncoding sequences (CNSs) that are enriched with transcription factor binding sites. The association of upstream CNSs with H3K27me3-marked genes represents the first genome-wide connection between H3K27me3 and potential regulatory elements in plants. Thus, we propose that CNSs likely function as part of the PRC2 recruitment in plants. PMID:24567304

JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors

PubMed Central

Kochat, Veena; Equbal, Zaffar; Baligar, Prakash; Kumar, Vikash; Srivastava, Madhulika; Mukhopadhyay, Asok

2017-01-01

The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

PubMed

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-09

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

H3K4 demethylase activities repress proliferative and postmitotic aging

PubMed Central

Alvares, Stacy M; Mayberry, Gaea A; Joyner, Ebony Y; Lakowski, Bernard; Ahmed, Shawn

2014-01-01

Homeostasis of postmitotic and proliferating cells is maintained by pathways that repress stress. We found that the Caenorhabditis elegans histone 3 lysine 4 (H3K4) demethylases RBR-2 and SPR-5 promoted postmitotic longevity of stress-resistant daf-2 adults, altered pools of methylated H3K4, and promoted silencing of some daf-2 target genes. In addition, RBR-2 and SPR-5 were required for germ cell immortality at a high temperature. Transgenerational proliferative aging was enhanced for spr-5; rbr-2 double mutants, suggesting that these histone demethylases may function sequentially to promote germ cell immortality by targeting distinct H3K4 methyl marks. RBR-2 did not play a comparable role in the maintenance of quiescent germ cells in dauer larvae, implying that it represses stress that occurs as a consequence of germ cell proliferation, rather than stress that accumulates in nondividing cells. We propose that H3K4 demethylase activities promote the maintenance of chromatin states during stressful growth conditions, thereby repressing postmitotic aging of somatic cells as well as proliferative aging of germ cells. PMID:24134677

Telobox motifs recruit CLF/SWN-PRC2 for H3K27me3 deposition via TRB factors in Arabidopsis.

PubMed

Zhou, Yue; Wang, Yuejun; Krause, Kristin; Yang, Tingting; Dongus, Joram A; Zhang, Yijing; Turck, Franziska

2018-05-01

Polycomb repressive complexes (PRCs) control organismic development in higher eukaryotes through epigenetic gene repression 1-4 . PRC proteins do not contain DNA-binding domains, thus prompting questions regarding how PRCs find their target loci 5 . Here we present genome-wide evidence of PRC2 recruitment by telomere-repeat-binding factors (TRBs) through telobox-related motifs in Arabidopsis. A triple trb1-2, trb2-1, and trb3-2 (trb1/2/3) mutant with a developmental phenotype and a transcriptome strikingly similar to those of strong PRC2 mutants showed redistribution of trimethyl histone H3 Lys27 (H3K27me3) marks and lower H3K27me3 levels, which were correlated with derepression of TRB1-target genes. TRB1-3 physically interacted with the PRC2 proteins CLF and SWN. A SEP3 reporter gene with a telobox mutation showed ectopic expression, which was correlated with H3K27me3 depletion, whereas tethering TRB1 to the mutated cis element partially restored repression. We propose that telobox-related motifs recruit PRC2 through the interaction between TRBs and CLF/SWN, a mechanism essential for H3K27me3 deposition at a subset of target genes.

Arsenic activates the expression of 3β-HSD in mouse Leydig cells through repression of histone H3K9 methylation.

PubMed

Alamdar, Ambreen; Xi, Guochen; Huang, Qingyu; Tian, Meiping; Eqani, Syed Ali Musstjab Akber Shah; Shen, Heqing

2017-07-01

Arsenic exposure has been associated with male reproductive dysfunction by disrupting steroidogenesis; however, the roles of epigenetic drivers, especially histone methylation in arsenic-induced steroidogenic toxicity remain not well documented. In this study, we investigated the role of histone H3 lysine 9 (H3K9) methylation in steroidogenesis disturbance in mouse Leydig cells (MLTC-1) due to arsenic exposure. Our results indicated that mRNA and protein expression levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) were both significantly up-regulated while the rest of key genes involved in steroidogenesis were down-regulated. Moreover, arsenic exposure significantly decreased the histone H3K9 di- and tri-methylation (H3K9me2/3) levels in MLTC-1 cells. Since H3K9 demethylation leads to gene activation, we further investigated whether the induction of 3β-HSD expression was ascribed to reduced H3K9 methylation. The results showed that H3K9me2/3 demethylase (JMJD2A) inhibitor, quercetin (Que) significantly attenuated the decrease of H3K9me2/3 and increase of 3β-HSD expression induced by arsenic. To further elucidate the mechanism for the activation of 3β-HSD, we determined the histone H3K9 methylation levels in Hsd3b gene promoter, which also showed significant decrease of H3K9me2/3 in the investigated region after arsenic exposure. Considering these results, we conclude that arsenic exposure induced 3β-HSD up-regulation by suppressing H3K9me2/3 status, which is suggested as a compensatory mechanism for steroidogenic disturbance in MLTC-1 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity.

PubMed

Xun, Jing; Wang, Dekun; Shen, Long; Gong, Junbo; Gao, Ruifang; Du, Lingfang; Chang, Antao; Song, Xiangrong; Xiang, Rong; Tan, Xiaoyue

2017-03-28

Epigenetic regulator JMJD3 plays an important role in both tumor progression and somatic cell reprogramming. Here, we explored the effect of JMJD3 on the stem cell-like characteristics of breast cancer and its underlying mechanism involving stemness-related transcription factor Oct4. Our data revealed that, in breast cancer cells lines and an orthotopic xenograph mouse model of breast cancer, ectopic overexpression of JMJD3 suppressed stem cell-like characteristics of breast cancer cells, whereas knockdown of JMJD3 promoted these characteristics. Oct4 mediated the suppressive effects of JMJD3 on the stemness of breast cancer cells. The inhibitory effect of JMJD3 on Oct4 was independent of demethylase activity, but mediated via degradation of PHF20. Furthermore, we applied an agonist of the vitamin D receptor, paricalcitol, and found that it induced JMJD3 in breast cancer cells. Our data showed that administration of paricalcitol suppressed stem cell-like characteristics and Oct4 expression. Taken together, JMJD3 inhibits the stem cell-like characteristics in breast cancer by suppression of stemness factor Oct4 in a PHF20-dependent manner. Administration of paricalcitol leads to upregulation of JMJD3 that suppresses Oct4 expression and the stem cell-like characteristics in breast cancer.

Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a/b.

PubMed

Ebata, Kevin T; Mesh, Kathryn; Liu, Shichong; Bilenky, Misha; Fekete, Alexander; Acker, Michael G; Hirst, Martin; Garcia, Benjamin A; Ramalho-Santos, Miguel

2017-01-01

Histone methylation patterns regulate gene expression and are highly dynamic during development. The erasure of histone methylation is carried out by histone demethylase enzymes. We had previously shown that vitamin C enhances the activity of Tet enzymes in embryonic stem (ES) cells, leading to DNA demethylation and activation of germline genes. We report here that vitamin C induces a remarkably specific demethylation of histone H3 lysine 9 dimethylation (H3K9me2) in naïve ES cells. Vitamin C treatment reduces global levels of H3K9me2, but not other histone methylation marks analyzed, as measured by western blot, immunofluorescence and mass spectrometry. Vitamin C leads to widespread loss of H3K9me2 at large chromosomal domains as well as gene promoters and repeat elements. Vitamin C-induced loss of H3K9me2 occurs rapidly within 24 h and is reversible. Importantly, we found that the histone demethylases Kdm3a and Kdm3b are required for vitamin C-induced demethylation of H3K9me2. Moreover, we show that vitamin C-induced Kdm3a/b-mediated H3K9me2 demethylation and Tet-mediated DNA demethylation are independent processes at specific loci. Lastly, we document Kdm3a/b are partially required for the upregulation of germline genes by vitamin C. These results reveal a specific role for vitamin C in histone demethylation in ES cells and document that DNA methylation and H3K9me2 cooperate to silence germline genes in pluripotent cells.

Arsenic activates the expression of 3β-HSD in mouse Leydig cells through repression of histone H3K9 methylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alamdar, Ambreen; Xi, Guochen

Arsenic exposure has been associated with male reproductive dysfunction by disrupting steroidogenesis; however, the roles of epigenetic drivers, especially histone methylation in arsenic-induced steroidogenic toxicity remain not well documented. In this study, we investigated the role of histone H3 lysine 9 (H3K9) methylation in steroidogenesis disturbance in mouse Leydig cells (MLTC-1) due to arsenic exposure. Our results indicated that mRNA and protein expression levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) were both significantly up-regulated while the rest of key genes involved in steroidogenesis were down-regulated. Moreover, arsenic exposure significantly decreased the histone H3K9 di- and tri-methylation (H3K9me2/3) levels in MLTC-1 cells. Sincemore » H3K9 demethylation leads to gene activation, we further investigated whether the induction of 3β-HSD expression was ascribed to reduced H3K9 methylation. The results showed that H3K9me2/3 demethylase (JMJD2A) inhibitor, quercetin (Que) significantly attenuated the decrease of H3K9me2/3 and increase of 3β-HSD expression induced by arsenic. To further elucidate the mechanism for the activation of 3β-HSD, we determined the histone H3K9 methylation levels in Hsd3b gene promoter, which also showed significant decrease of H3K9me2/3 in the investigated region after arsenic exposure. Considering these results, we conclude that arsenic exposure induced 3β-HSD up-regulation by suppressing H3K9me2/3 status, which is suggested as a compensatory mechanism for steroidogenic disturbance in MLTC-1 cells. - Highlights: • Epigenetic mechanisms of arsenic-induced male reproductive toxicity remain unclear. • Arsenic disturbs the expression of key steroidogenic genes in MLTC-1 cells. • Histone H3K9 di- and tri-methylation was suppressed in arsenic-exposed cells. • Arsenic activates 3β-HSD expression through repression of histone H3K9 methylation.« less

H3K27me3 forms BLOCs over silent genes and intergenic regions and specifies a histone banding pattern on a mouse autosomal chromosome

PubMed Central

Pauler, Florian M.; Sloane, Mathew A.; Huang, Ru; Regha, Kakkad; Koerner, Martha V.; Tamir, Ido; Sommer, Andreas; Aszodi, Andras; Jenuwein, Thomas; Barlow, Denise P.

2009-01-01

In mammals, genome-wide chromatin maps and immunofluorescence studies show that broad domains of repressive histone modifications are present on pericentromeric and telomeric repeats and on the inactive X chromosome. However, only a few autosomal loci such as silent Hox gene clusters have been shown to lie in broad domains of repressive histone modifications. Here we present a ChIP-chip analysis of the repressive H3K27me3 histone modification along chr 17 in mouse embryonic fibroblast cells using an algorithm named broad local enrichments (BLOCs), which allows the identification of broad regions of histone modifications. Our results, confirmed by BLOC analysis of a whole genome ChIP-seq data set, show that the majority of H3K27me3 modifications form BLOCs rather than focal peaks. H3K27me3 BLOCs modify silent genes of all types, plus flanking intergenic regions and their distribution indicates a negative correlation between H3K27me3 and transcription. However, we also found that some nontranscribed gene-poor regions lack H3K27me3. We therefore performed a low-resolution analysis of whole mouse chr 17, which revealed that H3K27me3 is enriched in mega-base-pair-sized domains that are also enriched for genes, short interspersed elements (SINEs) and active histone modifications. These genic H3K27me3 domains alternate with similar-sized gene-poor domains. These are deficient in active histone modifications, as well as H3K27me3, but are enriched for long interspersed elements (LINEs) and long-terminal repeat (LTR) transposons and H3K9me3 and H4K20me3. Thus, an autosome can be seen to contain alternating chromatin bands that predominantly separate genes from one retrotransposon class, which could offer unique domains for the specific regulation of genes or the silencing of autonomous retrotransposons. PMID:19047520

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27).

PubMed

McCabe, Michael T; Graves, Alan P; Ganji, Gopinath; Diaz, Elsie; Halsey, Wendy S; Jiang, Yong; Smitheman, Kimberly N; Ott, Heidi M; Pappalardi, Melissa B; Allen, Kimberly E; Chen, Stephanie B; Della Pietra, Anthony; Dul, Edward; Hughes, Ashley M; Gilbert, Seth A; Thrall, Sara H; Tummino, Peter J; Kruger, Ryan G; Brandt, Martin; Schwartz, Benjamin; Creasy, Caretha L

2012-02-21

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k(cat)/K(m) ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)

PubMed Central

McCabe, Michael T.; Graves, Alan P.; Ganji, Gopinath; Diaz, Elsie; Halsey, Wendy S.; Jiang, Yong; Smitheman, Kimberly N.; Ott, Heidi M.; Pappalardi, Melissa B.; Allen, Kimberly E.; Chen, Stephanie B.; Della Pietra, Anthony; Dul, Edward; Hughes, Ashley M.; Gilbert, Seth A.; Thrall, Sara H.; Tummino, Peter J.; Kruger, Ryan G.; Brandt, Martin; Schwartz, Benjamin; Creasy, Caretha L.

2012-01-01

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 kcat/Km ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 kcat/Km ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 kcat/Km ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2. PMID:22323599

Immunohistochemical analysis of H3K27me3 demonstrates global reduction in group-A childhood posterior fossa ependymoma and is a powerful predictor of outcome.

PubMed

Panwalkar, Pooja; Clark, Jonathan; Ramaswamy, Vijay; Hawes, Debra; Yang, Fusheng; Dunham, Christopher; Yip, Stephen; Hukin, Juliette; Sun, Yilun; Schipper, Matthew J; Chavez, Lukas; Margol, Ashley; Pekmezci, Melike; Chung, Chan; Banda, Adam; Bayliss, Jill M; Curry, Sarah J; Santi, Mariarita; Rodriguez, Fausto J; Snuderl, Matija; Karajannis, Matthias A; Saratsis, Amanda M; Horbinski, Craig M; Carret, Anne-Sophie; Wilson, Beverly; Johnston, Donna; Lafay-Cousin, Lucie; Zelcer, Shayna; Eisenstat, David; Silva, Marianna; Scheinemann, Katrin; Jabado, Nada; McNeely, P Daniel; Kool, Marcel; Pfister, Stefan M; Taylor, Michael D; Hawkins, Cynthia; Korshunov, Andrey; Judkins, Alexander R; Venneti, Sriram

2017-11-01

Posterior fossa ependymomas (EPN_PF) in children comprise two morphologically identical, but biologically distinct tumor entities. Group-A (EPN_PFA) tumors have a poor prognosis and require intensive therapy. In contrast, group-B tumors (EPN_PFB) exhibit excellent prognosis and the current consensus opinion recommends future clinical trials to test the possibility of treatment de-escalation in these patients. Therefore, distinguishing these two tumor subtypes is critical. EPN_PFA and EPN_PFB can be distinguished based on DNA methylation signatures, but these assays are not routinely available. We have previously shown that a subset of poorly prognostic childhood EPN_PF exhibits global reduction in H3K27me3. Therefore, we set out to determine whether a simple immunohistochemical assay for H3K27me3 could be used to segregate EPN_PFA from EPN_PFB tumors. We assembled a cohort of 230 childhood ependymomas and H3K27me3 immunohistochemistry was assessed as positive or negative in a blinded manner. H3K27me3 staining results were compared with DNA methylation-based subgroup information available in 112 samples [EPN_PFA (n = 72) and EPN_PFB tumors (n = 40)]. H3K27me3 staining was globally reduced in EPN_PFA tumors and immunohistochemistry showed 99% sensitivity and 100% specificity in segregating EPN_PFA from EPN_PFB tumors. Moreover, H3K27me3 immunostaining was sufficient to delineate patients with worse prognosis in two independent, non-overlapping cohorts (n = 133 and n = 97). In conclusion, immunohistochemical evaluation of H3K27me3 global reduction is an economic, easily available and readily adaptable method for defining high-risk EPN_PFA from low-risk posterior fossa EPN_PFB tumors to inform prognosis and to enable the design of future clinical trials.

EZH2-mediated H3K27 trimethylation mediates neurodegeneration in ataxia-telangiectasia

PubMed Central

Li, Jiali; Hart, Ronald P.; Mallimo, Elyse M.; Swerdel, Mavis R.; Kusnecov, Alexander; Herrup, Karl

2014-01-01

The symptoms of ataxia-telangiectasia (A-T) include a progressive neurodegeneration caused by ATM protein deficiency. We previously found that nuclear accumulation of histone deacetylase-4, HDAC4, contributes to this degeneration; we now report that increased histone H3K27 trimethylation (H3K27me3) mediated by polycomb repressive complex 2 (PRC2) also plays an important role in the A-T phenotype. Enhancer of zeste homolog 2 (EZH2), a core catalytic component of PRC2, is a new ATM kinase target, and ATM-mediated S734 phosphorylation of EZH2 reduces protein stability. Thus, PRC2 formation is elevated along with H3K27me3in ATM deficiency. ChIP-sequencing shows a significant increase in H3K27me3 ‘marks’ and a dramatic shift in their location. The change of H3K27me3 chromatin-binding pattern is directly related to cell cycle re-entry and cell death of ATM-deficient neurons. Lentiviral knockdown of EZH2 rescues Purkinje cell degeneration and behavioral abnormalities in Atm−/− mice, demonstrating that EZH2 hyperactivity is another key factor in A-T neurodegeneration. PMID:24162653

Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

PubMed Central

Zhen, Chao Yu; Tatavosian, Roubina; Huynh, Thao Ngoc; Duc, Huy Nguyen; Das, Raibatak; Kokotovic, Marko; Grimm, Jonathan B; Lavis, Luke D; Lee, Jun; Mejia, Frances J; Li, Yang; Yao, Tingting; Ren, Xiaojun

2016-01-01

The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes. DOI: http://dx.doi.org/10.7554/eLife.17667.001 PMID:27723458

The regulation of Jmjd3 upon the expression of NF-κB downstream inflammatory genes in LPS activated vascular endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Shaoqing; Graduate School of Medicine, Nanchang University, Nanchang; Chen, Xia

Inflammatory mediators and adhesion molecules have been implicated in a variety of diseases including atherosclerosis. As both the mediator-releasing and targeted cells, vascular endothelial cells play key role in pathological processes. NF-κB signaling regulates a cluster of inflammatory factors in LPS-activated vascular endothelial cells but the underlying mechanisms remain largely unknown. Here, we investigated the epigenetic regulation of LPS upon the expression of inflammatory mediators and adhesion molecules. We found that LPS treatment promoted jmjd3 expression, enhanced Jmjd3 nuclear accumulation in human vascular endothelial cells. In addition, LPS enhanced the demethylation of H3K27me3, a specific substrate of Jmjd3. LPS treatmentmore » recruited Jmjd3 and NF-κB to the promoter region of target genes, suggesting Jmjd3 synergizes with NF-κB to activate the expression of target genes. We further found that Jmjd3 attenuated the methylation status in promoter region of target genes, culminating in target gene expression. Our findings unveil epigenetic regulations of LPS upon NF-κB pathway and identify Jmjd3 as a critical modulator of NF-κB pathway and potential therapeutic target for NF-κB related diseases including atherosclerosis.« less

LHP1 Regulates H3K27me3 Spreading and Shapes the Three-Dimensional Conformation of the Arabidopsis Genome

PubMed Central

Ariel, Federico; Latrasse, David; Mariappan, Kiruthiga Gayathri; Kim, Soon-Kap; Crespi, Martin; Hirt, Heribert; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

2016-01-01

Precise expression patterns of genes in time and space are essential for proper development of multicellular organisms. Dynamic chromatin conformation and spatial organization of the genome constitute a major step in this regulation to modulate developmental outputs. Polycomb repressive complexes (PRCs) mediate stable or flexible gene repression in response to internal and environmental cues. In Arabidopsis thaliana, LHP1 co-localizes with H3K27me3 epigenetic marks throughout the genome and interacts with PRC1 and PRC2 members as well as with a long noncoding RNA. Here, we show that LHP1 is responsible for the spreading of H3K27me3 towards the 3’ end of the gene body. We also identified a subset of LHP1-activated genes and demonstrated that LHP1 shapes local chromatin topology in order to control transcriptional co-regulation. Our work reveals a general role of LHP1 from local to higher conformation levels of chromatin configuration to determine its accessibility to define gene expression patterns. PMID:27410265

Additional sex combs interacts with enhancer of zeste and trithorax and modulates levels of trimethylation on histone H3K4 and H3K27 during transcription of hsp70.

PubMed

Li, Taosui; Hodgson, Jacob W; Petruk, Svetlana; Mazo, Alexander; Brock, Hugh W

2017-09-19

Maintenance of cell fate determination requires the Polycomb group for repression; the trithorax group for gene activation; and the enhancer of trithorax and Polycomb (ETP) group for both repression and activation. Additional sex combs (Asx) is a genetically identified ETP for the Hox loci, but the molecular basis of its dual function is unclear. We show that in vitro, Asx binds directly to the SET domains of the histone methyltransferases (HMT) enhancer of zeste [E(z)] (H3K27me3) and Trx (H3K4me3) through a bipartite interaction site separated by 846 amino acid residues. In Drosophila S2 cell nuclei, Asx interacts with E(z) and Trx in vivo. Drosophila Asx is required for repression of heat-shock gene hsp70 and is recruited downstream of the hsp70 promoter. Changes in the levels of H3K4me3 and H3K27me3 downstream of the hsp70 promoter in Asx mutants relative to wild type show that Asx regulates H3K4 and H3K27 trimethylation. We propose that during transcription Asx modulates the ratio of H3K4me3 to H3K27me3 by selectively recruiting the antagonistic HMTs, E(z) and Trx or other nucleosome-modifying enzymes to hsp70.

The SUVR4 Histone Lysine Methyltransferase Binds Ubiquitin and Converts H3K9me1 to H3K9me3 on Transposon Chromatin in Arabidopsis

PubMed Central

Veiseth, Silje V.; Rahman, Mohummad A.; Yap, Kyoko L.; Fischer, Andreas; Egge-Jacobsen, Wolfgang; Reuter, Gunter; Zhou, Ming-Ming; Aalen, Reidunn B.; Thorstensen, Tage

2011-01-01

Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs) on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases) can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3) is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation–dependent and –independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity. PMID:21423664

Epigenetic regulation of facultative heterochromatinisation in Planococcus citri via the Me(3)K9H3-HP1-Me(3)K20H4 pathway.

PubMed

Bongiorni, Silvia; Pasqualini, Barbara; Taranta, Monia; Singh, Prim B; Prantera, Giorgio

2007-03-15

Using RNA interference (RNAi) we have conducted a functional analysis of the HP1-like chromobox gene pchet2 during embryogenesis of the mealybug Planococcus citri. Knocking down pchet2 expression results in decondensation of the male-specific chromocenter that normally arises from the developmentally-regulated facultative heterochromatinisation of the paternal chromosome complement. Together with the disappearance of the chromocenter the staining levels of two associated histone modifications, tri-methylated lysine 9 of histone H3 [Me(3)K9H3] and tri-methylated lysine 20 of histone H4 [Me(3)K20H4], are reduced to undetectable levels. Embryos treated with double-stranded RNA (dsRNA) targeting pchet2 also exhibit chromosome abnormalities, such as aberrant chromosome condensation, and also the presence of metaphases that contain 'lagging' chromosomes. We conclude that PCHET2 regulates chromosome behavior during metaphase and is a crucial component of a Me(3)K9H3-HP1-Me(3)K20H4 pathway involved in the facultative heterochromatinisation of the (imprinted) paternal chromosome set.

H3K4me3 breadth is linked to cell identity and transcriptional consistency.

PubMed

Benayoun, Bérénice A; Pollina, Elizabeth A; Ucar, Duygu; Mahmoudi, Salah; Karra, Kalpana; Wong, Edith D; Devarajan, Keerthana; Daugherty, Aaron C; Kundaje, Anshul B; Mancini, Elena; Hitz, Benjamin C; Gupta, Rakhi; Rando, Thomas A; Baker, Julie C; Snyder, Michael P; Cherry, J Michael; Brunet, Anne

2014-07-31

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a chromatin modification known to mark the transcription start sites of active genes. Here, we show that H3K4me3 domains that spread more broadly over genes in a given cell type preferentially mark genes that are essential for the identity and function of that cell type. Using the broadest H3K4me3 domains as a discovery tool in neural progenitor cells, we identify novel regulators of these cells. Machine learning models reveal that the broadest H3K4me3 domains represent a distinct entity, characterized by increased marks of elongation. The broadest H3K4me3 domains also have more paused polymerase at their promoters, suggesting a unique transcriptional output. Indeed, genes marked by the broadest H3K4me3 domains exhibit enhanced transcriptional consistency and [corrected] increased transcriptional levels, and perturbation of H3K4me3 breadth leads to changes in transcriptional consistency. Thus, H3K4me3 breadth contains information that could ensure transcriptional precision at key cell identity/function genes. Copyright © 2014 Elsevier Inc. All rights reserved.

Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains.

PubMed

Van Bortle, Kevin; Ramos, Edward; Takenaka, Naomi; Yang, Jingping; Wahi, Jessica E; Corces, Victor G

2012-11-01

Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions.

Delayed Accumulation of H3K27me3 on Nascent DNA Is Essential for Recruitment of Transcription Factors at Early Stages of Stem Cell Differentiation.

PubMed

Petruk, Svetlana; Cai, Jingli; Sussman, Robyn; Sun, Guizhi; Kovermann, Sina K; Mariani, Samanta A; Calabretta, Bruno; McMahon, Steven B; Brock, Hugh W; Iacovitti, Lorraine; Mazo, Alexander

2017-04-20

Recruitment of transcription factors (TFs) to repressed genes in euchromatin is essential to activate new transcriptional programs during cell differentiation. However, recruitment of all TFs, including pioneer factors, is impeded by condensed H3K27me3-containing chromatin. Single-cell and gene-specific analyses revealed that, during the first hours of induction of differentiation of mammalian embryonic stem cells (ESCs), accumulation of the repressive histone mark H3K27me3 is delayed after DNA replication, indicative of a decondensed chromatin structure in all regions of the replicating genome. This delay provides a critical "window of opportunity" for recruitment of lineage-specific TFs to DNA. Increasing the levels of post-replicative H3K27me3 or preventing S phase entry inhibited recruitment of new TFs to DNA and significantly blocked cell differentiation. These findings suggest that recruitment of lineage-specifying TFs occurs soon after replication and is facilitated by a decondensed chromatin structure. This insight may explain the developmental plasticity of stem cells and facilitate their exploitation for therapeutic purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

ATXR5 and ATXR6 are novel H3K27 monomethyltransferases required for chromatin structure and gene silencing

PubMed Central

Jacob, Yannick; Feng, Suhua; LeBlanc, Chantal A.; Bernatavichute, Yana V.; Stroud, Hume; Cokus, Shawn; Johnson, Lianna M.; Pellegrini, Matteo; Jacobsen, Steven E.; Michaels, Scott D.

2009-01-01

Constitutive heterochromatin in Arabidopsis thaliana is marked by repressive chromatin modifications including DNA methylation, histone H3 dimethylation at lysine 9 (H3K9me2), and monomethylation at lysine 27 (H3K27me1). The enzymes catalyzing DNA methylation and H3K9me2 have been identified and mutations in these proteins lead to the reactivation of silenced heterochromatic elements. The enzymes responsible for heterochromatic H3K27me1, in contrast, remain unknown. Here we show that the divergent SET-domain proteins ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6 exhibit H3K27 monomethyltransferase activity and double mutants have reduced H3K27me1 in vivo and show partial heterochromatin decondensation. Mutations in atxr5 and atxr6 also lead to transcriptional activation of repressed heterochromatic elements. Interestingly, H3K9me2 and DNA methylation are unaffected in the double mutant. These results indicate that ATXR5 and ATXR6 form a novel class of H3K27 methyltransferases and that H3K27me1 represents a new pathway required for transcriptional repression in Arabidopsis. PMID:19503079

Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains

PubMed Central

Van Bortle, Kevin; Ramos, Edward; Takenaka, Naomi; Yang, Jingping; Wahi, Jessica E.; Corces, Victor G.

2012-01-01

Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions. PMID:22722341

Somatic mutations of the histone H3K27 demethylase, UTX, in human cancer

PubMed Central

van Haaften, Gijs; Dalgliesh, Gillian L; Davies, Helen; Chen, Lina; Bignell, Graham; Greenman, Chris; Edkins, Sarah; Hardy, Claire; O’Meara, Sarah; Teague, Jon; Butler, Adam; Hinton, Jonathan; Latimer, Calli; Andrews, Jenny; Barthorpe, Syd; Beare, Dave; Buck, Gemma; Campbell, Peter J; Cole, Jennifer; Dunmore, Rebecca; Forbes, Simon; Jia, Mingming; Jones, David; Kok, Chai Yin; Leroy, Catherine; Lin, Meng-Lay; McBride, David J; Maddison, Mark; Maquire, Simon; McLay, Kirsten; Menzies, Andrew; Mironenko, Tatiana; Lee, Mulderrig; Mudie, Laura; Pleasance, Erin; Shepherd, Rebecca; Smith, Raffaella; Stebbings, Lucy; Stephens, Philip; Tang, Gurpreet; Tarpey, Patrick S; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Collins, V Peter; Ichimura, Koichi; Law, Simon; Wong, John; Yuen, Siu Tsan; Leung, Suet Yi; Tonon, Giovanni; DePinho, Ronald A; Tai, Yu-Tzu; Anderson, Kenneth C; Kahnoski, Richard J.; Massie, Aaron; Khoo, Sok Kean; Teh, Bin Tean; Stratton, Michael R; Futreal, P Andrew

2010-01-01

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase, UTX, pointing to histone H3 lysine methylation deregulation in multiple tumour types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene. PMID:19330029

H3K27 methylation and H3S28 phosphorylation-dependent transcriptional regulation by INHAT subunit SET/TAF-Iβ.

PubMed

Kim, Ji-Young; Kim, Kee-Beom; Son, Hye-Ju; Chae, Yun-Cheol; Oh, Si-Taek; Kim, Dong-Wook; Pak, Jhang Ho; Seo, Sang-Beom

2012-09-21

Significant progress has been made in understanding the relationship between histone modifications and 'reader' molecules and their effects on transcriptional regulation. A previously identified INHAT complex subunit, SET/TAF-Iβ, binds to histones and inhibits histone acetylation. To investigate the binding specificities of SET/TAF-Iβ to various histone modifications, we employed modified histone tail peptide array analyses. SET/TAF-Iβ strongly recognized PRC2-mediated H3K27me1/2/3; however, the bindings were completely disrupted by H3S28 phosphorylation. We have demonstrated that SET/TAF-Iβ is sequentially recruited to the target gene promoter ATF3 after the PRC2 complex via H3K27me recognition and may offer additive effects in the repression of the target gene. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3

PubMed Central

Chung, Ho-Ryun; Xu, Chao; Fuchs, Alisa; Mund, Andreas; Lange, Martin; Staege, Hannah; Schubert, Tobias; Bian, Chuanbing; Dunkel, Ilona; Eberharter, Anton; Regnard, Catherine; Klinker, Henrike; Meierhofer, David; Cozzuto, Luca; Winterpacht, Andreas; Di Croce, Luciano; Min, Jinrong; Will, Hans; Kinkley, Sarah

2016-01-01

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes. DOI: http://dx.doi.org/10.7554/eLife.10607.001 PMID:27223324

Transcription precedes loss of Xist coating and depletion of H3K27me3 during X-chromosome reprogramming in the mouse inner cell mass

PubMed Central

Williams, Lucy H.; Kalantry, Sundeep; Starmer, Joshua; Magnuson, Terry

2011-01-01

Repression of Xist RNA expression is considered a prerequisite to reversal of X-chromosome inactivation (XCI) in the mouse inner cell mass (ICM), and reactivation of X-linked genes is thought to follow loss of Xist RNA coating and heterochromatic markers of inactivation, such as methylation of histone H3. We analyzed X-chromosome activity in developing ICMs and show that reactivation of gene expression from the inactive-X initiates in the presence of Xist coating and H3K27me3. Furthermore, depletion of Xist RNA coating through forced upregulation of NANOG does not result in altered reactivation kinetics. Taken together, our observations suggest that in the ICM, X-linked gene transcription and Xist coating are uncoupled. These data fundamentally alter our perception of the reactivation process and support the existence of a mechanism to reactivate Xp-linked genes in the ICM that operates independently of loss of Xist RNA and H3K27me3 from the imprinted inactive-X. PMID:21471155

Analysis of H3K27me3 expression and DNA methylation at CCGG sites in smoking and non-smoking patients with non-small cell lung cancer and their clinical significance

PubMed Central

Zhu, Kunshou; Deng, Yujie; Weng, Guoxing; Hu, Dan; Huang, Cheng; Matsumoto, Keitaro; Nagayasu, Takeshi; Koji, Takehiko; Zheng, Xiongwei; Jiang, Wenhui; Lin, Gen; Cai, Yibin; Weng, Guibin; Chen, Xiaohui

2018-01-01

Smoking frequently leads to epigenetic alterations, including DNA methylation and histone modifications. The effect that smoking has on the DNA methylation levels at CCGG sites, the expression of trimethylation of histone H3 at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (EZH2), and their interactions in patients with non-small cell lung cancer (NSCLC) were analyzed. There were a total of 42 patients with NSCLC, 22 with adenocarcinomas and 20 with squamous cell carcinomas enrolled in the present study. Expression of H3K27me3, EZH2 and proliferating cellular nuclear antigen (PCNA) were immunohistochemically detected. DNA methylation at CCGG sites was evaluated via histoendonuclease-linked detection of DNA methylation sites. The apoptotic index of cancerous tissues obtained from patients of different smoking statuses was evaluated via the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling method. The association with clinicopathological data was calculated relative to different smoking statuses. Compared with the non-smokers, smokers with NSCLC exhibited a significantly lower apoptotic index (P<0.05), and frequently had a lower level of DNA methylation at CCGG sites, lower H3K27me3 expression and a higher EZH2 expression (P<0.05). DNA methylation levels at CCGG sites were negatively correlated to the Brinkman index (P=0.017). Furthermore, there was a parallel association between the H3K27me3 and EZH2 expression levels in the majority of smokers, whereas in the majority of non-smokers, there was a diverging association (P=0.015). There was a diverging association between the PCNA and EZH2 expression levels in the majority of smokers; however, in the majority of non-smokers, there was a parallel association (P=0.048). In addition, the association between the CCGG methylation ratio and immunohistochemical expression of H3K27me3 was a parallel association in the majority of smokers, while in the majority of non-smokers there was a

Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer.

PubMed

van Haaften, Gijs; Dalgliesh, Gillian L; Davies, Helen; Chen, Lina; Bignell, Graham; Greenman, Chris; Edkins, Sarah; Hardy, Claire; O'Meara, Sarah; Teague, Jon; Butler, Adam; Hinton, Jonathan; Latimer, Calli; Andrews, Jenny; Barthorpe, Syd; Beare, Dave; Buck, Gemma; Campbell, Peter J; Cole, Jennifer; Forbes, Simon; Jia, Mingming; Jones, David; Kok, Chai Yin; Leroy, Catherine; Lin, Meng-Lay; McBride, David J; Maddison, Mark; Maquire, Simon; McLay, Kirsten; Menzies, Andrew; Mironenko, Tatiana; Mulderrig, Lee; Mudie, Laura; Pleasance, Erin; Shepherd, Rebecca; Smith, Raffaella; Stebbings, Lucy; Stephens, Philip; Tang, Gurpreet; Tarpey, Patrick S; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Collins, V Peter; Ichimura, Koichi; Law, Simon; Wong, John; Yuen, Siu Tsan; Leung, Suet Yi; Tonon, Giovanni; DePinho, Ronald A; Tai, Yu-Tzu; Anderson, Kenneth C; Kahnoski, Richard J; Massie, Aaron; Khoo, Sok Kean; Teh, Bin Tean; Stratton, Michael R; Futreal, P Andrew

2009-05-01

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase gene UTX, pointing to histone H3 lysine methylation deregulation in multiple tumor types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene.

Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells.

PubMed

Fan, Lingling; Zhang, Fengbo; Xu, Songhui; Cui, Xiaolu; Hussain, Arif; Fazli, Ladan; Gleave, Martin; Dong, Xuesen; Qi, Jianfei

2018-05-15

Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR-V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mRNA for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy.

Increased H3K9me3 drives dedifferentiated phenotype via KLF6 repression in liposarcoma

PubMed Central

Keung, Emily Z.; Akdemir, Kadir C.; Al Sannaa, Ghadah A.; Garnett, Jeannine; Lev, Dina; Torres, Keila E.; Lazar, Alexander J.; Rai, Kunal; Chin, Lynda

2015-01-01

Liposarcoma (LPS) can be divided into 4 different subtypes, of which well-differentiated LPS (WDLPS) and dedifferentiated LPS (DDLPS) are the most common. WDLPS is typically low grade, whereas DDLPS is high grade, aggressive, and carries a worse prognosis. WDLPS and DDLPS frequently co-occur in patients. However, it is not clear whether DDLPS arises independently from WDLPS, or whether epigenomic alterations underly the histopathological differences of these subtypes. Here, we profiled 9 epigenetic marks in tumor samples from 151 patients with LPS and showed elevated trimethylation of histone H3 at Lys9 (H3K9me3) levels in DDLPS tumors. Integrated ChIP-seq and gene expression analyses of patient-derived cell lines revealed that H3K9me3 mediates differential regulation of genes involved in cellular differentiation and migration. Among these, Kruppel-like factor 6 (KLF6) was reduced in DDLPS, with increased H3K9me3 at associated regulatory regions. Pharmacologic inhibition of H3K9me3 with chaetocin decreased DDLPS proliferation and increased expression of the adipogenesis-associated factors PPARγ, CEBPα, and CEBPβ, suggesting that increased H3K9me3 may mediate DDLPS-associated aggressiveness and dedifferentiation properties. KLF6 overexpression partially phenocopied chaetocin treatment in DDLPS cells and induced phenotypic changes that were consistent with adipocytic differentiation, suggesting that the effects of increased H3K9me3 may be mediated through KLF6. In conclusion, we provide evidence of an epigenetic basis for the transition between WDLPS and DDLPS. PMID:26193637

Increased H3K9me3 drives dedifferentiated phenotype via KLF6 repression in liposarcoma.

PubMed

Keung, Emily Z; Akdemir, Kadir C; Al Sannaa, Ghadah A; Garnett, Jeannine; Lev, Dina; Torres, Keila E; Lazar, Alexander J; Rai, Kunal; Chin, Lynda

2015-08-03

Liposarcoma (LPS) can be divided into 4 different subtypes, of which well-differentiated LPS (WDLPS) and dedifferentiated LPS (DDLPS) are the most common. WDLPS is typically low grade, whereas DDLPS is high grade, aggressive, and carries a worse prognosis. WDLPS and DDLPS frequently co-occur in patients. However, it is not clear whether DDLPS arises independently from WDLPS, or whether epigenomic alterations underly the histopathological differences of these subtypes. Here, we profiled 9 epigenetic marks in tumor samples from 151 patients with LPS and showed elevated trimethylation of histone H3 at Lys9 (H3K9me3) levels in DDLPS tumors. Integrated ChIP-seq and gene expression analyses of patient-derived cell lines revealed that H3K9me3 mediates differential regulation of genes involved in cellular differentiation and migration. Among these, Kruppel-like factor 6 (KLF6) was reduced in DDLPS, with increased H3K9me3 at associated regulatory regions. Pharmacologic inhibition of H3K9me3 with chaetocin decreased DDLPS proliferation and increased expression of the adipogenesis-associated factors PPARγ, CEBPα, and CEBPβ, suggesting that increased H3K9me3 may mediate DDLPS-associated aggressiveness and dedifferentiation properties. KLF6 overexpression partially phenocopied chaetocin treatment in DDLPS cells and induced phenotypic changes that were consistent with adipocytic differentiation, suggesting that the effects of increased H3K9me3 may be mediated through KLF6. In conclusion, we provide evidence of an epigenetic basis for the transition between WDLPS and DDLPS.

JMJD1C Ensures Mouse Embryonic Stem Cell Self-Renewal and Somatic Cell Reprogramming through Controlling MicroRNA Expression.

PubMed

Xiao, Feng; Liao, Bing; Hu, Jing; Li, Shuang; Zhao, Haixin; Sun, Ming; Gu, Junjie; Jin, Ying

2017-09-12

The roles of histone demethylases (HDMs) for the establishment and maintenance of pluripotency are incompletely characterized. Here, we show that JmjC-domain-containing protein 1c (JMJD1C), an H3K9 demethylase, is required for mouse embryonic stem cell (ESC) self-renewal. Depletion of Jmjd1c leads to the activation of ERK/MAPK signaling and epithelial-to-mesenchymal transition (EMT) to induce differentiation of ESCs. Inhibition of ERK/MAPK signaling rescues the differentiation phenotype caused by Jmjd1c depletion. Mechanistically, JMJD1C, with the help of pluripotency factor KLF4, maintains ESC identity at least in part by regulating the expression of the miR-200 family and miR-290/295 cluster to suppress the ERK/MAPK signaling and EMT. Additionally, we uncover that JMJD1C ensures efficient generation and maintenance of induced pluripotent stem cells, at least partially through controlling the expression of microRNAs. Collectively, we propose an integrated model of epigenetic and transcriptional control mediated by the H3K9 demethylase for ESC self-renewal and somatic cell reprogramming. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation

PubMed Central

Grandy, Rodrigo A.; Whitfield, Troy W.; Wu, Hai; Fitzgerald, Mark P.; VanOudenhove, Jennifer J.; Zaidi, Sayyed K.; Montecino, Martin A.; Lian, Jane B.; van Wijnen, André J.; Stein, Janet L.

2015-01-01

Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. PMID:26644406

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation.

PubMed

Grandy, Rodrigo A; Whitfield, Troy W; Wu, Hai; Fitzgerald, Mark P; VanOudenhove, Jennifer J; Zaidi, Sayyed K; Montecino, Martin A; Lian, Jane B; van Wijnen, André J; Stein, Janet L; Stein, Gary S

2016-02-15

Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Super-Enhancers and Broad H3K4me3 Domains Form Complex Gene Regulatory Circuits Involving Chromatin Interactions.

PubMed

Cao, Fan; Fang, Yiwen; Tan, Hong Kee; Goh, Yufen; Choy, Jocelyn Yeen Hui; Koh, Bryan Thean Howe; Hao Tan, Jiong; Bertin, Nicolas; Ramadass, Aroul; Hunter, Ewan; Green, Jayne; Salter, Matthew; Akoulitchev, Alexandre; Wang, Wilson; Chng, Wee Joo; Tenen, Daniel G; Fullwood, Melissa J

2017-05-19

Stretched histone regions, such as super-enhancers and broad H3K4me3 domains, are associated with maintenance of cell identity and cancer. We connected super-enhancers and broad H3K4me3 domains in the K562 chronic myelogenous leukemia cell line as well as the MCF-7 breast cancer cell line with chromatin interactions. Super-enhancers and broad H3K4me3 domains showed higher association with chromatin interactions than their typical counterparts. Interestingly, we identified a subset of super-enhancers that overlap with broad H3K4me3 domains and show high association with cancer-associated genes including tumor suppressor genes. Besides cell lines, we could observe chromatin interactions by a Chromosome Conformation Capture (3C)-based method, in primary human samples. Several chromatin interactions involving super-enhancers and broad H3K4me3 domains are constitutive and can be found in both cancer and normal samples. Taken together, these results reveal a new layer of complexity in gene regulation by super-enhancers and broad H3K4me3 domains.

MINA controls proliferation and tumorigenesis of glioblastoma by epigenetically regulating cyclins and CDKs via H3K9me3 demethylation.

PubMed

Huang, M-Y; Xuan, F; Liu, W; Cui, H-J

2017-01-19

It is generally known that histone demethylases regulate gene transcription by altering the methylate status on histones, but their roles in cancers and the underlying molecular mechanisms still remain unclear. MYC-induced nuclear antigen (MINA) is reported to be a histone demethylase and highly expressed in many cancers. Here, for the first time, we show that MINA is involved in glioblastoma carcinogenesis and reveal the probable mechanisms of it in cell-cycle control. Kaplan-Meier analysis of progression-free survival showed that high MINA expression was strongly correlated with poor outcome and advancing tumor stage. MINA knockdown significantly repressed the cell proliferation and tumorigenesis abilities of glioblastoma cells in vitro and in vivo that were rescued by overexpressing the full-length MINA afterwards. Microarray analysis after knockdown of MINA revealed that MINA probably regulated glioblastoma carcinogenesis through the predominant cell-cycle pathways. Further investigation showed that MINA deficiency led to a cell-cycle arrest in G1 and G2 phases. And among the downstream genes, we found that cyclins and cyclin-dependent kinases were directly activated by MINA via the demethylation of H3K9me3.

Epigenomic analysis in a cell-based model reveals the roles of H3K9me3 in breast cancer transformation.

PubMed

Li, Qing-Lan; Lei, Pin-Ji; Zhao, Quan-Yi; Li, Lianyun; Wei, Gang; Wu, Min

2017-08-01

Epigenetic marks are critical regulators of chromatin and gene activity. Their roles in normal physiology and disease states, including cancer development, still remain elusive. Herein, the epigenomic change of H3K9me3, as well as its potential impacts on gene activity and genome stability, was investigated in an in vitro breast cancer transformation model. The global H3K9me3 level was studied with western blotting. The distribution of H3K9me3 on chromatin and gene expression was studied with ChIP-Seq and RNA-Seq, respectively. The global H3K9me3 level decreases during transformation and its distribution on chromatin is reprogrammed. By combining with TCGA data, we identified 67 candidate oncogenes, among which five genes are totally novel. Our analysis further links H3K9me3 with transposon activity, and suggests H3K9me3 reduction increases the cell's sensitivity to DNA damage reagents. H3K9me3 reduction is possibly related with breast cancer transformation by regulating gene expression and chromatin stability during transformation.

Induction of H3K9me3 and DNA methylation by tethered heterochromatin factors in Neurospora crassa

PubMed Central

Selker, Eric U.

2017-01-01

Functionally different chromatin domains display distinct chemical marks. Constitutive heterochromatin is commonly associated with trimethylation of lysine 9 on histone H3 (H3K9me3), hypoacetylated histones, and DNA methylation, but the contributions of and interplay among these features are not fully understood. To dissect the establishment of heterochromatin, we investigated the relationships among these features using an in vivo tethering system in Neurospora crassa. Artificial recruitment of the H3K9 methyltransferase DIM-5 (defective in methylation-5) induced H3K9me3 and DNA methylation at a normally active, euchromatic locus but did not bypass the requirement of DIM-7, previously implicated in the localization of DIM-5, indicating additional DIM-7 functionality. Tethered heterochromatin protein 1 (HP1) induced H3K9me3, DNA methylation, and gene silencing. The induced heterochromatin required histone deacetylase 1 (HDA-1), with an intact catalytic domain, but HDA-1 was not essential for de novo heterochromatin formation at native heterochromatic regions. Silencing did not require H3K9me3 or DNA methylation. However, DNA methylation contributed to establishment of H3K9me3 induced by tethered HP1. Our analyses also revealed evidence of regulatory mechanisms, dependent on HDA-1 and DIM-5, to control the localization and catalytic activity of the DNA methyltransferase DIM-2. Our study clarifies the interrelationships among canonical aspects of heterochromatin and supports a central role of HDA-1–mediated histone deacetylation in heterochromatin spreading and gene silencing. PMID:29078403

Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor suppressor genes

PubMed Central

Chen, Kaifu; Chen, Zhong; Wu, Dayong; Zhang, Lili; Lin, Xueqiu; Su, Jianzhong; Rodriguez, Benjamin; Xi, Yuanxin; Xia, Zheng; Chen, Xi; Shi, Xiaobing; Wang, Qianben; Li, Wei

2016-01-01

Tumor suppressors are mostly defined by inactivating mutations in tumors, yet little is known about their epigenetic features in normal cells. Through integrative analysis of 1,134 genome-wide epigenetic profiles, mutations from >8,200 tumor-normal pairs, and our experimental data from clinical samples, we discovered broad H3K4me3 (wider than 4 kb) as the first epigenetic signature for tumor suppressors in normal cells. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity together leading to exceptionally high gene expression, and is distinct from other broad epigenetic features, such as super-enhancers. Broad H3K4me3 conserved across normal cells may represent pan-cancer tumor suppressors, such as P53 and PTEN, whereas cell-type-specific broad H3K4me3 may indicate cell-identity genes and cell-type-specific tumor suppressors. Furthermore, widespread shortening of broad H3K4me3 in cancers is associated with repression of tumor suppressors. Together, the broad H3K4me3 epigenetic signature provides mutation-independent information for the discovery and characterization of novel tumor suppressors. PMID:26301496

Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor-suppressor genes.

PubMed

Chen, Kaifu; Chen, Zhong; Wu, Dayong; Zhang, Lili; Lin, Xueqiu; Su, Jianzhong; Rodriguez, Benjamin; Xi, Yuanxin; Xia, Zheng; Chen, Xi; Shi, Xiaobing; Wang, Qianben; Li, Wei

2015-10-01

Tumor suppressors are mostly defined by inactivating mutations in tumors, yet little is known about their epigenetic features in normal cells. Through integrative analysis of 1,134 genome-wide epigenetic profiles, mutations from >8,200 tumor-normal pairs and our experimental data from clinical samples, we discovered broad peaks for trimethylation of histone H3 at lysine 4 (H3K4me3; wider than 4 kb) as the first epigenetic signature for tumor suppressors in normal cells. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity, which together lead to exceptionally high gene expression, and is distinct from other broad epigenetic features, such as super-enhancers. Genes with broad H3K4me3 peaks conserved across normal cells may represent pan-cancer tumor suppressors, such as TP53 and PTEN, whereas genes with cell type-specific broad H3K4me3 peaks may represent cell identity genes and cell type-specific tumor suppressors. Furthermore, widespread shortening of broad H3K4me3 peaks in cancers is associated with repression of tumor suppressors. Thus, the broad H3K4me3 epigenetic signature provides mutation-independent information for the discovery and characterization of new tumor suppressors.

ATRX binds to atypical chromatin domains at the 3′ exons of zinc finger genes to preserve H3K9me3 enrichment

PubMed Central

Chowdhury, Asif H.; Hasson, Dan; Dyer, Michael A.

2016-01-01

ABSTRACT ATRX is a SWI/SNF chromatin remodeler proposed to govern genomic stability through the regulation of repetitive sequences, such as rDNA, retrotransposons, and pericentromeric and telomeric repeats. However, few direct ATRX target genes have been identified and high-throughput genomic approaches are currently lacking for ATRX. Here we present a comprehensive ChIP-sequencing study of ATRX in multiple human cell lines, in which we identify the 3′ exons of zinc finger genes (ZNFs) as a new class of ATRX targets. These 3′ exonic regions encode the zinc finger motifs, which can range from 1–40 copies per ZNF gene and share large stretches of sequence similarity. These regions often contain an atypical chromatin signature: they are transcriptionally active, contain high levels of H3K36me3, and are paradoxically enriched in H3K9me3. We find that these ZNF 3′ exons are co-occupied by SETDB1, TRIM28, and ZNF274, which form a complex with ATRX. CRISPR/Cas9-mediated loss-of-function studies demonstrate (i) a reduction of H3K9me3 at the ZNF 3′ exons in the absence of ATRX and ZNF274 and, (ii) H3K9me3 levels at atypical chromatin regions are particularly sensitive to ATRX loss compared to other H3K9me3-occupied regions. As a consequence of ATRX or ZNF274 depletion, cells with reduced levels of H3K9me3 show increased levels of DNA damage, suggesting that ATRX binds to the 3′ exons of ZNFs to maintain their genomic stability through preservation of H3K9me3. PMID:27029610

A case report of adult cerebellar high-grade glioma with H3.1 K27M mutation: a rare example of an H3 K27M mutant cerebellar tumor.

PubMed

Funata, Nobuaki; Nobusawa, Sumihito; Nakata, Satoshi; Yamazaki, Tatsuya; Takabagake, Kazuhiko; Koike, Tsukasa; Maegawa, Tatsuya; Yamada, Ryoji; Shinoura, Nobusada; Mine, Yutaka

2018-01-01

Diffuse midline glioma, H3 K27M mutant, is newly recognized as a distinct category, which usually arises in the brain stem, thalamus or spinal cord of children, and young adults. The oncogenic H3 K27M mutation involves H3.3 (encoded by H3F3A) or H3.1 (encoded by HIST1H3B/HIST1H3C), and the incidence of each mutation differs among the primary sites. Recently, several papers have reported that cerebellar high-grade gliomas in both children and adults also harbor H3 K27 mutation. With the exception of one pediatric case, all of the cases carried the mutation in H3.3. We herein present the case of an adult cerebellar high-grade astrocytic tumor with H3.1 K27M mutation in a 45-year-old man, which also involvedTP53 mutation and was immunonegative for ATRX. Some groups have reported that H3.3 and H3.1 K27M mutations define subgroups of diffuse intrinsic pontine gliomas (DIPGs) with different phenotypes as well as genetic alterations. On comparing the findings of the present case, particularly TP53 mutation status and ATRX expression, to the findings of the previous studies on DIPGs, our case seems unusual among the H3.1 K27M mutant subgroup. Further studies are needed to clarify the exact frequency, clinicopathological characteristics, and genomic alterations of cerebellar gliomas harboring H3 K27M mutation.

High expression of trimethylated histone H3 at lysine 27 predicts better prognosis in non-small cell lung cancer.

PubMed

Chen, Xiaohui; Song, Ning; Matsumoto, Keitaro; Nanashima, Atsushi; Nagayasu, Takeshi; Hayashi, Tomayoshi; Ying, Mingang; Endo, Daisuke; Wu, Zhiren; Koji, Takehiko

2013-11-01

Epigenetic parameters such as DNA methylation and histone modifications play pivotal roles in carcinogenesis. Global histone modification patterns have been implicated as possible predictors of cancer recurrence and prognoses in a great variety of tumor entities. Our study was designed to evaluate the association among trimethylated histone H3 at lysine 27 (H3K27me3), clinicopathological variables and outcome in early-stage non-small cell lung cancer (NSCLC). The expression of H3K27me3 and its methyl-transferase, enhancer of zeste homolog 2 (EZH2) together with proliferating cell nuclear antigen (PCNA) were evaluated by immunohistochemistry in normal lung tissue (n=5) and resected NSCLC patients (n=42). In addition, the specificity of antibody for H3K27me3 was tested by western blot analysis. The optimal cut-off point of H3K27me3 expression for prognosis was determined by the X-tile program. The prognostic significance was determined by means of Kaplan-Meier survival estimates and log-rank tests. As a result, enhanced trimethylation of H3K27me3 was correlated with longer overall survival (OS) and better prognosis (P<0.05). Moreover, both univariate and multivariate analyses indicated that H3K27me3 level was a significant and independent predictor of better survival (hazard ratio, 0.187; 95% confidence interval, 0.066-0.531, P=0.002). Furthermore, H3K27me3 expression was positively correlated with DNA methylation level at CCGG sites while reversely related to EZH2 expression (P<0.05). In conclusion, H3K27me3 level defines unrecognized subgroups of NSCLC patients with distinct epigenetic phenotype and clinical outcome, and can probably be used as a novel predictor for better prognosis in NSCLC patients.

H3K9me2/3 Binding of the MBT Domain Protein LIN-61 Is Essential for Caenorhabditis elegans Vulva Development

PubMed Central

Koester-Eiserfunke, Nora; Fischle, Wolfgang

2011-01-01

MBT domain proteins are involved in developmental processes and tumorigenesis. In vitro binding and mutagenesis studies have shown that individual MBT domains within clustered MBT repeat regions bind mono- and dimethylated histone lysine residues with little to no sequence specificity but discriminate against the tri- and unmethylated states. However, the exact function of promiscuous histone methyl-lysine binding in the biology of MBT domain proteins has not been elucidated. Here, we show that the Caenorhabditis elegans four MBT domain protein LIN-61, in contrast to other MBT repeat factors, specifically interacts with histone H3 when methylated on lysine 9, displaying a strong preference for di- and trimethylated states (H3K9me2/3). Although the fourth MBT repeat is implicated in this interaction, H3K9me2/3 binding minimally requires MBT repeats two to four. Further, mutagenesis of residues conserved with other methyl-lysine binding MBT regions in the fourth MBT repeat does not abolish interaction, implicating a distinct binding mode. In vivo, H3K9me2/3 interaction of LIN-61 is required for C. elegans vulva development within the synMuvB pathway. Mutant LIN-61 proteins deficient in H3K9me2/3 binding fail to rescue lin-61 synMuvB function. Also, previously identified point mutant synMuvB alleles are deficient in H3K9me2/3 interaction although these target residues that are outside of the fourth MBT repeat. Interestingly, lin-61 genetically interacts with two other synMuvB genes, hpl-2, an HP1 homologous H3K9me2/3 binding factor, and met-2, a SETDB1 homologous H3K9 methyl transferase (H3K9MT), in determining C. elegans vulva development and fertility. Besides identifying the first sequence specific and di-/trimethylation binding MBT domain protein, our studies imply complex multi-domain regulation of ligand interaction of MBT domains. Our results also introduce a mechanistic link between LIN-61 function and biology, and they establish interplay of the H3K9me2/3

Histone demethylase JMJD2B is required for tumor cell proliferation and survival and is overexpressed in gastric cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wenjuan; Zhao, Li; Zang, Wen

2011-12-16

Highlights: Black-Right-Pointing-Pointer JMJD2B is required for cell proliferation and in vivo tumorigenesis. Black-Right-Pointing-Pointer JMJD2B depletion induces apoptosis and/or cell cycle arrest. Black-Right-Pointing-Pointer JMJD2B depletion activates DNA damage response and enhances p53 stabilization. Black-Right-Pointing-Pointer JMJD2B is overexpressed in human primary gastric cancer. -- Abstract: Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Jumonji domain containing 2B (JMJD2B) is a newly identified histone demethylase that regulates chromatin structure or gene expression by removing methyl residues from trimethylated lysine 9 on histone H3. Recent observations have shown oncogenic activity of JMJD2B. We explored the functional role ofmore » JMJD2B in cancer cell proliferation, survival and tumorigenesis, and determined its expression profile in gastric cancer. Knocking down JMJD2B expression by small interfering RNA (siRNA) in gastric and other cancer cells inhibited cell proliferation and/or induced apoptosis and elevated the expression of p53 and p21{sup CIP1} proteins. The enhanced p53 expression resulted from activation of the DNA damage response pathway. JMJD2B knockdown markedly suppressed xenograft tumor growth in vivo in mice. Moreover, JMJD2B expression was increased in primary gastric-cancer tissues of humans. Thus, JMJD2B is required for sustained proliferation and survival of tumor cells in vitro and in vivo, and its aberrant expression may contribute to the pathogenesis of gastric cancer.« less

Compromised JMJD6 histone demethylase activity impacts on VHL gene repression in preeclampsia.

PubMed

Alahari, Sruthi; Post, Martin; Rolfo, Alessandro; Weksberg, Rosanna; Caniggia, Isabella

2018-01-24

The von Hippel Lindau (VHL) protein is a key executor of the cellular hypoxic response that is compromised in preeclampsia, a serious disorder complicating 5-7% of pregnancies. To date, the mechanisms controlling VHL gene expression in the human placenta remain elusive. We examined VHL epigenetic regulation in normal pregnancy and in preeclampsia, a pathology characterized by placental hypoxia. Placentae were obtained from early-onset (E-PE: n=56; <34 weeks of gestation) and late onset preeclampsia (L-PE: n=19; ≥ 34 weeks of gestation). Placentae from healthy normotensive age-matched preterm and term pregnancies (PTC: n=43; TC: n=23) were included as controls. We measured the activity of Jumonji domain containing protein 6 (JMJD6), a Fe2+ and oxygen-dependent histone demethylase, and examined its function in the epigenetic control of VHL. JMJD6 regulates VHL gene expression in the human placenta. VHL downregulation in preeclampsia is dependent on decreased JMJD6 demethylase activity due to hypoxia and reduced Fe2+ bioavailability. Chromatin immunoprecipitation assays revealed decreased association of JMJD6 and its histone targets with the VHL promoter. Findings in preeclampsia were corroborated in a murine model of pharmacological hypoxia using FG-4592. Placentae from FG-4592 treated mice exhibited reduced VHL levels, accompanied by placental morphological alterations and reduced pup weights. Notably, Fe2+ supplementation rescued JMJD6 histone demethylase activity in histone from E-PE and FG-4592-treated mice. Our study uncovers novel epigenetic regulation of VHL and its functional consequences for altered oxygen and iron homeostasis in preeclampsia. Copyright © 2018 Endocrine Society

H3K9me3 Inhibition Improves Memory, Promotes Spine Formation, and Increases BDNF Levels in the Aged Hippocampus

PubMed Central

Prieto, G. Aleph; Petrosyan, Arpine; Loertscher, Brad M.; Dieskau, André P.; Overman, Larry E.; Cotman, Carl W.

2016-01-01

An increasing number of studies show that an altered epigenetic landscape may cause impairments in regulation of learning and memory-related genes within the aged hippocampus, eventually resulting in cognitive deficits in the aged brain. One such epigenetic repressive mark is trimethylation of H3K9 (H3K9me3), which is typically implicated in gene silencing. Here, we identify, for the first time, an essential role for H3K9me3 and its histone methyl transferase (SUV39H1) in mediating hippocampal memory functions. Pharmacological inhibition of SUV39H1 using a novel and selective inhibitor decreased levels of H3K9me3 in the hippocampus of aged mice, and improved performance in the objection location memory and fear conditioning tasks and in a complex spatial environment learning task. The inhibition of SUV39H1 induced an increase in spine density of thin and stubby but not mushroom spines in the hippocampus of aged animals and increased surface GluR1 levels in hippocampal synaptosomes, a key index of spine plasticity. Furthermore, there were changes at BDNF exon I gene promoter, in concert with overall BDNF levels in the hippocampus of drug-treated animals compared with control animals. Together, these data demonstrate that SUV39H1 inhibition and the concomitant H3K9me3 downregulation mediate gene transcription in the hippocampus and reverse age-dependent deficits in hippocampal memory. SIGNIFICANCE STATEMENT Cognitive decline is a debilitating condition associated with not only neurodegenerative diseases but also aging in general. However, effective treatments have been slow to emerge so far. In this study, we demonstrate that epigenetic regulation of key synaptic proteins may be an underlying, yet reversible, cause of this decline. Our findings suggest that histone 3 trimethylation is a probable target for pharmacological intervention that can counteract cognitive decline in the aging brain. Finally, we provide support to the hypothesis that, by manipulating the

Activation of Bmp2-Smad1 Signal and Its Regulation by Coordinated Alteration of H3K27 Trimethylation in Ras-Induced Senescence

PubMed Central

Kaneda, Atsushi; Fujita, Takanori; Anai, Motonobu; Yamamoto, Shogo; Nagae, Genta; Morikawa, Masato; Tsuji, Shingo; Oshima, Masanobu; Miyazono, Kohei; Aburatani, Hiroyuki

2011-01-01

Cellular senescence involves epigenetic alteration, e.g. loss of H3K27me3 in Ink4a-Arf locus. Using mouse embryonic fibroblast (MEF), we here analyzed transcription and epigenetic alteration during Ras-induced senescence on genome-wide scale by chromatin immunoprecipitation (ChIP)-sequencing and microarray. Bmp2 was the most activated secreted factor with H3K4me3 gain and H3K27me3 loss, whereas H3K4me3 loss and de novo formation of H3K27me3 occurred inversely in repression of nine genes, including two BMP-SMAD inhibitors Smad6 and Noggin. DNA methylation alteration unlikely occurred. Ras-activated cells senesced with nuclear accumulation of phosphorylated SMAD1/5/8. Senescence was bypassed in Ras-activated cells when Bmp2/Smad1 signal was blocked by Bmp2 knockdown, Smad6 induction, or Noggin induction. Senescence was induced when recombinant BMP2 protein was added to Bmp2-knocked-down Ras-activated cells. Downstream Bmp2-Smad1 target genes were then analyzed genome-wide by ChIP-sequencing using anti-Smad1 antibody in MEF that was exposed to BMP2. Smad1 target sites were enriched nearby transcription start sites of genes, which significantly correlated to upregulation by BMP2 stimulation. While Smad6 was one of Smad1 target genes to be upregulated by BMP2 exposure, Smad6 repression in Ras-activated cells with increased enrichment of Ezh2 and gain of H3K27me3 suggested epigenetic disruption of negative feedback by Polycomb. Among Smad1 target genes that were upregulated in Ras-activated cells without increased repressive mark, Parvb was found to contribute to growth inhibition as Parvb knockdown lead to escape from senescence. It was revealed through genome-wide analyses in this study that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence. PMID:22072987

Association between H3K4 methylation and cancer prognosis: A meta-analysis.

PubMed

Li, Simin; Shen, Luyan; Chen, Ke-Neng

2018-05-08

Histone H3 lysine 4 methylation (H3K4 methylation), including mono-methylation (H3K4me1), di-methylation (H3K4me2), or tri-methylation (H3K4me3), is one of the epigenetic modifications to histone proteins, which are related to the transcriptional activation of genes. H3K4 methylation has both tumor inhibiting and promoting effects, and the prognostic value of H3K4 methylation in cancer remains controversial. Therefore, we performed a systematic review and meta-analysis to examine the association between H3K4 methylation and cancer prognosis. A comprehensive search of PubMed, Web of Science, ScienceDirect, Embase, and Ovid databases was conducted to identify studies investigating the association between H3K4 methylation and prognosis of patients with malignant tumors. The data and characteristics of each study were extracted, and the hazard ratio (HR) at a 95% confidence interval (CI) was calculated to estimate the effect. A total of 1474 patients in 10 studies were enrolled in this meta-analysis. The pooled HR of 1.52 (95% CI 1.02-2.26) indicated that patients with a lower level of H3K4me2 expression were expected to have shorter overall survival, while the pooled HR of 0.45 (95% CI 0.27-0.74) indicated that patients with a lower level of H3K4me3 expression were expected to have longer overall survival. This meta-analysis indicates that increased H3K4me3 expression and decreased H3K4me2 expression might be predictive factors of poor prognosis in cancer. Further large cohort studies are needed to confirm these findings. © 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase

PubMed Central

Bettridge, John; Na, Chan Hyun; Desiderio, Stephen

2017-01-01

V(D)J recombination is initiated by the recombination-activating gene (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates substrate binding and catalysis, which are functions of RAG-1. This has suggested an allosteric mechanism in which information regarding occupancy of the RAG-2 PHD is transmitted to RAG-1. To determine whether the conformational distribution of RAG is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols by differential isotopic chemical footprinting. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within a DNA-binding domain and in the ZnH2 domain, which acts as a scaffold for the catalytic center. Thus, engagement of H3K4me3 by the RAG-2 PHD is associated with dynamic conformational changes in RAG-1, consistent with allosteric control by active chromatin. PMID:28174273

The Dynamics and Regulatory Mechanism of Pronuclear H3k9me2 Asymmetry in Mouse Zygotes

PubMed Central

Ma, Xue-Shan; Chao, Shi-Bin; Huang, Xian-Ju; Lin, Fei; Qin, Ling; Wang, Xu-Guang; Meng, Tie-Gang; Zhu, Cheng-Cheng; Schatten, Heide; Liu, Hong-Lin; Sun, Qing-Yuan

2015-01-01

H3K9 methylation is an important histone modification that is correlated with gene transcription repression. The asymmetric H3K9 dimethylation (H3K9me2) pattern between paternal and maternal genomes is generated soon after fertilization. In the present study, we carefully determined the dynamics of H3K9me2 changes in mouse zygotes, and investigated the regulatory mechanisms. The results indicated that histone methyltransferase G9a, but not GLP, was involved in the regulation of asymmetric H3K9me2, and G9a was the methyltransferase that induced the appearance of H3K9me2 in the male pronucleus of the zygote treated with cycloheximide. We found that there were two distinct mechanisms that regulate H3K9me2 in the male pronucleus. Before 8 h of in vitro fertilization (IVF), a mechanism exists that inhibits the association of G9a with the H3K9 sites. After 10 h of IVF the inhibition of G9a activity depends on yet unknown novel protein(s) synthesis. The two mechanisms of transfer take place between 8–10 h of IVF, and the novel protein failed to inhibit G9a activity in time, resulting in the appearance of a low level de novo H3K9me2 in the male pronucleus. PMID:26639638

MLL4 Is Required to Maintain Broad H3K4me3 Peaks and Super-Enhancers at Tumor Suppressor Genes.

PubMed

Dhar, Shilpa S; Zhao, Dongyu; Lin, Tao; Gu, Bingnan; Pal, Khusboo; Wu, Sarah J; Alam, Hunain; Lv, Jie; Yun, Kyuson; Gopalakrishnan, Vidya; Flores, Elsa R; Northcott, Paul A; Rajaram, Veena; Li, Wei; Shilatifard, Ali; Sillitoe, Roy V; Chen, Kaifu; Lee, Min Gyu

2018-06-07

Super-enhancers are large clusters of enhancers that activate gene expression. Broad trimethyl histone H3 lysine 4 (H3K4me3) often defines active tumor suppressor genes. However, how these epigenomic signatures are regulated for tumor suppression is little understood. Here we show that brain-specific knockout of the H3K4 methyltransferase MLL4 (a COMPASS-like enzyme, also known as KMT2D) in mice spontaneously induces medulloblastoma. Mll4 loss upregulates oncogenic Ras and Notch pathways while downregulating neuronal gene expression programs. MLL4 enhances DNMT3A-catalyzed DNA methylation and SIRT1/BCL6-mediated H4K16 deacetylation, which antagonize expression of Ras activators and Notch pathway components, respectively. Notably, Mll4 loss downregulates tumor suppressor genes (e.g., Dnmt3a and Bcl6) by diminishing broad H3K4me3 and super-enhancers and also causes widespread impairment of these epigenomic signatures during medulloblastoma genesis. These findings suggest an anti-tumor role for super-enhancers and provide a unique tumor-suppressive mechanism in which MLL4 is necessary to maintain broad H3K4me3 and super-enhancers at tumor suppressor genes. Copyright © 2018 Elsevier Inc. All rights reserved.

H3S10ph broadly marks early-replicating domains in interphase ESCs and shows reciprocal antagonism with H3K9me2.

PubMed

Chen, Carol C L; Goyal, Preeti; Karimi, Mohammad M; Abildgaard, Marie H; Kimura, Hiroshi; Lorincz, Matthew C

2018-01-01

Phosphorylation of histone H3 at serine 10 (H3S10ph) by Aurora kinases plays an important role in mitosis; however, H3S10ph also marks regulatory regions of inducible genes in interphase mammalian cells, implicating mitosis-independent functions. Using the fluorescent ubiquitin-mediated cell cycle indicator (FUCCI), we found that 30% of the genome in interphase mouse embryonic stem cells (ESCs) is marked with H3S10ph. H3S10ph broadly demarcates gene-rich regions in G1 and is positively correlated with domains of early DNA replication timing (RT) but negatively correlated with H3K9me2 and lamin-associated domains (LADs). Consistent with mitosis-independent kinase activity, this pattern was preserved in ESCs treated with Hesperadin, a potent inhibitor of Aurora B/C kinases. Disruption of H3S10ph by expression of nonphosphorylatable H3.3S10A results in ectopic spreading of H3K9me2 into adjacent euchromatic regions, mimicking the phenotype observed in Drosophila JIL-1 kinase mutants . Conversely, interphase H3S10ph domains expand in Ehmt1 (also known as Glp ) null ESCs, revealing that H3S10ph deposition is restricted by H3K9me2. Strikingly, spreading of H3S10ph at RT transition regions (TTRs) is accompanied by aberrant transcription initiation of genes co-oriented with the replication fork in Ehmt1 -/- and Ehmt2 -/- ESCs, indicating that establishment of repressive chromatin on the leading strand following DNA synthesis may depend upon these lysine methyltransferases. H3S10ph is also anti-correlated with H3K9me2 in interphase murine embryonic fibroblasts (MEFs) and is restricted to intragenic regions of actively transcribing genes by EHMT2. Taken together, these observations reveal that H3S10ph may play a general role in restricting the spreading of repressive chromatin in interphase mammalian cells. © 2018 Chen et al.; Published by Cold Spring Harbor Laboratory Press.

The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo.

PubMed

Powers, Natalie R; Parvanov, Emil D; Baker, Christopher L; Walker, Michael; Petkov, Petko M; Paigen, Kenneth

2016-06-01

In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo

PubMed Central

Powers, Natalie R.; Parvanov, Emil D.; Baker, Christopher L.; Walker, Michael; Petkov, Petko M.; Paigen, Kenneth

2016-01-01

In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway*

PubMed Central

Foda, Bardees M.; Singh, Upinder

2015-01-01

RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5′-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

Epigenetics and sex differences in the brain: A genome-wide comparison of histone-3 lysine-4 trimethylation (H3K4me3) in male and female mice.

PubMed

Shen, Erica Y; Ahern, Todd H; Cheung, Iris; Straubhaar, Juerg; Dincer, Aslihan; Houston, Isaac; de Vries, Geert J; Akbarian, Schahram; Forger, Nancy G

2015-06-01

Many neurological and psychiatric disorders exhibit gender disparities, and sex differences in the brain likely explain some of these effects. Recent work in rodents points to a role for epigenetics in the development or maintenance of neural sex differences, although genome-wide studies have so far been lacking. Here we review the existing literature on epigenetics and brain sexual differentiation and present preliminary analyses on the genome-wide distribution of histone-3 lysine-4 trimethylation in a sexually dimorphic brain region in male and female mice. H3K4me3 is a histone mark primarily organized as 'peaks' surrounding the transcription start site of active genes. We microdissected the bed nucleus of the stria terminalis and preoptic area (BNST/POA) in adult male and female mice and used ChIP-Seq to compare the distribution of H3K4me3 throughout the genome. We found 248 genes and loci with a significant sex difference in H3K4me3. Of these, the majority (71%) had larger H3K4me3 peaks in females. Comparisons with existing databases indicate that genes and loci with increased H3K4me3 in females are associated with synaptic function and with expression atlases from related brain areas. Based on RT-PCR, only a minority of genes with a sex difference in H3K4me3 has detectable sex differences in expression at baseline conditions. Together with previous findings, our data suggest that there may be sex biases in the use of epigenetic marks. Such biases could underlie sex differences in vulnerabilities to drugs or diseases that disrupt specific epigenetic processes. Copyright © 2014 Elsevier Inc. All rights reserved.

Epigenetics and Sex Differences in the Brain: A Genome-Wide Comparison of Histone-3 Lysine-4 Trimethylation (H3K4me3) in Male and Female Mice

PubMed Central

Shen, Erica Y.; Ahern, Todd H.; Cheung, Iris; Straubhaar, Juerg; Dincer, Aslihan; Houston, Isaac; de Vries, Geert J.; Akbarian, Schahram; Forger, Nancy G.

2014-01-01

Many neurological and psychiatric disorders exhibit gender disparities, and sex differences in the brain likely explain some of these effects. Recent work in rodents points to a role for epigenetics in the development or maintenance of neural sex differences, although genome-wide studies have so far been lacking. Here we review the existing literature on epigenetics and brain sexual differentiation and present preliminary analyses on the genome-wide distribution of histone-3 lysine-4 trimethylation in a sexually dimorphic brain region in male and female mice. H3K4me3 is a histone mark primarily organized as ‘peaks’ surrounding the transcription start site of active genes. We microdissected the bed nucleus of the stria terminalis and preoptic area (BNST/POA) in adult male and female mice and used ChIP-Seq to compare the distribution of H3K4me3 throughout the genome. We found 248 genes and loci with a significant sex difference in H3K4me3. Of these, the majority (71%) had larger H3K4me3 peaks in females. Comparisons with existing databases indicate that genes and loci with increased H3K4me3 in females are associated with synaptic function and with expression atlases from related brain areas. Based on RT-PCR, only a minority of genes with a sex difference in H3K4me3 has detectable sex differences in expression at baseline conditions. Together with previous findings, our data suggest there may be sex biases in the use of epigenetic marks. Such biases could underlie sex differences in vulnerabilities to drugs or diseases that disrupt specific epigenetic processes. PMID:25131640

Menin regulates Inhbb expression through an Akt/Ezh2-mediated H3K27 histone modification.

PubMed

Gherardi, Samuele; Ripoche, Doriane; Mikaelian, Ivan; Chanal, Marie; Teinturier, Romain; Goehrig, Delphine; Cordier-Bussat, Martine; Zhang, Chang X; Hennino, Ana; Bertolino, Philippe

2017-04-01

Although Men1 is a well-known tumour suppressor gene, little is known about the functions of Menin, the protein it encodes for. Since few years, numerous publications support a major role of Menin in the control of epigenetics gene regulation. While Menin interaction with MLL complex favours transcriptional activation of target genes through H3K4me3 marks, Menin also represses gene expression via mechanisms involving the Polycomb repressing complex (PRC). Interestingly, Ezh2, the PRC-methyltransferase that catalyses H3K27me3 repressive marks and Menin have been shown to co-occupy a large number of promoters. However, lack of binding between Menin and Ezh2 suggests that another member of the PRC complex is mediating this indirect interaction. Having found that ActivinB - a TGFβ superfamily member encoded by the Inhbb gene - is upregulated in insulinoma tumours caused by Men1 invalidation, we hypothesize that Menin could directly participate in the epigenetic-repression of Inhbb gene expression. Using Animal model and cell lines, we report that loss of Menin is directly associated with ActivinB-induced expression both in vivo and in vitro. Our work further reveals that ActivinB expression is mediated through a direct modulation of H3K27me3 marks on the Inhbb locus in Menin-KO cell lines. More importantly, we show that Menin binds on the promoter of Inhbb gene where it favours the recruitment of Ezh2 via an indirect mechanism involving Akt-phosphorylation. Our data suggests therefore that Menin could take an important part to the Ezh2-epigenetic repressive landscape in many cells and tissues through its capacity to modulate Akt phosphorylation. Copyright © 2017 Elsevier B.V. All rights reserved.

The Hypoxia-Inducible Epigenetic Regulators Jmjd1a and G9a Provide a Mechanistic Link between Angiogenesis and Tumor Growth

PubMed Central

Ho, Jolene Caifeng; Lee, Kian Leong; Kitajima, Shojiro; Yang, Henry; Sun, Wendi; Fukuhara, Noriko; Zaiden, Norazean; Chan, Shing Leng; Tachibana, Makoto; Shinkai, Yoichi; Kato, Hiroyuki

2014-01-01

Hypoxia promotes stem cell maintenance and tumor progression, but it remains unclear how it regulates long-term adaptation toward these processes. We reveal a striking downregulation of the hypoxia-inducible histone H3 lysine 9 (H3K9) demethylase JMJD1A as a hallmark of clinical human germ cell-derived tumors, such as seminomas, yolk sac tumors, and embryonal carcinomas. Jmjd1a was not essential for stem cell self-renewal but played a crucial role as a tumor suppressor in opposition to the hypoxia-regulated oncogenic H3K9 methyltransferase G9a. Importantly, loss of Jmjd1a resulted in increased tumor growth, whereas loss of G9a produced smaller tumors. Pharmacological inhibition of G9a also resulted in attenuation of tumor growth, offering a novel therapeutic strategy for germ cell-derived tumors. Finally, Jmjd1a and G9a drive mutually opposing expression of the antiangiogenic factor genes Robo4, Igfbp4, Notch4, and Tfpi accompanied by changes in H3K9 methylation status. Thus, we demonstrate a novel mechanistic link whereby hypoxia-regulated epigenetic changes are instrumental for the control of tumor growth through coordinated dysregulation of antiangiogenic gene expression. PMID:25071150

Clipping of arginine-methylated histone tails by JMJD5 and JMJD7

PubMed Central

Liu, Haolin; Wang, Chao; Lee, Schuyler; Deng, Yu; Wither, Matthew; Oh, Sangphil; Ning, Fangkun; Dege, Carissa; Zhang, Qianqian; Liu, Xinjian; Johnson, Aaron M.; Zang, Jianye; Janknecht, Ralf; Hansen, Kirk; Marrack, Philippa; Li, Chuan-Yuan; Kappler, John W.; Hagman, James; Zhang, Gongyi

2017-01-01

Two of the unsolved, important questions about epigenetics are: do histone arginine demethylases exist, and is the removal of histone tails by proteolysis a major epigenetic modification process? Here, we report that two orphan Jumonji C domain (JmjC)-containing proteins, JMJD5 and JMJD7, have divalent cation-dependent protease activities that preferentially cleave the tails of histones 2, 3, or 4 containing methylated arginines. After the initial specific cleavage, JMJD5 and JMJD7, acting as aminopeptidases, progressively digest the C-terminal products. JMJD5-deficient fibroblasts exhibit dramatically increased levels of methylated arginines and histones. Furthermore, depletion of JMJD7 in breast cancer cells greatly decreases cell proliferation. The protease activities of JMJD5 and JMJD7 represent a mechanism for removal of histone tails bearing methylated arginine residues and define a potential mechanism of transcription regulation. PMID:28847961

Chromatin organization regulated by EZH2-mediated H3K27me3 is required for OPN-induced migration of bone marrow-derived mesenchymal stem cells.

PubMed

Liu, Lingling; Luo, Qing; Sun, Jinghui; Ju, Yang; Morita, Yasuyuki; Song, Guanbin

2018-03-01

Osteopontin (OPN) is a chemokine-like extracellular matrix-associated protein involved in the migration of bone marrow-derived mesenchymal stem cells (BMSCs). An increasing number of studies have found that chromatin organization may affect cellular migration. However, whether OPN regulates chromatin organization is not understood, nor are the underlying molecular mechanisms. In this study, we investigated the link between chromatin organization and BMSC migration and demonstrated that OPN-mediated BMSC migration leads to elevated levels of heterochromatin marker histone H3 lysine 27 trimethylation (H3K27me3) through the methyltransferase EZH2. The expression of EZH2 reorganizes the chromatin structure of BMSCs. Pharmacological inhibition or depletion of EZH2 blocks BMSC migration. Moreover, using an atomic force microscope (AFM), we found that chromatin decondensation alters the mechanical properties of the nucleus. In addition, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signals represses OPN-promoted chromatin condensation and cell migration. Thus, our results identify a mechanism by which ERK1/2 signalling drives specific chromatin modifications in BMSCs, which alters chromatin organization and thereby enables OPN-mediated BMSC migration. Copyright © 2018 Elsevier Ltd. All rights reserved.

ERRα induces H3K9 demethylation by LSD1 to promote cell invasion

PubMed Central

Carnesecchi, Julie; Forcet, Christelle; Zhang, Ling; Tribollet, Violaine; Barenton, Bruno; Boudra, Rafik; Cerutti, Catherine; Billas, Isabelle M. L.; Sérandour, Aurélien A.; Carroll, Jason S.; Beaudoin, Claude; Vanacker, Jean-Marc

2017-01-01

Lysine Specific Demethylase 1 (LSD1) removes mono- and dimethyl groups from lysine 4 of histone H3 (H3K4) or H3K9, resulting in repressive or activating (respectively) transcriptional histone marks. The mechanisms that control the balance between these two antagonist activities are not understood. We here show that LSD1 and the orphan nuclear receptor estrogen-related receptor α (ERRα) display commonly activated genes. Transcriptional activation by LSD1 and ERRα involves H3K9 demethylation at the transcriptional start site (TSS). Strikingly, ERRα is sufficient to induce LSD1 to demethylate H3K9 in vitro. The relevance of this mechanism is highlighted by functional data. LSD1 and ERRα coregulate several target genes involved in cell migration, including the MMP1 matrix metallo-protease, also activated through H3K9 demethylation at the TSS. Depletion of LSD1 or ERRα reduces the cellular capacity to invade the extracellular matrix, a phenomenon that is rescued by MMP1 reexpression. Altogether our results identify a regulatory network involving a direct switch in the biochemical activities of a histone demethylase, leading to increased cell invasion. PMID:28348226

ERRα induces H3K9 demethylation by LSD1 to promote cell invasion.

PubMed

Carnesecchi, Julie; Forcet, Christelle; Zhang, Ling; Tribollet, Violaine; Barenton, Bruno; Boudra, Rafik; Cerutti, Catherine; Billas, Isabelle M L; Sérandour, Aurélien A; Carroll, Jason S; Beaudoin, Claude; Vanacker, Jean-Marc

2017-04-11

Lysine Specific Demethylase 1 (LSD1) removes mono- and dimethyl groups from lysine 4 of histone H3 (H3K4) or H3K9, resulting in repressive or activating (respectively) transcriptional histone marks. The mechanisms that control the balance between these two antagonist activities are not understood. We here show that LSD1 and the orphan nuclear receptor estrogen-related receptor α (ERRα) display commonly activated genes. Transcriptional activation by LSD1 and ERRα involves H3K9 demethylation at the transcriptional start site (TSS). Strikingly, ERRα is sufficient to induce LSD1 to demethylate H3K9 in vitro. The relevance of this mechanism is highlighted by functional data. LSD1 and ERRα coregulate several target genes involved in cell migration, including the MMP1 matrix metallo-protease, also activated through H3K9 demethylation at the TSS. Depletion of LSD1 or ERRα reduces the cellular capacity to invade the extracellular matrix, a phenomenon that is rescued by MMP1 reexpression. Altogether our results identify a regulatory network involving a direct switch in the biochemical activities of a histone demethylase, leading to increased cell invasion.

Enrichment of H3K9me2 on Unsynapsed Chromatin in Caenorhabditis elegans Does Not Target de Novo Sites

PubMed Central

Guo, Yiqing; Yang, Bing; Li, Yini; Maine, Eleanor M.

2015-01-01

Many organisms alter the chromatin state of unsynapsed chromosomes during meiotic prophase, a phenomenon hypothesized to function in maintaining germline integrity. In Caenorhabditis elegans, histone H3 lysine 9 dimethylation (H3K9me2) is detected by immunolabeling as enriched on unsynapsed meiotic chromosomes. Loss of the SET domain protein, MET-2, greatly reduces H3K9me2 abundance and results in germline mortality. Here, we used him-8 mutations to disable X chromosome synapsis and performed a combination of molecular assays to map the sites of H3K9me2 accumulation, evaluate H3K9me2 abundance in germline vs. whole animals, and evaluate the impact of H3K9me2 loss on the germline transcriptome. Our data indicate that H3K9me2 is elevated broadly across the X chromosome and at defined X chromosomal sites in him-8 adults compared with controls. H3K9me2 levels are also elevated to a lesser degree at sites on synapsed chromosomes in him-8 adults compared with controls. These results suggest that MET-2 activity is elevated in him-8 mutants generally as well as targeted preferentially to the unsynapsed X. Abundance of H3K9me2 and other histone H3 modifications is low in germline chromatin compared with whole animals, which may facilitate genome reprogramming during gametogenesis. Loss of H3K9me2 has a subtle impact on the him-8 germline transcriptome, suggesting H3K9me2 may not be a major regulator of developmental gene expression in C. elegans. We hypothesize H3K9me2 may have a structural function critical for germline immortality, and a greater abundance of these marks may be required when a chromosome does not synapse. PMID:26156747

Genomic architecture of histone 3 lysine 27 trimethylation during late ovine skeletal muscle development.

PubMed

Byrne, K; McWilliam, S; Vuocolo, T; Gondro, C; Cockett, N E; Tellam, R L

2014-06-01

The ruminant developmental transition from late foetus to lamb is associated with marked changes in skeletal muscle structure and function that reflect programming for new physiological demands following birth. To determine whether epigenetic changes are involved in this transition, we investigated the genomic architecture of the chromatin modification, histone 3 lysine 27 trimethylation (H3K27me3), which typically regulates early life developmental processes; however, its role in later life processes is unclear. Chromatin immunoprecipitation coupled with next-generation sequencing was used to map H3K27me3 nucleosomes in ovine longissimus lumborum skeletal muscle at 100 days of gestation and 12 weeks post-partum. In both states, H3K27me3 modification was associated with genes, transcription start sites and CpG islands and with transcriptional silencing. The H3K27me3 peaks consisted of two major categories, promoter specific and regional, with the latter the dominant feature. Genes encoding homeobox transcription factors regulating early life development and genes involved in neural functions, particularly gated ion channels, were strongly modified by H3K27me3. Gene promoters differentially modified by H3K27me3 in the foetus and lamb were enriched for gated ion channels, which may reflect changes in neuromuscular function. However, most modified genes showed no changes, indicating that H3K27me3 does not have a large role in late muscle maturation. Notably, promyogenic transcription factors were strongly modified with H3K27me3 but showed no differences between the late gestation foetus and lamb, likely reflecting their lack of involvement in the myofibre fusion process occurring in this transition. H3K27me3 is a major architectural feature of the epigenetic landscape of ruminant skeletal muscle, and it comments on gene transcription and gene function in the context of late skeletal muscle development. © 2014 The Authors. Animal Genetics published by John Wiley

PLANT HOMOLOGOUS TO PARAFIBROMIN is a component of the PAF1 complex and assists in regulating expression of genes within H3K27ME3-enriched chromatin.

PubMed

Park, Sunchung; Oh, Sookyung; Ek-Ramos, Julissa; van Nocker, Steven

2010-06-01

The human Paf1 complex (Paf1C) subunit Parafibromin assists in mediating output from the Wingless/Int signaling pathway, and dysfunction of the encoding gene HRPT2 conditions specific cancer-related disease phenotypes. Here, we characterize the organismal and molecular roles of PLANT HOMOLOGOUS TO PARAFIBROMIN (PHP), the Arabidopsis (Arabidopsis thaliana) homolog of Parafibromin. PHP resides in an approximately 670-kD protein complex in nuclear extracts, and physically interacts with other known Paf1C-related proteins in vivo. In striking contrast to the developmental pleiotropy conferred by mutation in other plant Paf1C component genes in Arabidopsis, loss of PHP specifically conditioned accelerated phase transition from vegetative growth to flowering and resulted in misregulation of a very limited subset of genes that included the flowering repressor FLOWERING LOCUS C. Those genes targeted by PHP were distinguished from the bulk of Arabidopsis genes and other plant Paf1C targets by strong enrichment for trimethylation of lysine-27 on histone H3 (H3K27me3) within chromatin. These findings suggest that PHP is a component of a plant Paf1C protein in Arabidopsis, but has a more specialized role in modulating expression of a subset of Paf1C targets.

Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas.

PubMed

Sneeringer, Christopher J; Scott, Margaret Porter; Kuntz, Kevin W; Knutson, Sarah K; Pollock, Roy M; Richon, Victoria M; Copeland, Robert A

2010-12-07

EZH2, the catalytic subunit of the PRC2 complex, catalyzes the mono- through trimethylation of lysine 27 on histone H3 (H3K27). Histone H3K27 trimethylation is a mechanism for suppressing transcription of specific genes that are proximal to the site of histone modification. Point mutations of the EZH2 gene (Tyr641) have been reported to be linked to subsets of human B-cell lymphoma. The mutant allele is always found associated with a wild-type allele (heterozygous) in disease cells, and the mutations were reported to ablate the enzymatic activity of the PRC2 complex for methylating an unmodified peptide substrate. Here we demonstrate that the WT enzyme displays greatest catalytic efficiency (k(cat)/K) for the zero to monomethylation reaction of H3K27 and diminished efficiency for subsequent (mono- to di- and di- to trimethylation) reactions. In stark contrast, the disease-associated Y641 mutations display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to the WT enzyme. These results imply that the malignant phenotype of disease requires the combined activities of a H3K27 monomethylating enzyme (PRC2 containing WT EZH2 or EZH1) together with the mutant PRC2s for augmented conversion of H3K27 to the trimethylated form. To our knowledge, this is the first example of a human disease that is dependent on the coordinated activities of normal and disease-associated mutant enzymatic function.

Epigenetic regulation of NFE2 overexpression in myeloproliferative neoplasms.

PubMed

Peeken, Jan C; Jutzi, Jonas S; Wehrle, Julius; Koellerer, Christoph; Staehle, Hans F; Becker, Heiko; Schoenwandt, Elias; Seeger, Thalia S; Schanne, Daniel H; Gothwal, Monika; Ott, Christopher J; Gründer, Albert; Pahl, Heike L

2018-05-03

The transcription factor "nuclear factor erythroid 2" (NFE2) is overexpressed in the majority of patients with myeloproliferative neoplasms (MPNs). In murine models, elevated NFE2 levels cause an MPN phenotype with spontaneous leukemic transformation. However, both the molecular mechanisms leading to NFE2 overexpression and its downstream targets remain incompletely understood. Here, we show that the histone demethylase JMJD1C constitutes a novel NFE2 target gene. JMJD1C levels are significantly elevated in polycythemia vera (PV) and primary myelofibrosis patients; concomitantly, global H3K9me1 and H3K9me2 levels are significantly decreased. JMJD1C binding to the NFE2 promoter is increased in PV patients, decreasing both H3K9me2 levels and binding of the repressive heterochromatin protein-1α (HP1α). Hence, JMJD1C and NFE2 participate in a novel autoregulatory loop. Depleting JMJD1C expression significantly reduced cytokine-independent growth of an MPN cell line. Independently, NFE2 is regulated through the epigenetic JAK2 pathway by phosphorylation of H3Y41. This likewise inhibits HP1α binding. Treatment with decitabine lowered H3Y41ph and augmented H3K9me2 levels at the NFE2 locus in HEL cells, thereby increasing HP1α binding, which normalized NFE2 expression selectively in JAK2 V617F -positive cell lines. © 2018 by The American Society of Hematology.

Genome-wide ChIP-seq mapping and analysis of butyrate-induced H3K9 and H3K27 acetylation and epigenomic landscape alteration in bovine cells

USDA-ARS?s Scientific Manuscript database

Utilizing next-generation sequencing technology, combined with ChIP (Chromatin Immunoprecipitation) technology, we analyzed histone modification (acetylation) induced by butyrate and the large-scale mapping of the epigenomic landscape of normal histone H3 and acetylated histone H3K9 and H3K27. To d...

Inhalable Metal-Rich Air Particles and Histone H3K4 Dimethylation and H3K9 Acetylation in a Cross-sectional Study of Steel Workers

PubMed Central

Cantone, Laura; Nordio, Francesco; Hou, Lifang; Apostoli, Pietro; Bonzini, Matteo; Tarantini, Letizia; Angelici, Laura; Bollati, Valentina; Zanobetti, Antonella; Schwartz, Joel; Bertazzi, Pier A.

2011-01-01

Background: Epidemiology investigations have linked exposure to ambient and occupational air particulate matter (PM) with increased risk of lung cancer. PM contains carcinogenic and toxic metals, including arsenic and nickel, which have been shown in in vitro studies to induce histone modifications that activate gene expression by inducing open-chromatin states. Whether inhalation of metal components of PM induces histone modifications in human subjects is undetermined. Objectives: We investigated whether the metal components of PM determined activating histone modifications in 63 steel workers with well-characterized exposure to metal-rich PM. Methods: We determined histone 3 lysine 4 dimethylation (H3K4me2) and histone 3 lysine 9 acetylation (H3K9ac) on histones from blood leukocytes. Exposure to inhalable metal components (aluminum, manganese, nickel, zinc, arsenic, lead, iron) and to total PM was estimated for each study subject. Results: Both H3K4me2 and H3K9ac increased in association with years of employment in the plant (p-trend = 0.04 and 0.006, respectively). H3K4me2 increased in association with air levels of nickel [β = 0.16; 95% confidence interval (CI), 0.03–0.3], arsenic (β = 0.16; 95% CI, 0.02–0.3), and iron (β = 0.14; 95% CI, 0.01–0.26). H3K9ac showed nonsignificant positive associations with air levels of nickel (β = 0.24; 95% CI, –0.02 to 0.51), arsenic (β = 0.21; 95% CI, –0.06 to 0.48), and iron (β = 0.22; 95% CI, –0.03 to 0.47). Cumulative exposures to nickel and arsenic, defined as the product of years of employment by metal air levels, were positively correlated with both H3K4me2 (nickel: β = 0.16; 95% CI, 0.01–0.3; arsenic: β = 0.16; 95% CI, 0.03–0.29) and H3K9ac (nickel: β = 0.27; 95% CI, 0.01–0.54; arsenic: β = 0.28; 95% CI, 0.04–0.51). Conclusions: Our results indicate histone modifications as a novel epigenetic mechanism induced in human subjects by long-term exposure to inhalable nickel and arsenic. PMID

Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity.

PubMed

Lu, Jun; Zhang, Xiaoli; Shen, Tingting; Ma, Chao; Wu, Jun; Kong, Hualei; Tian, Jing; Shao, Zhifeng; Zhao, Xiaodong; Xu, Ling

2016-01-01

Traditional Chinese medicine Jinfukang (JFK) has been clinically used for treating lung cancer. To examine whether epigenetic modifications are involved in its anticancer activity, we performed a global profiling analysis of H3K4Me3, an epigenomic marker associated with active gene expression, in JFK-treated lung cancer cells. We identified 11,670 genes with significantly altered status of H3K4Me3 modification following JFK treatment (P < 0.05). Gene Ontology analysis indicates that these genes are involved in tumor-related pathways, including pathway in cancer, basal cell carcinoma, apoptosis, induction of programmed cell death, regulation of transcription (DNA-templated), intracellular signal transduction, and regulation of peptidase activity. In particular, we found that the levels of H3K4Me3 at the promoters of SUSD2, CCND2, BCL2A1, and TMEM158 are significantly altered in A549, NCI-H1975, NCI-H1650, and NCI-H2228 cells, when treated with JFK. Collectively, these findings provide the first evidence that the anticancer activity of JFK involves modulation of histone modification at many cancer-related gene loci.

Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity

PubMed Central

Lu, Jun; Zhang, Xiaoli; Shen, Tingting; Ma, Chao; Wu, Jun; Kong, Hualei; Tian, Jing; Shao, Zhifeng; Zhao, Xiaodong; Xu, Ling

2016-01-01

Traditional Chinese medicine Jinfukang (JFK) has been clinically used for treating lung cancer. To examine whether epigenetic modifications are involved in its anticancer activity, we performed a global profiling analysis of H3K4Me3, an epigenomic marker associated with active gene expression, in JFK-treated lung cancer cells. We identified 11,670 genes with significantly altered status of H3K4Me3 modification following JFK treatment (P < 0.05). Gene Ontology analysis indicates that these genes are involved in tumor-related pathways, including pathway in cancer, basal cell carcinoma, apoptosis, induction of programmed cell death, regulation of transcription (DNA-templated), intracellular signal transduction, and regulation of peptidase activity. In particular, we found that the levels of H3K4Me3 at the promoters of SUSD2, CCND2, BCL2A1, and TMEM158 are significantly altered in A549, NCI-H1975, NCI-H1650, and NCI-H2228 cells, when treated with JFK. Collectively, these findings provide the first evidence that the anticancer activity of JFK involves modulation of histone modification at many cancer-related gene loci. PMID:27087825

FOXP3 Orchestrates H4K16 Acetylation and H3K4 Tri-Methylation for Activation of Multiple Genes through Recruiting MOF and Causing Displacement of PLU-1

PubMed Central

Katoh, Hiroto; Qin, Zhaohui S.; Liu, Runhua; Wang, Lizhong; Li, Weiquan; Li, Xiangzhi; Wu, Lipeng; Du, Zhanwen; Lyons, Robert; Liu, Chang-Gong; Liu, Xiuping; Dou, Yali; Zheng, Pan; Liu, Yang

2011-01-01

SUMMARY Both H4K16 acetylation and H3K4 tri-methylation are required for gene activation. However, it is still largely unclear how these modifications are orchestrated by transcriptional factors. Here we analyzed the mechanism of the transcriptional activation by FOXP3, an X-linked suppressor of autoimmune diseases and cancers. FOXP3 binds near transcriptional start sites of its target genes. By recruiting MOF and displacing histone H3K4 demethylase PLU-1, FOXP3 increases both H4K16 acetylation and H3K4 tri-methylation at the FOXP3-associated chromatins of multiple FOXP3-activated genes. RNAi-mediated silencing of MOF reduced both gene activation and tumor suppression by FOXP3, while both somatic mutations in clinical cancer samples and targeted mutation of FOXP3 in mouse prostate epithelial disrupted nuclear localization of MOF. Our data demonstrate a pull-push model in which a single transcription factor orchestrates two epigenetic alterations necessary for gene activation and provide a mechanism for somatic inactivation of the FOXP3 protein function in cancer cells. PMID:22152480

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

PubMed Central

LeRoy, Gary; Bua, Dennis J; Garcia, Benjamin A; Gozani, Or; Richard, Stéphane

2010-01-01

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4me3) while heterochromatin contains the H3K9me3 mark. The H3K9me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP and SUV39H1 also fail to bind and to methylate H3K4me3 substrates. Accordingly, we provide in vivo evidence that H3K9me2-enriched histones are devoid of H3K4me2/3 and that histones depleted of H3K4me2/3 have elevated H3K9me2/3. The correlation between the loss of interaction of these KMTs with H3K4me3 and concomitant methylation impairment leads to the postulate that at least these four KMTs require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4me3 and H3K9me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners. PMID:21124070

SETDB1 modulates PRC2 activity at developmental genes independently of H3K9 trimethylation in mouse ES cells

PubMed Central

Fei, Qi; Yang, Xiaoqin; Jiang, Hua; Wang, Qian; Yu, Yanyan; Yu, Yiling; Yi, Wei; Zhou, Shaolian; Chen, Taiping; Lu, Chris; Atadja, Peter; Liu, Xiaole Shirley; Li, En; Zhang, Yong; Shou, Jianyong

2015-01-01

SETDB1, a histone methyltransferase responsible for methylation of histone H3 lysine 9 (H3K9), is involved in maintenance of embryonic stem (ES) cells and early embryonic development of the mouse. However, how SETDB1 regulates gene expression during development is largely unknown. Here, we characterized genome-wide SETDB1 binding and H3K9 trimethylation (H3K9me3) profiles in mouse ES cells and uncovered two distinct classes of SETDB1 binding sites, termed solo and ensemble peaks. The solo peaks were devoid of H3K9me3 and enriched near developmental regulators while the ensemble peaks were associated with H3K9me3. A subset of the SETDB1 solo peaks, particularly those near neural development–related genes, was found to be associated with Polycomb Repressive Complex 2 (PRC2) as well as PRC2-interacting proteins JARID2 and MTF2. Genetic deletion of Setdb1 reduced EZH2 binding as well as histone 3 lysine 27 (H3K27) trimethylation level at SETDB1 solo peaks and facilitated neural differentiation. Furthermore, we found that H3K27me3 inhibits SETDB1 methyltransferase activity. The currently identified reciprocal action between SETDB1 and PRC2 reveals a novel mechanism underlying ES cell pluripotency and differentiation regulation. PMID:26160163

Chromatin interaction networks revealed unique connectivity patterns of broad H3K4me3 domains and super enhancers in 3D chromatin.

PubMed

Thibodeau, Asa; Márquez, Eladio J; Shin, Dong-Guk; Vera-Licona, Paola; Ucar, Duygu

2017-10-31

Broad domain promoters and super enhancers are regulatory elements that govern cell-specific functions and harbor disease-associated sequence variants. These elements are characterized by distinct epigenomic profiles, such as expanded deposition of histone marks H3K27ac for super enhancers and H3K4me3 for broad domains, however little is known about how they interact with each other and the rest of the genome in three-dimensional chromatin space. Using network theory methods, we studied chromatin interactions between broad domains and super enhancers in three ENCODE cell lines (K562, MCF7, GM12878) obtained via ChIA-PET, Hi-C, and Hi-CHIP assays. In these networks, broad domains and super enhancers interact more frequently with each other compared to their typical counterparts. Network measures and graphlets revealed distinct connectivity patterns associated with these regulatory elements that are robust across cell types and alternative assays. Machine learning models showed that these connectivity patterns could effectively discriminate broad domains from typical promoters and super enhancers from typical enhancers. Finally, targets of broad domains in these networks were enriched in disease-causing SNPs of cognate cell types. Taken together these results suggest a robust and unique organization of the chromatin around broad domains and super enhancers: loci critical for pathologies and cell-specific functions.

Citrullination/Methylation Crosstalk on Histone H3 Regulates ER-Target Gene Transcription.

PubMed

Clancy, Kathleen W; Russell, Anna-Maria; Subramanian, Venkataraman; Nguyen, Hannah; Qian, Yuewei; Campbell, Robert M; Thompson, Paul R

2017-06-16

Posttranslational modifications of histone tails are a key contributor to epigenetic regulation. Histone H3 Arg26 and Lys27 are both modified by multiple enzymes, and their modifications have profound effects on gene expression. Citrullination of H3R26 by PAD2 and methylation of H3K27 by PRC2 have opposing downstream impacts on gene regulation; H3R26 citrullination activates gene expression, and H3K27 methylation represses gene expression. Both of these modifications are drivers of a variety of cancers, and their writer enzymes, PAD2 and EZH2, are the targets of drug therapies. After biochemical and cell-based analysis of these modifications, a negative crosstalk interaction is observed. Methylation of H3K27 slows citrullination of H3R26 30-fold, whereas citrullination of H3R26 slows methylation 30,000-fold. Examination of the mechanism of this crosstalk interaction uncovered a change in structure of the histone tail upon citrullination which prevents methylation by the PRC2 complex. This mechanism of crosstalk is reiterated in cell lines using knockdowns and inhibitors of both enzymes. Based our data, we propose a model in which, after H3 Cit26 formation, H3K27 demethylases are recruited to the chromatin to activate transcription. In total, our studies support the existence of crosstalk between citrullination of H3R26 and methylation of H3K27.

Quantitative Histone Mass Spectrometry Identifies Elevated Histone H3 Lysine 27 (Lys27) Trimethylation in Melanoma*

PubMed Central

Sengupta, Deepanwita; Byrum, Stephanie D.; Avaritt, Nathan L.; Davis, Lauren; Shields, Bradley; Mahmoud, Fade; Reynolds, Matthew; Orr, Lisa M.; Mackintosh, Samuel G.; Shalin, Sara C.; Tackett, Alan J.

2016-01-01

Normal cell growth is characterized by a regulated epigenetic program that drives cellular activities such as gene transcription, DNA replication, and DNA damage repair. Perturbation of this epigenetic program can lead to events such as mis-regulation of gene transcription and diseases such as cancer. To begin to understand the epigenetic program correlated to the development of melanoma, we performed a novel quantitative mass spectrometric analysis of histone post-translational modifications mis-regulated in melanoma cell culture as well as patient tumors. Aggressive melanoma cell lines as well as metastatic melanoma were found to have elevated histone H3 Lys27 trimethylation (H3K27me3) accompanied by overexpressed methyltransferase EZH2 that adds the specific modification. The altered epigenetic program that led to elevated H3K27me3 in melanoma cell culture was found to directly silence transcription of the tumor suppressor genes RUNX3 and E-cadherin. The EZH2-mediated silencing of RUNX3 and E-cadherin transcription was also validated in advanced stage human melanoma tissues. This is the first study focusing on the detailed epigenetic mechanisms leading to EZH2-mediated silencing of RUNX3 and E-cadherin tumor suppressors in melanoma. This study underscores the utility of using high resolution mass spectrometry to identify mis-regulated epigenetic programs in diseases such as cancer, which could ultimately lead to the identification of biological markers for diagnostic and prognostic applications. PMID:26621846

Diffuse high-grade gliomas with H3 K27M mutations carry a dismal prognosis independent of tumor location.

PubMed

Karremann, Michael; Gielen, Gerrit H; Hoffmann, Marion; Wiese, Maria; Colditz, Niclas; Warmuth-Metz, Monika; Bison, Brigitte; Claviez, Alexander; van Vuurden, Dannis G; von Bueren, André O; Gessi, Marco; Kühnle, Ingrid; Hans, Volkmar H; Benesch, Martin; Sturm, Dominik; Kortmann, Rolf-Dieter; Waha, Andreas; Pietsch, Torsten; Kramm, Christof M

2018-01-10

The novel entity of "diffuse midline glioma, H3 K27M-mutant" has been defined in the 2016 revision of the World Health Organization (WHO) classification of tumors of the central nervous system (CNS). Tumors of this entity arise in CNS midline structures of predominantly pediatric patients and are associated with an overall dismal prognosis. They are defined by K27M mutations in H3F3A or HIST1H3B/C, encoding for histone 3 variants H3.3 and H3.1, respectively, which are considered hallmark events driving gliomagenesis. Here, we characterized 85 centrally reviewed diffuse gliomas on midline locations enrolled in the nationwide pediatric German HIT-HGG registry regarding tumor site, histone 3 mutational status, WHO grade, age, sex, and extent of tumor resection. We found 56 H3.3 K27M-mutant tumors (66%), 6 H3.1 K27M-mutant tumors (7%), and 23 H3-wildtype tumors (27%). H3 K27M-mutant gliomas shared an aggressive clinical course independent of their anatomic location. Multivariate regression analysis confirmed the significant impact of the H3 K27M mutation as the only independent parameter predictive of overall survival (P = 0.009). In H3 K27M-mutant tumors, neither anatomic midline location nor histopathological grading nor extent of tumor resection had an influence on survival. These results substantiate the clinical significance of considering diffuse midline glioma, H3 K27M-mutant, as a distinct entity corresponding to WHO grade IV, carrying a universally fatal prognosis. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

In silico probing and biological evaluation of SETDB1/ESET-targeted novel compounds that reduce tri-methylated histone H3K9 (H3K9me3) level

NASA Astrophysics Data System (ADS)

Park, Insun; Hwang, Yu Jin; Kim, TaeHun; Viswanath, Ambily Nath Indu; Londhe, Ashwini M.; Jung, Seo Yun; Sim, Kyoung Mi; Min, Sun-Joon; Lee, Ji Eun; Seong, Jihye; Kim, Yun Kyung; No, Kyoung Tai; Ryu, Hoon; Pae, Ae Nim

2017-10-01

ERG-associated protein with the SET domain (ESET/SET domain bifurcated 1/SETDB1/KMT1E) is a histone lysine methyltransferase (HKMT) and it preferentially tri-methylates lysine 9 of histone H3 (H3K9me3). SETDB1/ESET leads to heterochromatin condensation and epigenetic gene silencing. These functional changes are reported to correlate with Huntington's disease (HD) progression and mood-related disorders which make SETDB1/ESET a viable drug target. In this context, the present investigation was performed to identify novel peptide-competitive small molecule inhibitors of the SETDB1/ESET by a combined in silico-in vitro approach. A ligand-based pharmacophore model was built and employed for the virtual screening of ChemDiv and Asinex database. Also, a human SETDB1/ESET homology model was constructed to supplement the data further. Biological evaluation of the selected 21 candidates singled out 5 compounds exhibiting a notable reduction of the H3K9me3 level via inhibitory potential of SETDB1/ESET activity in SETDB1/ESET-inducible cell line and HD striatal cells. Later on, we identified two compounds as final hits that appear to have neuronal effects without cytotoxicity based on the result from MTT assay. These compounds hold the calibre to become the future lead compounds and can provide structural insights into more SETDB1/ESET-focused drug discovery research. Moreover, these SETDB1/ESET inhibitors may be applicable for the preclinical study to ameliorate neurodegenerative disorders via epigenetic regulation.

In silico probing and biological evaluation of SETDB1/ESET-targeted novel compounds that reduce tri-methylated histone H3K9 (H3K9me3) level.

PubMed

Park, Insun; Hwang, Yu Jin; Kim, TaeHun; Viswanath, Ambily Nath Indu; Londhe, Ashwini M; Jung, Seo Yun; Sim, Kyoung Mi; Min, Sun-Joon; Lee, Ji Eun; Seong, Jihye; Kim, Yun Kyung; No, Kyoung Tai; Ryu, Hoon; Pae, Ae Nim

2017-10-01

ERG-associated protein with the SET domain (ESET/SET domain bifurcated 1/SETDB1/KMT1E) is a histone lysine methyltransferase (HKMT) and it preferentially tri-methylates lysine 9 of histone H3 (H3K9me3). SETDB1/ESET leads to heterochromatin condensation and epigenetic gene silencing. These functional changes are reported to correlate with Huntington's disease (HD) progression and mood-related disorders which make SETDB1/ESET a viable drug target. In this context, the present investigation was performed to identify novel peptide-competitive small molecule inhibitors of the SETDB1/ESET by a combined in silico-in vitro approach. A ligand-based pharmacophore model was built and employed for the virtual screening of ChemDiv and Asinex database. Also, a human SETDB1/ESET homology model was constructed to supplement the data further. Biological evaluation of the selected 21 candidates singled out 5 compounds exhibiting a notable reduction of the H3K9me3 level via inhibitory potential of SETDB1/ESET activity in SETDB1/ESET-inducible cell line and HD striatal cells. Later on, we identified two compounds as final hits that appear to have neuronal effects without cytotoxicity based on the result from MTT assay. These compounds hold the calibre to become the future lead compounds and can provide structural insights into more SETDB1/ESET-focused drug discovery research. Moreover, these SETDB1/ESET inhibitors may be applicable for the preclinical study to ameliorate neurodegenerative disorders via epigenetic regulation.

The PR-Set7 binding domain of Riz1 is required for the H4K20me1-H3K9me1 trans-tail 'histone code' and Riz1 tumor suppressor function.

PubMed

Congdon, Lauren M; Sims, Jennifer K; Tuzon, Creighton T; Rice, Judd C

2014-04-01

PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail 'histone code'. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail 'histone code'. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function.

Nucleosome eviction along with H3K9ac deposition enhances Sox2 binding during human neuroectodermal commitment

PubMed Central

Du, Yanhua; Liu, Zhenping; Cao, Xinkai; Chen, Xiaolong; Chen, Zhenyu; Zhang, Xiaobai; Zhang, Xiaoqing; Jiang, Cizhong

2017-01-01

Neuroectoderm is an important neural precursor. However, chromatin remodeling and its epigenetic regulatory roles during the differentiation of human neuroectodermal cells (hNECs) from human embryonic stem cells (hESCs) remain largely unexplored. Here, we obtained hNECs through directed differentiation from hESCs, and determined chromatin states in the two cell types. Upon differentiation, H2A.Z-mediated nucleosome depletion leads to an open chromatin structure in promoters and upregulates expression of neuroectodermal genes. Increase in H3K9ac signals and decrease in H3K27me3 signals in promoters result in an active chromatin state and activate neuroectodermal genes. Conversely, decrease in H3K9ac signals and increase in H3K27me3 signals in promoters repress pluripotency genes. Moreover, H3K9ac signals facilitate the pluripotency factor Sox2 binding to target sites unique to hNECs. Knockdown of the acetyltransferase Kat2b erases H3K9ac signals, disrupts Sox2 binding, and fails the differentiation. Our results demonstrate a hierarchy of epigenetic regulation of gene expression during the differentiation of hNECs from hESCs through chromatin remodeling. PMID:28475175

Immunohistochemical Analysis of Histone H3 Modifications in Germ Cells during Mouse Spermatogenesis

PubMed Central

Song, Ning; Liu, Jie; An, Shucai; Nishino, Tomoya; Hishikawa, Yoshitaka; Koji, Takehiko

2011-01-01

Histone modification has been implicated in the regulation of mammalian spermatogenesis. However, the association of differently modified histone H3 with a specific stage of germ cells during spermatogenesis is not fully understood. In this study, we examined the localization of variously modified histone H3 in paraffin-embedded sections of adult mouse testis immunohistochemically, focusing on acetylation at lysine 9 (H3K9ac), lysine 18 (H3K18ac), and lysine 23 (H3K23ac); tri-methylation at lysine 4 (H3K4me3) and lysine 27 (H3K27me3); and phosphorylation at serine 10 (H3S10phos). As a result, we found that there was a significant fluctuation in the modifications; in spermatogonia, the stainings for H3K9ac, H3K18ac, and H3K23ac were strong while that for H3K4me3 was weak. In spermatocytes, the stainings for H3K9ac, H3K18ac, H3K23ac, and H3K4me3 were reduced in the preleptotene to pachytene stage, but in diplotene stage the stainings for H3K18ac, H3K23ac, and H3K4me3 seemed to become intense again. The staining for H3K27me3 was nearly constant throughout these stages. In the ensuing spermiogenesis, a dramatic acetylation and methylation of histone H3 was found in the early elongated spermatids and then almost all signals disappeared in the late elongated spermatids, in parallel with the replacement from histones to protamines. In addition, we confirmed that the staining of histone H3S10phos was exclusively associated with mitotic and meiotic cell division. Based upon the above results, we indicated that the modification pattern of histone H3 is subject to dynamic change and specific to a certain stage of germ cell differentiation during mouse spermatogenesis. PMID:21927517

Immunohistochemical Analysis of Histone H3 Modifications in Germ Cells during Mouse Spermatogenesis.

PubMed

Song, Ning; Liu, Jie; An, Shucai; Nishino, Tomoya; Hishikawa, Yoshitaka; Koji, Takehiko

2011-08-27

Histone modification has been implicated in the regulation of mammalian spermatogenesis. However, the association of differently modified histone H3 with a specific stage of germ cells during spermatogenesis is not fully understood. In this study, we examined the localization of variously modified histone H3 in paraffin-embedded sections of adult mouse testis immunohistochemically, focusing on acetylation at lysine 9 (H3K9ac), lysine 18 (H3K18ac), and lysine 23 (H3K23ac); tri-methylation at lysine 4 (H3K4me3) and lysine 27 (H3K27me3); and phosphorylation at serine 10 (H3S10phos). As a result, we found that there was a significant fluctuation in the modifications; in spermatogonia, the stainings for H3K9ac, H3K18ac, and H3K23ac were strong while that for H3K4me3 was weak. In spermatocytes, the stainings for H3K9ac, H3K18ac, H3K23ac, and H3K4me3 were reduced in the preleptotene to pachytene stage, but in diplotene stage the stainings for H3K18ac, H3K23ac, and H3K4me3 seemed to become intense again. The staining for H3K27me3 was nearly constant throughout these stages. In the ensuing spermiogenesis, a dramatic acetylation and methylation of histone H3 was found in the early elongated spermatids and then almost all signals disappeared in the late elongated spermatids, in parallel with the replacement from histones to protamines. In addition, we confirmed that the staining of histone H3S10phos was exclusively associated with mitotic and meiotic cell division. Based upon the above results, we indicated that the modification pattern of histone H3 is subject to dynamic change and specific to a certain stage of germ cell differentiation during mouse spermatogenesis.

Structural Investigations of the Nickel-Induced Inhibition of Truncated Constructs of the JMJD2 Family of Histone Demethylases Using X-ray Absorption Spectroscopy

PubMed Central

Giri, Nitai Charan; Passantino, Lisa; Sun, Hong; Zoroddu, Maria Antonietta; Costa, Max; Maroney, Michael J.

2013-01-01

Occupational and/or environmental exposure to nickel has been implicated in various types of cancer, and in vitro exposure to nickel compounds results in accumulation of Ni(II) ions in cells. One of the major targets of Ni(II) ions inside the cell is Fe(II)- and αKG-dependent dioxygenases. Using JMJD2A and JMJD2C as examples, we show that JMJD2 family of histone demethylases, which are products of putative oncogenes as well as Fe(II)- and αKG-dependent dioxygenases, are highly sensitive to inhibition by Ni(II) ions. In this work, X-ray absorption spectroscopy (XAS) has been used to investigate the Fe(II) active site of truncated JMJD2A and JMJD2C (1 – 350 aa) in the presence and absence of αKG and/or substrate to obtain mechanistic details of the early steps in catalysis that precede O2 binding in histone demethylation by the JMJD2 family of histone demethylases. Zinc K-edge XAS has been performed on the resting JMJD2A (with iron in the active site) to confirm the presence of the expected structural zinc site. XAS of the Ni(II)-substituted enzymes has also been performed to investigate the inhibition of these enzymes by Ni(II) ions. Our XAS results indicate that the five-coordinate Fe(II) center in the resting enzyme is retained in the binary and ternary complexes. In contrast, the Ni(II) center is six-coordinate in the resting enzyme, binary and ternary complexes. XAS results indicate that both Fe(II) and Ni(II) bind αKG in the binary and ternary complexes. The electron density build-up that is observed at the Fe(II) center in the presence of αKG and substrate is not observed at the Ni(II) center. Thus, both electronic and steric factors are responsible for Ni-induced inhibition of the JMJD2 family of histone demethylases. Ni-induced inhibition of these enzymes may explain the alteration of the epigenetic mechanism of gene expression that is responsible for Ni-induced carcinogenesis. PMID:23692052

Ezh1 and Ezh2 differentially regulate PSD-95 gene transcription in developing hippocampal neurons.

PubMed

Henriquez, Berta; Bustos, Fernando J; Aguilar, Rodrigo; Becerra, Alvaro; Simon, Felipe; Montecino, Martin; van Zundert, Brigitte

2013-11-01

Polycomb Repressive Complex 2 (PRC2) mediates transcriptional silencing by catalyzing histone H3 lysine 27 trimethylation (H3K27me3), but its role in the maturation of postmitotic mammalian neurons remains largely unknown. We report that the PRC2 paralogs Ezh1 and Ezh2 are differentially expressed during hippocampal development. We show that depletion of Ezh2 leads to increased expression of PSD-95, a critical plasticity gene, and that reduced PSD-95 gene transcription is correlated with enrichment of Ezh2 at the PSD-95 gene promoter; however, the H3K27me3 epigenetic mark is not present at the PSD-95 gene promoter, likely due to the antagonizing effects of the H3S28P and H3K27Ac marks and the activity of the H3K27 demethylases JMJD3 and UTX. In contrast, increased PSD-95 gene transcription is accompanied by the presence of Ezh1 and elongation-engaged RNA Polymerase II complexes at the PSD-95 gene promoter, while knock-down of Ezh1 reduces PSD-95 transcription. These results indicate that Ezh1 and Ezh2 have antagonistic roles in regulating PSD-95 transcription. © 2013.

Mitochondrial control through nutritionally regulated global histone H3 lysine-4 demethylation

PubMed Central

Soloveychik, Maria; Xu, Mengshu; Zaslaver, Olga; Lee, Kwanyin; Narula, Ashrut; Jiang, River; Rosebrock, Adam P.; Caudy, Amy A.; Meneghini, Marc D.

2016-01-01

Histone demethylation by Jumonji-family proteins is coupled with the decarboxylation of α-ketoglutarate (αKG) to yield succinate, prompting hypotheses that their activities are responsive to levels of these metabolites in the cell. Consistent with this paradigm we show here that the Saccharomyces cerevisiae Jumonji demethylase Jhd2 opposes the accumulation of H3K4me3 in fermenting cells only when they are nutritionally manipulated to contain an elevated αKG/succinate ratio. We also find that Jhd2 opposes H3K4me3 in respiratory cells that do not exhibit such an elevated αKG/succinate ratio. While jhd2∆ caused only limited gene expression defects in fermenting cells, transcript profiling and physiological measurements show that JHD2 restricts mitochondrial respiratory capacity in cells grown in non-fermentable carbon in an H3K4me-dependent manner. In association with these phenotypes, we find that JHD2 limits yeast proliferative capacity under physiologically challenging conditions as measured by both replicative lifespan and colony growth on non-fermentable carbon. JHD2’s impact on nutrient response may reflect an ancestral role of its gene family in mediating mitochondrial regulation. PMID:27897198

Genome-wide Analysis of the H3K4 Histone Demethylase RBP2 Reveals a Transcriptional Program Controlling Differentiation

PubMed Central

Lopez-Bigas, Nuria; Kisiel, Tomasz A.; DeWaal, Dannielle C.; Holmes, Katie B.; Volkert, Tom L.; Gupta, Sumeet; Love, Jennifer; Murray, Heather L.; Young, Richard A.; Benevolenskaya, Elizaveta V.

2010-01-01

SUMMARY Retinoblastoma protein (pRB) mediates cell-cycle withdrawal and differentiation by interacting with a variety of proteins. RB-Binding Protein 2 (RBP2) has been shown to be a key effector. We sought to determine transcriptional regulation by RBP2 genome-wide by using location analysis and gene expression profiling experiments. We describe that RBP2 shows high correlation with the presence of H3K4me3 and its target genes are separated into two functionally distinct classes: differentiation-independent and differentiation-dependent genes. The former class is enriched by genes that encode mitochondrial proteins, while the latter is represented by cell-cycle genes. We demonstrate the role of RBP2 in mitochondrial biogenesis, which involves regulation of H3K4me3-modified nucleosomes. Analysis of expression changes upon RBP2 depletion depicted genes with a signature of differentiation control, analogous to the changes seen upon reintroduction of pRB. We conclude that, during differentiation, RBP2 exerts inhibitory effects on multiple genes through direct interaction with their promoters. PMID:18722178

Lgr4 promotes prostate tumorigenesis through the Jmjd2a/AR signaling pathway.

PubMed

Zhang, Jianwei; Li, Qi; Zhang, Shaojin; Xu, Quanquan; Wang, Tianen

2016-11-15

Lgr4 (leucine-rich repeat domain containing G protein-coupled receptor 4) is implicated in the transcriptional regulation of multiple histone demethylases in the progression of diverse cancers, but there are few reports concerning the molecular mechanism by which Lgr4 regulates histone demethylase activation in prostate cancer (PCa) progression. As Jmjd2a is a histone demethylase, in the current study, we investigated the relationship between interaction Lgr4 with Jmjd 2a and Jmjd2a/androgen receptor (AR) signaling pathway in PCa progression. Firstly, Lgr4 was overexpressed by transfecting pcDNA3.1(+)/Lgr4 plasmids into PCa (LNCaP and PC-3) cell lines. Next, we found that Lgr4 overexpression promoted Jmjd2a mRNA expression, reduced cell apoptosis and arrested cell cycle in the S phase, these effects were reversed by Jmjd2a silencing. Moreover, Lgr4 overexpression markedly elevated AR levels and its interaction with Jmjd2a, which was tested by co-immunoprecipitation and luciferase reporter assays. Furthermore, interaction AR with PSA promoter (containing an AR response element) was obviously improved by Lgr4 overexpression, and PSA silencing reduced Lgr4-induced cell apoptosis and cell cycle arrest in PCa cells. Taken together, Lgr4 may be a novel tumor marker providing new mechanistic insights into PCa progression. Lgr4 activates Jmjd2a/AR signaling pathway to promote interaction AR with PSA promoter, causing reduction of PCa apoptosis and cell cycle arrest. Copyright © 2016 Elsevier Inc. All rights reserved.

Both H4K20 mono-methylation and H3K56 acetylation mark transcription-dependent histone turnover in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Hanna; Kwon, Chang Seob; Choi, Yoonjung, E-mail: jjungii@kaist.ac.kr

Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5′ promoter and 3′ termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNAmore » production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast.« less

The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy

PubMed Central

Aronsen, Jan Magnus; Ferrini, Arianna; Brien, Patrick; Alkass, Kanar; Tomasso, Antonio; Agrawal, Asmita; Bergmann, Olaf; Reik, Wolf; Roderick, Hywel Llewelyn

2016-01-01

Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217–mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217–mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease. PMID:27893464

SMYD5 regulates H4K20me3-marked heterochromatin to safeguard ES cell self-renewal and prevent spurious differentiation.

PubMed

Kidder, Benjamin L; Hu, Gangqing; Cui, Kairong; Zhao, Keji

2017-01-01

Epigenetic regulation of chromatin states is thought to control the self-renewal and differentiation of embryonic stem (ES) cells. However, the roles of repressive histone modifications such as trimethylated histone 4 lysine 20 (H4K20me3) in pluripotency and development are largely unknown. Here, we show that the histone lysine methyltransferase SMYD5 mediates H4K20me3 at heterochromatin regions. Depletion of SMYD5 leads to compromised self-renewal, including dysregulated expression of OCT4 targets, and perturbed differentiation. SMYD5-bound regions are enriched with repetitive DNA elements. Knockdown of SMYD5 results in a global decrease of H4K20me3 levels, a redistribution of heterochromatin constituents including H3K9me3/2, G9a, and HP1α, and de-repression of endogenous retroelements. A loss of SMYD5-dependent silencing of heterochromatin nearby genic regions leads to upregulated expression of lineage-specific genes, thus contributing to the decreased self-renewal and perturbed differentiation of SMYD5-depleted ES cells. Altogether, these findings implicate a role for SMYD5 in regulating ES cell self-renewal and H4K20me3-marked heterochromatin.

Epigenetic signatures of autism: trimethylated H3K4 landscapes in prefrontal neurons.

PubMed

Shulha, Hennady P; Cheung, Iris; Whittle, Catheryne; Wang, Jie; Virgil, Daniel; Lin, Cong L; Guo, Yin; Lessard, Andree; Akbarian, Schahram; Weng, Zhiping

2012-03-01

Neuronal dysfunction in cerebral cortex and other brain regions could contribute to the cognitive and behavioral defects in autism. To characterize epigenetic signatures of autism in prefrontal cortex neurons. We performed fluorescence-activated sorting and separation of neuronal and nonneuronal nuclei from postmortem prefrontal cortex, digested the chromatin with micrococcal nuclease, and deeply sequenced the DNA from the mononucleosomes with trimethylated H3K4 (H3K4me3), a histone mark associated with transcriptional regulation. Approximately 15 billion base pairs of H3K4me3-enriched sequences were collected from 32 brains. Academic medical center. A total of 16 subjects diagnosed as having autism and 16 control subjects ranging in age from 0.5 to 70 years. Identification of genomic loci showing autism-associated H3K4me3 changes in prefrontal cortex neurons. Subjects with autism showed no evidence for generalized disruption of the developmentally regulated remodeling of the H3K4me3 landscape that defines normal prefrontal cortex neurons in early infancy. However, excess spreading of H3K4me3 from the transcription start sites into downstream gene bodies and upstream promoters was observed specifically in neuronal chromatin from 4 of 16 autism cases but not in controls. Variable subsets of autism cases exhibit altered H3K4me3 peaks at numerous genes regulating neuronal connectivity, social behaviors, and cognition, often in conjunction with altered expression of the corresponding transcripts. Autism-associated H3K4me3 peaks were significantly enriched in genes and loci implicated in neurodevelopmental diseases. Prefrontal cortex neurons from subjects with autism show changes in chromatin structures at hundreds of loci genome-wide, revealing considerable overlap between genetic and epigenetic risk maps of developmental brain disorders.

Enhanced H3K4me3 modifications are involved in the transactivation of DNA damage responsive genes in workers exposed to low-level benzene.

PubMed

Li, Jie; Xing, Xiumei; Zhang, Xinjie; Liang, Boxuan; He, Zhini; Gao, Chen; Wang, Shan; Wang, Fangping; Zhang, Haiyan; Zeng, Shan; Fan, Junling; Chen, Liping; Zhang, Zhengbao; Zhang, Bo; Liu, Caixia; Wang, Qing; Lin, Weiwei; Dong, Guanghui; Tang, Huanwen; Chen, Wen; Xiao, Yongmei; Li, Daochuan

2018-03-01

In this study, we explore whether altered global histone modifications respond to low-level benzene exposure as well as their association with the hematotoxicity. We recruited 147 low-level benzene-exposed workers and 122 control workers from a petrochemical factory in Maoming City, Guangdong Province, China. The internal exposure marker level, urinary S-phenylmercapturic acid (SPMA), in benzene-exposed workers was 1.81-fold higher than that of the controls (P < 0.001). ELISA method was established to examine the specific histone modifications in human peripheral blood lymphocytes (PBLCs) of workers. A decrease in the counts of white blood cells (WBC), neutrophils, lymphocytes, and monocytes appeared in the benzene-exposed group (all P < 0.05) compared to the control group. Global trimethylated histone 3 lysine 4 (H3K4me3) modification was enhanced in the benzene-exposed group (P < 0.05) and was positively associated with the concentration of urinary SPMA (β = 0.103, P = 0.045) and the extent of DNA damage (% Tail DNA: β = 0.181, P = 0.022), but was negatively associated with the leukocyte count (WBC: β = -0.038, P = 0.023). The in vitro study revealed that H3K4me3 mark was enriched in the promoters of several DNA damage responsive (DDR) genes including CRY1, ERCC2, and TP53 in primary human lymphocytes treated with hydroquinone. Particularly, H3K4me3 modification was positively correlated with the expression of CRY1 in the PBLCs of benzene-exposed workers. These observations indicate that H3K4me3 modification might mediate the transcriptional regulation of DDR genes in response to low-dose benzene exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mutation of C. elegans demethylase spr-5 extends transgenerational longevity

PubMed Central

Greer, Eric Lieberman; Becker, Ben; Latza, Christian; Antebi, Adam; Shi, Yang

2016-01-01

Complex organismal properties such as longevity can be transmitted across generations by non-genetic factors. Here we demonstrate that deletion of the C. elegans histone H3 lysine 4 dimethyl (H3K4me2) demethylase, spr-5, causes a trans-generational increase in lifespan. We identify a chromatin-modifying network, which regulates this lifespan extension. We further show that this trans-generational lifespan extension is dependent on a hormonal signaling pathway involving the steroid dafachronic acid, an activator of the nuclear receptor DAF-12. These findings suggest that loss of the demethylase SPR-5 causes H3K4me2 mis-regulation and activation of a known lifespan-regulating signaling pathway, leading to trans-generational lifespan extension. PMID:26691751

Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4

PubMed Central

Dhar, Shilpa S.; Lee, Sung-Hun; Kan, Pu-Yeh; Voigt, Philipp; Ma, Li; Shi, Xiaobing; Reinberg, Danny; Lee, Min Gyu

2012-01-01

Mixed-lineage leukemia 4 (MLL4; also called MLL2 and ALR) enzymatically generates trimethylated histone H3 Lys 4 (H3K4me3), a hallmark of gene activation. However, how MLL4-deposited H3K4me3 interplays with other histone marks in epigenetic processes remains largely unknown. Here, we show that MLL4 plays an essential role in differentiating NT2/D1 stem cells by activating differentiation-specific genes. A tandem plant homeodomain (PHD4–6) of MLL4 recognizes unmethylated or asymmetrically dimethylated histone H4 Arg 3 (H4R3me0 or H4R3me2a) and is required for MLL4's nucleosomal methyltransferase activity and MLL4-mediated differentiation. Kabuki syndrome mutations in PHD4–6 reduce PHD4–6's binding ability and MLL4's catalytic activity. PHD4–6's binding strength is inhibited by H4R3 symmetric dimethylation (H4R3me2s), a gene-repressive mark. The protein arginine methyltransferase 7 (PRMT7), but not PRMT5, represses MLL4 target genes by up-regulating H4R3me2s levels and antagonizes MLL4-mediated differentiation. Consistently, PRMT7 knockdown increases MLL4-catalyzed H3K4me3 levels. During differentiation, decreased H4R3me2s levels are associated with increased H3K4me3 levels at a cohort of genes, including many HOXA and HOXB genes. These findings indicate that the trans-tail inhibition of MLL4-generated H3K4me3 by PRMT7-regulated H4R3me2s may result from H4R3me2s's interference with PHD4–6's binding activity and is a novel epigenetic mechanism that underlies opposing effects of MLL4 and PRMT7 on cellular differentiation. PMID:23249737

Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4.

PubMed

Dhar, Shilpa S; Lee, Sung-Hun; Kan, Pu-Yeh; Voigt, Philipp; Ma, Li; Shi, Xiaobing; Reinberg, Danny; Lee, Min Gyu

2012-12-15

Mixed-lineage leukemia 4 (MLL4; also called MLL2 and ALR) enzymatically generates trimethylated histone H3 Lys 4 (H3K4me3), a hallmark of gene activation. However, how MLL4-deposited H3K4me3 interplays with other histone marks in epigenetic processes remains largely unknown. Here, we show that MLL4 plays an essential role in differentiating NT2/D1 stem cells by activating differentiation-specific genes. A tandem plant homeodomain (PHD(4-6)) of MLL4 recognizes unmethylated or asymmetrically dimethylated histone H4 Arg 3 (H4R3me0 or H4R3me2a) and is required for MLL4's nucleosomal methyltransferase activity and MLL4-mediated differentiation. Kabuki syndrome mutations in PHD(4-6) reduce PHD(4-6)'s binding ability and MLL4's catalytic activity. PHD(4-6)'s binding strength is inhibited by H4R3 symmetric dimethylation (H4R3me2s), a gene-repressive mark. The protein arginine methyltransferase 7 (PRMT7), but not PRMT5, represses MLL4 target genes by up-regulating H4R3me2s levels and antagonizes MLL4-mediated differentiation. Consistently, PRMT7 knockdown increases MLL4-catalyzed H3K4me3 levels. During differentiation, decreased H4R3me2s levels are associated with increased H3K4me3 levels at a cohort of genes, including many HOXA and HOXB genes. These findings indicate that the trans-tail inhibition of MLL4-generated H3K4me3 by PRMT7-regulated H4R3me2s may result from H4R3me2s's interference with PHD(4-6)'s binding activity and is a novel epigenetic mechanism that underlies opposing effects of MLL4 and PRMT7 on cellular differentiation.

Dynamic association of epigenetic H3K4me3 and DNA 5hmC marks in the dorsal hippocampus and anterior cingulate cortex following reactivation of a fear memory.

PubMed

Webb, William M; Sanchez, Richard G; Perez, Gabriella; Butler, Anderson A; Hauser, Rebecca M; Rich, Megan C; O'Bierne, Aidan L; Jarome, Timothy J; Lubin, Farah D

2017-07-01

Epigenetic mechanisms such as DNA methylation and histone methylation are critical regulators of gene transcription changes during memory consolidation. However, it is unknown how these epigenetic modifications coordinate control of gene expression following reactivation of a previously consolidated memory. Here, we found that retrieval of a recent contextual fear conditioned memory increased global levels of H3 lysine 4-trimethylation (H3K4me3) and DNA 5-hydroxymethylation (5hmC) in area CA1 of the dorsal hippocampus. Further experiments revealed increased levels of H3K4me3 and DNA 5hmC within a CpG-enriched coding region of the Npas4, but not c-fos, gene. Intriguingly, retrieval of a 30-day old memory increased H3K4me3 and DNA 5hmC levels at a CpG-enriched coding region of c-fos, but not Npas4, in the anterior cingulate cortex, suggesting that while these two epigenetic mechanisms co-occur following the retrieval of a recent or remote memory, their gene targets differ depending on the brain region. Additionally, we found that in vivo siRNA-mediated knockdown of the H3K4me3 methyltransferase Mll1 in CA1 abolished retrieval-induced increases in DNA 5hmC levels at the Npas4 gene, suggesting that H3K4me3 couples to DNA 5hmC mechanisms. Consistent with this, loss of Mll1 prevented retrieval-induced increases in Npas4 mRNA levels in CA1 and impaired fear memory. Collectively, these findings suggest an important link between histone methylation and DNA hydroxymethylation mechanisms in the epigenetic control of de novo gene transcription triggered by memory retrieval. Copyright © 2017 Elsevier Inc. All rights reserved.

A demonstration of the H3 trimethylation ChIP-seq analysis of galline follicular mesenchymal cells and male germ cells.

PubMed

Chokeshaiusaha, Kaj; Puthier, Denis; Nguyen, Catherine; Sananmuang, Thanida

2018-06-01

Trimethylation of histone 3 (H3) at 4th lysine N-termini (H3K4me3) in gene promoter region was the universal marker of active genes specific to cell lineage. On the contrary, coexistence of trimethylation at 27th lysine (H3K27me3) in the same loci-the bivalent H3K4m3/H3K27me3 was known to suspend the gene transcription in germ cells, and could also be inherited to the developed stem cell. In galline species, throughout example of H3K4m3 and H3K27me3 ChIP-seq analysis was still not provided. We therefore designed and demonstrated such procedures using ChIP-seq and mRNA-seq data of chicken follicular mesenchymal cells and male germ cells. Analytical workflow was designed and provided in this study. ChIP-seq and RNA-seq datasets of follicular mesenchymal cells and male germ cells were acquired and properly preprocessed. Peak calling by Model-based analysis of ChIP-seq 2 was performed to identify H3K4m3 or H3K27me3 enriched regions (Fold-change≥2, FDR≤0.01) in gene promoter regions. Integrative genomics viewer was utilized for cellular retinoic acid binding protein 1 ( CRABP1 ), growth differentiation factor 10 ( GDF10 ), and gremlin 1 ( GREM1 ) gene explorations. The acquired results indicated that follicular mesenchymal cells and germ cells shared several unique gene promoter regions enriched with H3K4me3 (5,704 peaks) and also unique regions of bivalent H3K4m3/H3K27me3 shared between all cell types and germ cells (1,909 peaks). Subsequent observation of follicular mesenchyme-specific genes- CRABP1 , GDF10 , and GREM1 correctly revealed vigorous transcriptions of these genes in follicular mesenchymal cells. As expected, bivalent H3K4m3/H3K27me3 pattern was manifested in gene promoter regions of germ cells, and thus suspended their transcriptions. According the results, an example of chicken H3K4m3/H3K27me3 ChIP-seq data analysis was successfully demonstrated in this study. Hopefully, the provided methodology should hereby be useful for galline ChIP-seq data

The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid composition

PubMed Central

Singhal, N. K.; Huang, H.; Li, S.; Clements, R.; Gadd, J.; Daniels, A.; Kooijman, E. E.; Bannerman, P.; Burns, T.; Guo, F.; Pleasure, D.; Freeman, E.; Shriver, L.

2017-01-01

The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metabolism of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and high-performance thin-layer chromatography (HPTLC) to identify changes in myelin lipid composition in postmortem MS brains and in NAT8L knockout (NAT8L−/−) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amounts of sphingomyelin and sulfatide lipids in the brains of NAT8L−/− mice compared to controls. Metabolomic analysis of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metabolism, and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was associated with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination. PMID:27709268

The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid composition.

PubMed

Singhal, N K; Huang, H; Li, S; Clements, R; Gadd, J; Daniels, A; Kooijman, E E; Bannerman, P; Burns, T; Guo, F; Pleasure, D; Freeman, E; Shriver, L; McDonough, J

2017-01-01

The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metabolism of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance thin-layer chromatography (HPTLC) to identify changes in myelin lipid composition in postmortem MS brains and in NAT8L knockout (NAT8L -/- ) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amounts of sphingomyelin and sulfatide lipids in the brains of NAT8L -/- mice compared to controls. Metabolomic analysis of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metabolism, and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was associated with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination.

Molecular basis for the role of oncogenic histone mutations in modulating H3K36 methylation

DOE PAGES

Zhang, Yinglu; Shan, Chun -Min; Wang, Jiyong; ...

2017-03-03

Histone H3 lysine 36 methylation (H3K36me) is critical for epigenetic regulation and mutations at or near H3K36 are associated with distinct types of cancers. H3K36M dominantly inhibits H3K36me on wild-type histones, whereas H3G34R/V selectively affects H3K36me on the same histone tail. Here we report the crystal structures of SETD2 SET domain in complex with an H3K36M peptide and SAM or SAH. There are large conformational changes in the substrate binding regions of the SET domain, and the K36M residue interacts with the catalytic pocket of SETD2. H3G34 is surrounded by a very narrow tunnel, which excludes larger amino acid sidemore » chains. H3P38 is in the trans configuration, and the cis configuration is incompatible with SETD2 binding. Lastly, mutations of H3G34 or H3P38 alleviate the inhibitory effects of H3K36M on H3K36me, demonstrating that the stable interaction of H3K36M with SETD2 is critical for its inhibitory effects.« less

P110β Inhibition Reduces Histone H3K4 Di-Methylation in Prostate Cancer.

PubMed

Pang, Jun; Yang, Yue-Wu; Huang, Yiling; Yang, Jun; Zhang, Hao; Chen, Ruibao; Dong, Liang; Huang, Yan; Wang, Dongying; Liu, Jihong; Li, Benyi

2017-02-01

Epigenetic alteration plays a major role in the development and progression of human cancers, including prostate cancer. Histones are the key factors in modulating gene accessibility to transcription factors and post-translational modification of the histone N-terminal tail including methylation is associated with either transcriptional activation (H3K4me2) or repression (H3K9me3). Furthermore, phosphoinositide 3-kinase (PI3 K) signaling and the androgen receptor (AR) are the key determinants in prostate cancer development and progression. We recently showed that prostate-targeted nano-micelles loaded with PI3 K/p110beta specific inhibitor TGX221 blocked prostate cancer growth in vitro and in vivo. Our objective of this study was to determine the role of PI3 K signaling in histone methylation in prostate cancer, with emphasis on histone H3K4 methylation. PI3 K non-specific inhibitor LY294002 and p110beta-specific inhibitor TGX221 were used to block PI3 K/p110beta signaling. The global levels of H3K4 and H3K9 methylation in prostate cancer cells and tissue specimens were evaluated by Western blot assay and immunohistochemical staining. A synthetic androgen R1881 was used to stimulate AR activity in prostate cancer cells. A castration-resistant prostate cancer (CRPC) specific human tissue microarray (TMA) was used to assess the global levels of H3K4me2 methylation by immunostaining approach. Our data revealed that H3K4me2 levels were significantly elevated after androgen stimulation. With RNA silencing and pharmacology approaches, we further defined that inhibition of PI3 K/p110beta activity through gene-specific knocking down and small chemical inhibitor TGX221 abolished androgen-stimulated H3K4me2 methylation. Consistently, prostate cancer-targeted delivery of TGX221 in vivo dramatically reduced the global levels of H3K4me2 as assessed by immunohistochemical staining on tissue section of mouse xenografts from CRPC cell lines 22RV1 and C4-2. Finally

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Dandan; Perkins, Jordan T.; Department of Animal and Food Sciences, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY 40536

Epigenetic modifications of DNA and histones alter cellular phenotypes without changing genetic codes. Alterations of epigenetic marks can be induced by exposure to environmental pollutants and may contribute to associated disease risks. Here we test the hypothesis that endothelial cell dysfunction induced by exposure to polychlorinated biphenyls (PCBs) is mediated in part though histone modifications. In this study, human vascular endothelial cells were exposed to physiologically relevant concentrations of several PCBs congeners (e.g., PCBs 77, 118, 126 and 153) followed by quantification of inflammatory gene expression and changes of histone methylation. Only exposure to coplanar PCBs 77 and 126 inducedmore » the expression of histone H3K9 trimethyl demethylase jumonji domain-containing protein 2B (JMJD2B) and nuclear factor-kappa B (NF-κB) subunit p65, activated NF-κB signaling as evidenced by nuclear translocation of p65, and up-regulated p65 target inflammatory genes, such as interleukin (IL)-6, C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and IL-1α/β. The increased accumulation of JMJD2B in the p65 promoter led to a depletion of H3K9me3 repression mark, which accounts for the observed up-regulation of p65 and associated inflammatory genes. JMJD2B gene knockdown confirmed a critical role for this histone demethylase in mediating PCB-induced inflammation of the vascular endothelium. Finally, it was determined, via chemical inhibition, that PCB-induced up-regulation of JMJD2B was estrogen receptor-alpha (ER-α) dependent. These data suggest that coplanar PCBs may exert endothelial cell toxicity through changes in histone modifications. - Highlights: • Coplanar PCBs significantly induced histone demethylase JMJD2B expression. • Coplanar PCBs activated NF-κB through p65 up-regulation and nuclear translocation. • Histone H3K4 and K9 modifications were mediated by ER-α/JMJD2B/MLL2

Development of Multiple Cell-Based Assays for the Detection of Histone H3 Lys27 Trimethylation (H3K27me3)

PubMed Central

Lu, Lihui; Wu, Jianghong

2013-01-01

Abstract Posttranslational modification of histone proteins in eukaryotes plays an important role in gene transcription and chromatin structure. Dysregulation of the enzymes involved in histone modification has been linked to many cancer forms, making this target class a potential new area for therapeutics. A reliable assay to monitor small-molecule inhibition of various epigenetic enzymes should play a critical role in drug discovery to fight cancer. However, it has been challenging to develop cell-based assays for high-throughput screening (HTS) and compound profiling. Recently, two homogeneous cell-based assay kits using the AlphaLISA® and LanthaScreen® technologies to detect trimethyl histone H3 Lysine 27 have become commercially available, and a heterogeneous cell assay with modified dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA®) format has been reported. To compare their pros and cons, we evaluated, optimized, and validated these three assay formats in three different cell lines and compared their activities with traditional Western blot detection of histone methylation inhibition by using commercial and in-house small-molecule inhibitors. Our data indicate that, although all four formats produced acceptable results, the homogeneous AlphaLISA assay was best suited for HTS and compound profiling due to its wider window and ease of automation. The DELFIA and Western blot assays were useful as validation tools to confirm the cell activities and eliminate potential false-positive compounds. PMID:23992119

Ischemic Preconditioning Confers Epigenetic Repression of Mtor and Induction of Autophagy Through G9a-Dependent H3K9 Dimethylation.

PubMed

Gidlöf, Olof; Johnstone, Andrea L; Bader, Kerstin; Khomtchouk, Bohdan B; O'Reilly, Jiaqi J; Celik, Selvi; Van Booven, Derek J; Wahlestedt, Claes; Metzler, Bernhard; Erlinge, David

2016-12-22

Ischemic preconditioning (IPC) protects the heart from prolonged ischemic insult and reperfusion injury through a poorly understood mechanism. Post-translational modifications of histone residues can confer rapid and drastic switches in gene expression in response to various stimuli, including ischemia. The aim of this study was to investigate the effect of histone methylation in the response to cardiac ischemic preconditioning. We used cardiac biopsies from mice subjected to IPC to quantify global levels of 3 of the most well-studied histone methylation marks (H3K9me2, H3K27me3, and H3K4me3) with Western blot and found that H3K9me2 levels were significantly increased in the area at risk compared to remote myocardium. In order to assess which genes were affected by the increase in H3K9me2 levels, we performed ChIP-Seq and transcriptome profiling using microarray. Two hundred thirty-seven genes were both transcriptionally repressed and enriched in H3K9me2 in the area at risk of IPC mice. Of these, Mtor (Mechanistic target of rapamycin) was chosen for mechanistic studies. Knockdown of the major H3K9 methyltransferase G9a resulted in a significant decrease in H3K9me2 levels across Mtor, increased Mtor expression, as well as decreased autophagic activity in response to rapamycin and serum starvation. IPC confers an increase of H3K9me2 levels throughout the Mtor gene-a master regulator of cellular metabolism and a key player in the cardioprotective effect of IPC-leading to transcriptional repression via the methyltransferase G9a. The results of this study indicate that G9a has an important role in regulating cardiac autophagy and the cardioprotective effect of IPC. © 2016 The Authors and University of Miami Miller School of Medicine. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Synthesis and reactivity of bis(tetramethylcyclopentadienyl) yttrium metallocenes including the reduction of Me(3)SiN(3) to [(Me(3)Si)(2)N](-) with [(C(5)Me(4)H)(2)Y(THF)](2)(mu-eta(2):eta(2)-N(2)).

PubMed

Lorenz, Sara E; Schmiege, Benjamin M; Lee, David S; Ziller, Joseph W; Evans, William J

2010-07-19

The metallocene precursors needed to provide the tetramethylcyclopentadienyl yttrium complexes (C(5)Me(4)H)(3)Y, [(C(5)Me(4)H)(2)Y(THF)](2)(mu-eta(2):eta(2)-N(2)), and [(C(5)Me(4)H)(2)Y(mu-H)](2) for reactivity studies have been synthesized and fully characterized, and their reaction chemistry has led to an unexpected conversion of an azide to an amide. (C(5)Me(4)H)(2)Y(mu-Cl)(2)K(THF)(x), 1, synthesized from YCl(3) and KC(5)Me(4)H reacts with allylmagnesium chloride to make (C(5)Me(4)H)(2)Y(eta(3)-C(3)H(5)), 2, which is converted to [(C(5)Me(4)H)(2)Y][(mu-Ph)(2)BPh(2)], 3, with [Et(3)NH][BPh(4)]. Complex 3 reacts with KC(5)Me(4)H to form (C(5)Me(4)H)(3)Y, 4. The reduced dinitrogen complex, [(C(5)Me(4)H)(2)Y(THF)](2)(mu-eta(2):eta(2)-N(2)), 5, can be synthesized from either [(C(5)Me(4)H)(2)Y](2)[(mu-Ph)(2)BPh(2)], 3, or (C(5)Me(4)H)(3)Y, 4, with potassium graphite under a dinitrogen atmosphere. The (15)N labeled analogue, [(C(5)Me(4)H)(2)Y(THF)](2)(mu-eta(2):eta(2)-(15)N(2)), 5-(15)N, has also been prepared, and the (15)N NMR data have been compared to previously characterized reduced dinitrogen complexes. Complex 2 reacts with H(2) to form the corresponding hydride, [(C(5)Me(4)H)(2)Y(mu-H)](2), 6. Complex 5 displays similar reactivity to that of the analogous [(C(5)Me(4)H)(2)Ln(THF)](2)(mu-eta(2):eta(2)-N(2)) complexes (Ln = La, Lu), with substrates such as phenazine, anthracene, and CO(2). In addition, 5 reduces Me(3)SiN(3) to form (C(5)Me(4)H)(2)Y[N(SiMe(3))(2)], 7.

Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae

PubMed Central

Sorenson, Matthew R.; Jha, Deepak K.; Ucles, Stefanie A.; Flood, Danielle M.; Strahl, Brian D.; Stevens, Scott W.; Kress, Tracy L.

2016-01-01

ABSTRACT Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) – a residue methylated by Set2 during transcription elongation – exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast. PMID:26821844

Histone H3.3 mutations drive paediatric glioblastoma through upregulation of MYCN

PubMed Central

Bjerke, Lynn; Mackay, Alan; Nandhabalan, Meera; Burford, Anna; Jury, Alexa; Popov, Sergey; Bax, Dorine A; Carvalho, Diana; Taylor, Kathryn R; Vinci, Maria; Bajrami, Ilirjana; McGonnell, Imelda M; Lord, Christopher J; Reis, Rui M; Hargrave, Darren; Ashworth, Alan; Workman, Paul; Jones, Chris

2013-01-01

Glioblastomas of children and young adults have a median survival of only 12-15months and are clinically and biologically distinct from histologically similar cancers in older adults1. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A2, occurring either at or close to key residues marked by methylation for regulation of transcription – K27 and G34. Here we show that the cerebral hemispheric-specific G34 mutation drives a distinct expression signature through differential genomic binding of the K36 trimethylation mark (H3K36me3). The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem cell maintenance, cell fate decisions and self-renewal. Critically, H3F3A G34 mutations cause profound upregulation of MYCN, a potent oncogene which is causative of glioblastomas when expressed in the correct developmental context. This driving aberration is selectively targetable in this patient population by inhibiting kinases responsible for stabilisation of the protein. PMID:23539269

Effects of Nickel Treatment on H3K4 Trimethylation and Gene Expression

PubMed Central

Tchou-Wong, Kam-Meng; Kluz, Thomas; Arita, Adriana; Smith, Phillip R.; Brown, Stuart; Costa, Max

2011-01-01

Occupational exposure to nickel compounds has been associated with lung and nasal cancers. We have previously shown that exposure of the human lung adenocarcinoma A549 cells to NiCl2 for 24 hr significantly increased global levels of trimethylated H3K4 (H3K4me3), a transcriptional activating mark that maps to the promoters of transcribed genes. To further understand the potential epigenetic mechanism(s) underlying nickel carcinogenesis, we performed genome-wide mapping of H3K4me3 by chromatin immunoprecipitation and direct genome sequencing (ChIP-seq) and correlated with transcriptome genome-wide mapping of RNA transcripts by massive parallel sequencing of cDNA (RNA-seq). The effect of NiCl2 treatment on H3K4me3 peaks within 5,000 bp of transcription start sites (TSSs) on a set of genes highly induced by nickel in both A549 cells and human peripheral blood mononuclear cells were analyzed. Nickel exposure increased the level of H3K4 trimethylation in both the promoters and coding regions of several genes including CA9 and NDRG1 that were increased in expression in A549 cells. We have also compared the extent of the H3K4 trimethylation in the absence and presence of formaldehyde crosslinking and observed that crosslinking of chromatin was required to observe H3K4 trimethylation in the coding regions immediately downstream of TSSs of some nickel-induced genes including ADM and IGFBP3. This is the first genome-wide mapping of trimethylated H3K4 in the promoter and coding regions of genes induced after exposure to NiCl2. This study may provide insights into the epigenetic mechanism(s) underlying the carcinogenicity of nickel compounds. PMID:21455298