Tabela com principais espec.
|Species Reactivity||Key Applications|
|H, M||WB, ChIP|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||1 year at -20°C from date of shipment|
|Material Size||25 assays|
|Material Package||25 assays per kit, ~4μL per chromatin immunoprecipitation.|
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|A gene expression signature identifying transient DNMT1 depletion as a causal factor of cancer-germline gene activation in melanoma. |
Cannuyer, J; Van Tongelen, A; Loriot, A; De Smet, C
Clinical epigenetics 7 114 2015
Many human tumors show aberrant activation of a group of germline-specific genes, termed cancer-germline (CG) genes, several of which appear to exert oncogenic functions. Although activation of CG genes in tumors has been linked to promoter DNA demethylation, the mechanisms underlying this epigenetic alteration remain unclear. Two main processes have been proposed: awaking of a gametogenic program directing demethylation of target DNA sequences via specific regulators, or general deficiency of DNA methylation activities resulting from mis-targeting or down-regulation of the DNMT1 methyltransferase.By the analysis of transcriptomic data, we searched to identify gene expression changes associated with CG gene activation in melanoma cells. We found no evidence linking CG gene activation with differential expression of gametogenic regulators. Instead, CG gene activation correlated with decreased expression of a set of mitosis/division-related genes (ICCG genes). Interestingly, a similar gene expression signature was previously associated with depletion of DNMT1. Consistently, analysis of a large set of melanoma tissues revealed that DNMT1 expression levels were often lower in samples showing activation of multiple CG genes. Moreover, by using immortalized melanocytes and fibroblasts carrying an inducible anti-DNMT1 small hairpin RNA (shRNA), we demonstrate that transient depletion of DNMT1 can lead to long-term activation of CG genes and repression of ICCG genes at the same time. For one of the ICCG genes (CDCA7L), we found that its down-regulation in melanoma cells was associated with deposition of repressive chromatin marks, including H3K27me3.Together, our observations point towards transient DNMT1 depletion as a causal factor of CG gene activation in vivo in melanoma.
|Brg1 modulates enhancer activation in mesoderm lineage commitment. |
Alexander, JM; Hota, SK; He, D; Thomas, S; Ho, L; Pennacchio, LA; Bruneau, BG
Development (Cambridge, England) 142 1418-30 2015
The interplay between different levels of gene regulation in modulating developmental transcriptional programs, such as histone modifications and chromatin remodeling, is not well understood. Here, we show that the chromatin remodeling factor Brg1 is required for enhancer activation in mesoderm induction. In an embryonic stem cell-based directed differentiation assay, the absence of Brg1 results in a failure of cardiomyocyte differentiation and broad deregulation of lineage-specific gene expression during mesoderm induction. We find that Brg1 co-localizes with H3K27ac at distal enhancers and is required for robust H3K27 acetylation at distal enhancers that are activated during mesoderm induction. Brg1 is also required to maintain Polycomb-mediated repression of non-mesodermal developmental regulators, suggesting cooperativity between Brg1 and Polycomb complexes. Thus, Brg1 is essential for modulating active and repressive chromatin states during mesoderm lineage commitment, in particular the activation of developmentally important enhancers. These findings demonstrate interplay between chromatin remodeling complexes and histone modifications that, together, ensure robust and broad gene regulation during crucial lineage commitment decisions.
|D-2-hydroxyglutarate is essential for maintaining oncogenic property of mutant IDH-containing cancer cells but dispensable for cell growth. |
Ma, S; Jiang, B; Deng, W; Gu, ZK; Wu, FZ; Li, T; Xia, Y; Yang, H; Ye, D; Xiong, Y; Guan, KL
Oncotarget 6 8606-20 2015
Cancer-associated isocitrate dehydrogenase (IDH) 1 and 2 mutations gain a new activity of reducing α-KG to produce D-2-hydroxyglutarate (D-2-HG), which is proposed to function as an oncometabolite by inhibiting α-KG dependent dioxygenases. We investigated the function of D-2-HG in tumorigenesis using IDH1 and IDH2 mutant cancer cell lines. Inhibition of D-2-HG production either by specific deletion of the mutant IDH1-R132C allele or overexpression of D-2-hydroxyglutarate dehydrogenase (D2HGDH) increases α-KG and related metabolites, restores the activity of some α-KG-dependent dioxygenases, and selectively alters gene expression. Ablation of D-2-HG production has no significant effect on cell proliferation and migration, but strongly inhibits anchorage independent growth in vitro and tumor growth in xenografted mouse models. Our study identifies a new activity of oncometabolite D-2-HG in promoting tumorigenesis.
|Epigenetics of Notch1 regulation in pulmonary microvascular rarefaction following extrauterine growth restriction. |
Tang, LL; Zhang, LY; Lao, LJ; Hu, QY; Gu, WZ; Fu, LC; Du, LZ
Respiratory research 16 66 2015
Extrauterine growth restriction (EUGR) plays an important role in the developmental origin of adult cardiovascular diseases. In an EUGR rat model, we reported an elevated pulmonary arterial pressure in adults and genome-wide epigenetic modifications in pulmonary vascular endothelial cells (PVECs). However, the underlying mechanism of the early nutritional insult that results in pulmonary vascular consequences later in life remains unclear.A rat model was used to investigate the physiological and structural effect of EUGR on early pulmonary vasculature by evaluating right ventricular systolic pressure and pulmonary vascular density in male rats. Epigenetic modifications of the Notch1 gene in PVECs were evaluated.EUGR decreased pulmonary vascular density with no significant impact on right ventricular systolic pressure at 3 weeks. Decreased transcription of Notch1 was observed both at 3 and 9 weeks, in association with decreased downstream target gene, Hes-1. Chromatin immunoprecipitation and bisulfite sequencing were performed to analyze the epigenetic modifications of the Notch1 gene promoter in PVECs. EUGR caused a significantly increased H3K27me3 in the proximal Notch1 gene promoter, and increased methylation of single CpG sites in the distal Notch1 gene promoter, both at 3 and 9 weeks.We conclude that EUGR results in decreased pulmonary vascular growth in association with decreased Notch1 in PVECs. This may be mediated by increased CpG methylation and H3K27me3 in the Notch1 gene promoter region.
|IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells. |
Wan, CK; Li, P; Spolski, R; Oh, J; Andraski, AB; Du, N; Yu, ZX; Dillon, CP; Green, DR; Leonard, WJ
Nature communications 6 7988 2015
The canonical pathway for IL-1β production requires TLR-mediated NF-κB-dependent Il1b gene induction, followed by caspase-containing inflammasome-mediated processing of pro-IL-1β. Here we show that IL-21 unexpectedly induces IL-1β production in conventional dendritic cells (cDCs) via a STAT3-dependent but NF-κB-independent pathway. IL-21 does not induce Il1b expression in CD4(+) T cells, with differential histone marks present in these cells versus cDCs. IL-21-induced IL-1β processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s). Moreover, STAT3-dependent IL-1β expression in cDCs at least partially explains the IL-21-mediated pathologic response occurring during infection with pneumonia virus of mice. These results demonstrate lineage-restricted IL-21-induced IL-1β via a non-canonical pathway and provide evidence for its importance in vivo.
|Epigenetic memory gained by priming with osteogenic induction medium improves osteogenesis and other properties of mesenchymal stem cells. |
Rui, Y; Xu, L; Chen, R; Zhang, T; Lin, S; Hou, Y; Liu, Y; Meng, F; Liu, Z; Ni, M; Tsang, KS; Yang, F; Wang, C; Chan, HC; Jiang, X; Li, G
Scientific reports 5 11056 2015
Mesenchymal stem cells (MSCs) are highly plastic cells that are able to transdifferentiate or dedifferentiate under appropriate conditions. In the present study, we reported here that after in vitro induction of osteogenic differentiation, MSCs could be reverted to a primitive stem cell population (dedifferentiated osteogenic MSCs, De-Os-MSCs) with improved cell survival, colony formation, osteogenic potential, migratory capacity and increased expression of Nanog, Oct4 and Sox2. Most importantly, our results showed great superiority of the De-Os-MSCs over untreated MSCs in ectopic bone formation in vivo. Furthermore, Nanog-knockdown in MSCs could reverse these enhanced properties in De-Os-MSCs in vitro, indicating a central role of Nanog in the transcriptional network. In addition, epigenetic regulations including DNA methylation and histone modifications may play important roles in regulating the de-osteogenic differentiation process. And we found decreased methylation and promoter accrual of activating histone marks, such as H3K4me3 and H4ac on both Nanog and Oct4 gene promoters. Taken together, our study demonstrated that epigenetic memory in De-Os-MSCs gained by priming with osteogenic induction medium favored their differentiation along osteoblastic lineage with improved cell survival and migratory abilities, which may have application potential in enhancing their regenerative capacity in mammals.
|Histone H3 Lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation. |
Manna, S; Kim, JK; Baugé, C; Cam, M; Zhao, Y; Shetty, J; Vacchio, MS; Castro, E; Tran, B; Tessarollo, L; Bosselut, R
Nature communications 6 8152 2015
Although histone H3 lysine 27 trimethylation (H3K27Me3) is associated with gene silencing, whether H3K27Me3 demethylation affects transcription and cell differentiation in vivo has remained elusive. To investigate this, we conditionally inactivated the two H3K27Me3 demethylases, Jmjd3 and Utx, in non-dividing intrathymic CD4(+) T-cell precursors. Here we show that both enzymes redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Thymocyte expression of S1pr1 was not rescued in Jmjd3- and Utx-deficient male mice, which carry the catalytically inactive Utx homolog Uty, supporting the conclusion that it requires H3K27Me3 demethylase activity. These findings demonstrate that Jmjd3 and Utx are required for T-cell development, and point to a requirement for their H3K27Me3 demethylase activity in cell differentiation.
|In Ovo injection of betaine affects hepatic cholesterol metabolism through epigenetic gene regulation in newly hatched chicks. |
Hu, Y; Sun, Q; Li, X; Wang, M; Cai, D; Li, X; Zhao, R
PloS one 10 e0122643 2015
Betaine is reported to regulate hepatic cholesterol metabolism in mammals. Chicken eggs contain considerable amount of betaine, yet it remains unknown whether and how betaine in the egg affects hepatic cholesterol metabolism in chicks. In this study, eggs were injected with betaine at 2.5 mg/egg and the hepatic cholesterol metabolism was investigated in newly hatched chicks. Betaine did not affect body weight or liver weight, but significantly increased the serum concentration (P less than 0.05) and the hepatic content (P less than 0.01) of cholesterol. Accordingly, the cholesterol biosynthetic enzyme HMGCR was up-regulated (P less than 0.05 for both mRNA and protein), while CYP7A1 which converts cholesterol to bile acids was down-regulated (P less than 0.05 for mRNA and P = 0.07 for protein). Moreover, hepatic protein content of the sterol-regulatory element binding protein 1 which regulates cholesterol and lipid biosynthesis, and the mRNA abundance of ATP binding cassette sub-family A member 1 (ABCA1) which mediates cholesterol counter transport were significantly (P less than 0.05) increased in betaine-treated chicks. Meanwhile, hepatic protein contents of DNA methyltransferases 1 and adenosylhomocysteinase-like 1 were increased (P less than 0.05), which was associated with global genomic DNA hypermethylation (P less than 0.05) and diminished gene repression mark histone H3 lysine 27 trimethylation (P less than 0.05). Furthermore, CpG methylation level on gene promoters was found to be increased (P less than 0.05) for CYP7A1 yet decreased (P less than 0.05) for ABCA1. These results indicate that in ovo betaine injection regulates hepatic cholesterol metabolism in chicks through epigenetic mechanisms including DNA and histone methylations.
|Acute ethanol alters multiple histone modifications at model gene promoters in the cerebral cortex. |
Finegersh, A; Homanics, GE
Alcoholism, clinical and experimental research 38 1865-73 2014
Ethanol (EtOH) exposure alters gene expression in the cerebral cortex (CCx); however, mechanisms of EtOH-induced gene regulation are not well understood. We hypothesized that EtOH regulates gene expression by differentially altering histone modifications at gene promoters that are up- and down-regulated by EtOH. Such epigenetic mechanisms may ultimately contribute to EtOH-induced neuro-adaptations that underlie tolerance, dependence, and EtOH-use disorders.Eight-week-old, male C57BL/6J mice were treated with 3 g/kg EtOH (intraperitoneally) or saline and sacrificed 6 hours after injection; the CCx and hippocampus (HC) were immediately removed and flash frozen. Chromatin immunoprecipitation was used to study the association of model gene promoters with histone modifications. Western blot was used to detect global changes in the histone modifications studied. We also used a polymerase chain reaction (PCR) array to identify changes in expression of chromatin-modifying enzymes.In CCx, acute EtOH decreased expression of Gad1, Hdac2, and Hdac11, which was associated with decreased histone acetylation at the Gad1 and Hdac2 promoters; we also identified increased expression of Mt1, Mt2, Egr1, which was associated with increased H3K4me3 levels at the Mt2 promoter and decreased H3K27me3 levels at the Mt1 promoter. We identified an increase in global levels of H3K4me3 in CCx as well as a global increase in H3K9ac and H3K14ac in HC. The PCR array identified decreased expression of Csrp2 bp, Hdac2, and Hdac11 as well as increased expression of Kat2b in CCx.Acute EtOH induces chromatin remodeling at model up- and down-regulated genes in CCx. Different patterns of histone modifications at these gene promoters indicate that EtOH may be acting through multiple histone-modifying enzymes to alter gene expression; in particular, differential expression of Kat2b, Hdac2, Hdac11, and Csrp2 bp in CCx may mediate EtOH-induced chromatin remodeling. Additional studies are necessary to determine the relationship between EtOH-induced changes in histone-modifying enzymes, specific EtOH-induced histone modifications, and gene expression.
|Loss of the polycomb mark from bivalent promoters leads to activation of cancer-promoting genes in colorectal tumors. |
Hahn, MA; Li, AX; Wu, X; Yang, R; Drew, DA; Rosenberg, DW; Pfeifer, GP
Cancer research 74 3617-29 2014
In colon tumors, the transcription of many genes becomes deregulated by poorly defined epigenetic mechanisms that have been studied mainly in established cell lines. In this study, we used frozen human colon tissues to analyze patterns of histone modification and DNA cytosine methylation in cancer and matched normal mucosa specimens. DNA methylation is strongly targeted to bivalent H3K4me3- and H3K27me3-associated promoters, which lose both histone marks and acquire DNA methylation. However, we found that loss of the Polycomb mark H3K27me3 from bivalent promoters was accompanied often by activation of genes associated with cancer progression, including numerous stem cell regulators, oncogenes, and proliferation-associated genes. Indeed, we found many of these same genes were also activated in patients with ulcerative colitis where chronic inflammation predisposes them to colon cancer. Based on our findings, we propose that a loss of Polycomb repression at bivalent genes combined with an ensuing selection for tumor-driving events plays a major role in cancer progression.
|Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci. |
Noordermeer, D; Leleu, M; Schorderet, P; Joye, E; Chabaud, F; Duboule, D
eLife 3 e02557 2014
Hox genes are essential regulators of embryonic development. Their step-wise transcriptional activation follows their genomic topology and the various states of activation are subsequently memorized into domains of progressively overlapping gene products. We have analyzed the 3D chromatin organization of Hox clusters during their early activation in vivo, using high-resolution circular chromosome conformation capture. Initially, Hox clusters are organized as single chromatin compartments containing all genes and bivalent chromatin marks. Transcriptional activation is associated with a dynamic bi-modal 3D organization, whereby the genes switch autonomously from an inactive to an active compartment. These local 3D dynamics occur within a framework of constitutive interactions within the surrounding Topological Associated Domains, indicating that this regulation process is mostly cluster intrinsic. The step-wise progression in time is fixed at various body levels and thus can account for the chromatin architectures previously described at a later stage for different anterior to posterior levels.DOI: http://dx.doi.org/10.7554/eLife.02557.001.
|Polycomb repressive complex 2 and H3K27me3 cooperate with H3K9 methylation to maintain heterochromatin protein 1α at chromatin. |
Boros, J; Arnoult, N; Stroobant, V; Collet, JF; Decottignies, A
Molecular and cellular biology 34 3662-74 2014
Methylation of histone H3 on lysine 9 or 27 is crucial for heterochromatin formation. Previously considered hallmarks of, respectively, constitutive and facultative heterochromatin, recent evidence has accumulated in favor of coexistence of these two marks and their cooperation in gene silencing maintenance. H3K9me2/3 ensures anchorage at chromatin of heterochromatin protein 1α (HP1α), a main component of heterochromatin. HP1α chromoshadow domain, involved in dimerization and interaction with partners, has additional but still unclear roles in HP1α recruitment to chromatin. Because of previously suggested links between polycomb repressive complex 2 (PRC2), which catalyzes H3K27 methylation, and HP1α, we tested whether PRC2 may regulate HP1α abundance at chromatin. We found that the EZH2 and SUZ12 subunits of PRC2 are required for HP1α stability, as knockdown of either protein led to HP1α degradation. Similar results were obtained upon overexpression of H3K27me2/3 demethylases. We further showed that binding of HP1α/β/γ to H3K9me3 peptides is greatly increased in the presence of H3K27me3, and this is dependent on PRC2. These data fit with recent proteomic studies identifying PRC2 as an indirect H3K9me3 binder in mouse tissues and suggest the existence of a cooperative mechanism of HP1α anchorage at chromatin involving H3 methylation on both K9 and K27 residues.
|Diffuse large B-cell lymphoma with combined TP53 mutation and MIR34A methylation: Another "double hit" lymphoma with very poor outcome? |
Asmar, F; Hother, C; Kulosman, G; Treppendahl, MB; Nielsen, HM; Ralfkiaer, U; Pedersen, A; Møller, MB; Ralfkiaer, E; de Nully Brown, P; Grønbæk, K
Oncotarget 5 1912-25 2014
MiR34A, B and C have been implicated in lymphomagenesis, but information on their role in normal CD19+ B-cells (PBL-B) and de novo diffuse large B-cell lymphoma (DLBCL) is limited. We show that in normal and activated B-cells miR34A-5p plays a dominant role compared to other miR34 family members. Only miR34A-5p is expressed in PBL-B, and significantly induced in activated B-cells and reactive lymph nodes. In PBL-B, the MIR34A and MIR34B/C promoters are unmethylated, but the latter shows enrichment for the H3K4me3/H3K27me3 silencing mark. Nine de novo DLBCL cases (n=150) carry both TP53 mutation and MIR34A methylation ("double hit") and these patients have an exceedingly poor prognosis with a median survival of 9.4 months (Pless than 0.0001), while neither TP53 mutation, MIR34A or MIR34B/C promoter methylation alone ("single hit") influence on survival. The TP53/MIR34A "double-hit" is an independent negative prognostic factor for survival (P=0.0002). In 2 DLBCL-cell lines with both TP53 mutation and promoter methylation of MIR34A, miR34A-5p is upregulated by 5-aza-2'deoxycytidine. Thus, the TP53/MIR34A "double hit" characterizes a very aggressive subgroup of DLBCL, which may be treatable with epigenetic therapy prior to or in combination with conventional immunochemotherapy.
|Altered nucleosome positioning at the transcription start site and deficient transcriptional initiation in Friedreich ataxia. |
Chutake, YK; Costello, WN; Lam, C; Bidichandani, SI
The Journal of biological chemistry 289 15194-202 2014
Most individuals with Friedreich ataxia (FRDA) are homozygous for an expanded GAA triplet repeat (GAA-TR) mutation in intron 1 of the FXN gene, which results in deficiency of FXN transcript. Consistent with the expanded GAA-TR sequence as a cause of variegated gene silencing, evidence for heterochromatin has been detected in intron 1 in the immediate vicinity of the expanded GAA-TR mutation in FRDA. Transcriptional deficiency in FRDA is thought to result from deficient elongation through the expanded GAA-TR sequence because of repeat-proximal heterochromatin and abnormal DNA structures adopted by the expanded repeat. There is also evidence for deficient transcriptional initiation in FRDA, but its relationship to the expanded GAA-TR mutation remains unclear. We show that repressive chromatin extends from the expanded GAA-TR in intron 1 to the upstream regions of the FXN gene, involving the FXN transcriptional start site. Using a chromatin accessibility assay and a high-resolution nucleosome occupancy assay, we found that the major FXN transcriptional start site, which is normally in a nucleosome-depleted region, is rendered inaccessible by altered nucleosome positioning in FRDA. Consistent with the altered epigenetic landscape the FXN gene promoter, a typical CpG island promoter, was found to be in a transcriptionally non-permissive state in FRDA. Both metabolic labeling of nascent transcripts and an unbiased whole transcriptome analysis revealed a severe deficiency of transcriptional initiation in FRDA. Deficient transcriptional initiation, and not elongation, is the major cause of FXN transcriptional deficiency in FRDA, and it is related to the spread of repressive chromatin from the expanded GAA-TR mutation.
|PIAS1 regulates breast tumorigenesis through selective epigenetic gene silencing. |
Liu, B; Tahk, S; Yee, KM; Yang, R; Yang, Y; Mackie, R; Hsu, C; Chernishof, V; O'Brien, N; Jin, Y; Fan, G; Lane, TF; Rao, J; Slamon, D; Shuai, K
PloS one 9 e89464 2014
Epigenetic gene silencing by histone modifications and DNA methylation is essential for cancer development. The molecular mechanism that promotes selective epigenetic changes during tumorigenesis is not understood. We report here that the PIAS1 SUMO ligase is involved in the progression of breast tumorigenesis. Elevated PIAS1 expression was observed in breast tumor samples. PIAS1 knockdown in breast cancer cells reduced the subpopulation of tumor-initiating cells, and inhibited breast tumor growth in vivo. PIAS1 acts by delineating histone modifications and DNA methylation to silence the expression of a subset of clinically relevant genes, including breast cancer DNA methylation signature genes such as cyclin D2 and estrogen receptor, and breast tumor suppressor WNT5A. Our studies identify a novel epigenetic mechanism that regulates breast tumorigenesis through selective gene silencing.
|Different epigenetic alterations are associated with abnormal IGF2/Igf2 upregulation in neural tube defects. |
Bai, B; Zhang, Q; Liu, X; Miao, C; Shangguan, S; Bao, Y; Guo, J; Wang, L; Zhang, T; Li, H
PloS one 9 e113308 2014
The methylation status of DNA methylation regions (DMRs) of the imprinted gene IGF2/Igf2 is associated with neural tube defects (NTDs), which are caused by a failure of the neural tube to fold and close and are the second-most common birth defect; however, the characterization of the expression level of IGF2/Igf2 in neural tissue from human fetuses affected with NTDs remains elusive. More importantly, whether abnormal chromatin structure also influences IGF2/Igf2 expression in NTDs is unclear. Here, we investigated the transcriptional activity of IGF2/Igf2 in normal and NTD spinal cord tissues, the methylation status of different DMRs, and the chromatin structure of the promoter. Our data indicated that in NTD samples from both human fetuses and retinoic acid (RA)-treated mouse fetuses, the expression level of IGF2/Igf2 was upregulated 6.41-fold and 1.84-fold, respectively, compared to controls. H19 DMR1, but not IGF2 DMR0, was hypermethylated in human NTD samples. In NTD mice, h19 DMR1 was stable, whereas the chromatin structure around the promoter of Igf2 might be loosened, which was displayed by higher H3K4 acetylation and lower H3K27 trimethylation. Therefore, the data revealed that IGF2/Igf2 expression can be ectopically up-regulated by dual epigenetic factors in NTDs. In detail, the upregulation of IGF2/Igf2 is likely controlled by hypermethylation of H19 DMR1 in human NTDs, however, in acute external RA-induced NTD mice it is potentially determined by more open chromatin structure.
|A genetic approach to the recruitment of PRC2 at the HoxD locus. |
Schorderet, P; Lonfat, N; Darbellay, F; Tschopp, P; Gitto, S; Soshnikova, N; Duboule, D
PLoS genetics 9 e1003951 2013
Polycomb group (PcG) proteins are essential for the repression of key factors during early development. In Drosophila, the polycomb repressive complexes (PRC) associate with defined polycomb response DNA elements (PREs). In mammals, however, the mechanisms underlying polycomb recruitment at targeted loci are poorly understood. We have used an in vivo approach to identify DNA sequences of importance for the proper recruitment of polycomb proteins at the HoxD locus. We report that various genomic re-arrangements of the gene cluster do not strongly affect PRC2 recruitment and that relatively small polycomb interacting sequences appear necessary and sufficient to confer polycomb recognition and targeting to ectopic loci. In addition, a high GC content, while not sufficient to recruit PRC2, may help its local spreading. We discuss the importance of PRC2 recruitment over Hox gene clusters in embryonic stem cells, for their subsequent coordinated transcriptional activation during development.
|HDAC inhibitor confers radiosensitivity to prostate stem-like cells. |
Frame, FM; Pellacani, D; Collins, AT; Simms, MS; Mann, VM; Jones, GD; Meuth, M; Bristow, RG; Maitland, NJ
British journal of cancer 109 3023-33 2013
Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction.Primary cultures were derived from patients undergoing resection for prostate cancer or benign prostatic hyperplasia. After short-term culture, three populations of cells were sorted, reflecting the prostate epithelial hierarchy, namely stem-like cells (SCs, α2β1integrin(hi)/CD133(+)), transit-amplifying (TA, α2β1integrin(hi)/CD133(-)) and committed basal (CB, α2β1integrin(lo)) cells. Radiosensitivity was measured by colony-forming efficiency (CFE) and DNA damage by comet assay and DNA damage foci quantification. Immunofluorescence and flow cytometry were used to measure heterochromatin. The HDAC (histone deacetylase) inhibitor Trichostatin A was used as a radiosensitiser.Stem-like cells had increased CFE post irradiation compared with the more differentiated cells (TA and CB). The SC population sustained fewer lethal double-strand breaks than either TA or CB cells, which correlated with SCs being less proliferative and having increased levels of heterochromatin. Finally, treatment with an HDAC inhibitor sensitised the SCs to radiation.Prostate SCs are more radioresistant than more differentiated cell populations. We suggest that the primitive cells survive radiation therapy and that pre-treatment with HDAC inhibitors may sensitise this resistant fraction.
|A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells. |
Johnsson, P; Ackley, A; Vidarsdottir, L; Lui, WO; Corcoran, M; Grandér, D; Morris, KV
Nature structural & molecular biology 20 440-6 2013
PTEN is a tumor-suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1-encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, α and β. The α isoform functions in trans, localizes to the PTEN promoter and epigenetically modulates PTEN transcription by the recruitment of DNA methyltransferase 3a and Enhancer of Zeste. In contrast, the β isoform interacts with PTENpg1 through an RNA-RNA pairing interaction, which affects PTEN protein output through changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell-cycle arrest and sensitizes cells to doxorubicin, which suggests a biological function for the respective PTENpg1 expressed asRNAs.
|Epigenetic hierarchy within the MAGEA1 cancer-germline gene: promoter DNA methylation dictates local histone modifications. |
Cannuyer, J; Loriot, A; Parvizi, GK; De Smet, C
PloS one 8 e58743 2013
Gene MAGEA1 belongs to a group of human germline-specific genes that rely on DNA methylation for repression in somatic tissues. Many of these genes, termed cancer-germline (CG) genes, become demethylated and activated in a wide variety of tumors, where they encode tumor-specific antigens. The process leading to DNA demethylation of CG genes in tumors remains unclear. Previous data suggested that histone acetylation might be involved. Here, we investigated the relative contribution of DNA methylation and histone acetylation in the epigenetic regulation of gene MAGEA1. We show that MAGEA1 DNA hypomethylation in expressing melanoma cells is indeed correlated with local increases in histone H3 acetylation (H3ac). However, when MAGEA1-negative cells were exposed to a histone deacetylase inhibitor (TSA), we observed only short-term activation of the gene and detected no demethylation of its promoter. As a more sensitive assay, we used a cell clone harboring a methylated MAGEA1/hph construct, which confers resistance to hygromycin upon stable re-activation. TSA induced only transient de-repression of the transgene, and did not lead to the emergence of hygromycin-resistant cells. In striking contrast, transient depletion of DNA-methyltransferase-1 in the reporter cell clone gave rise to a hygromycin-resistant population, in which the re-activated MAGEA1/hph transgene displayed not only marked DNA hypomethylation, but also significant reversal of histone marks, including gains in H3ac and H3K4me2, and losses of H3K9me2. Collectively, our results indicate that DNA methylation has a dominant role in the epigenetic hierarchy governing MAGEA1 expression.
|Alcohol-induced epigenetic alterations to developmentally crucial genes regulating neural stemness and differentiation. |
Veazey, KJ; Carnahan, MN; Muller, D; Miranda, RC; Golding, MC
Alcoholism, clinical and experimental research 37 1111-22 2013
From studies using a diverse range of model organisms, we now acknowledge that epigenetic changes to chromatin structure provide a plausible link between environmental teratogens and alterations in gene expression leading to disease. Observations from a number of independent laboratories indicate that ethanol (EtOH) has the capacity to act as a powerful epigenetic disruptor and potentially derail the coordinated processes of cellular differentiation. In this study, we sought to examine whether primary neurospheres cultured under conditions maintaining stemness were susceptible to alcohol-induced alterations in the histone code. We focused our studies on trimethylated histone 3 lysine 4 and trimethylated histone 3 lysine 27, as these are 2 of the most prominent posttranslational histone modifications regulating stem cell maintenance and neural differentiation.Primary neurosphere cultures were maintained under conditions promoting the stem cell state and treated with EtOH for 5 days. Control and EtOH-treated cellular extracts were examined using a combination of quantitative RT-PCR and chromatin immunoprecipitation techniques.We find that the regulatory regions of genes controlling both neural precursor cell identity and processes of differentiation exhibited significant declines in the enrichment of the chromatin marks examined. Despite these widespread changes in chromatin structure, only a small subset of genes including Dlx2, Fabp7, Nestin, Olig2, and Pax6 displayed EtOH-induced alterations in transcription. Unexpectedly, the majority of chromatin-modifying enzymes examined including members of the Polycomb Repressive Complex displayed minimal changes in expression and localization. Only transcripts encoding Dnmt1, Uhrf1, Ehmt1, Ash2 l, Wdr5, and Kdm1b exhibited significant differences.Our results indicate that primary neurospheres maintained as stem cells in vitro are susceptible to alcohol-induced perturbation of the histone code and errors in the epigenetic program. These observations indicate that alterations to chromatin structure may represent a crucial component of alcohol teratogenesis and progress toward a better understanding of the developmental origins of fetal alcohol spectrum disorders.
|Chromatin remodeling of human subtelomeres and TERRA promoters upon cellular senescence: commonalities and differences between chromosomes. |
Thijssen, PE; Tobi, EW; Balog, J; Schouten, SG; Kremer, D; El Bouazzaoui, F; Henneman, P; Putter, H; Eline Slagboom, P; Heijmans, BT; van der Maarel, SM
Epigenetics 8 512-21 2013
Subtelomeres are patchworks of evolutionary conserved sequence blocks and harbor the transcriptional start sites for telomere repeat containing RNAs (TERRA). Recent studies suggest that the interplay between telomeres and subtelomeric chromatin is required for maintaining telomere function. To further characterize chromatin remodeling of subtelomeres in relation to telomere shortening and cellular senescence, we systematically quantified histone modifications and DNA methylation at the subtelomeres of chromosomes 7q and 11q in primary human WI-38 fibroblasts. Upon senescence, both subtelomeres were characterized by a decrease in markers of constitutive heterochromatin, suggesting relative chromatin relaxation. However, we did not find increased levels of markers of euchromatin or derepression of the 7q VIPR2 gene. The repressed state of the subtelomeres was maintained upon senescence, which could be attributed to a rise in levels of facultative heterochromatin markers at both subtelomeres. While senescence-induced subtelomeric chromatin remodeling was similar for both chromosomes, chromatin remodeling at TERRA promoters displayed chromosome-specific patterns. At the 7q TERRA promoter, chromatin structure was co-regulated with the more proximal subtelomere. In contrast, the 11q TERRA promoter, which was previously shown to be bound by CCCTC-binding factor CTCF, displayed lower levels of markers of constitutive heterochromatin that did not change upon senescence, whereas levels of markers of facultative heterochromatin decreased upon senescence. In line with the chromatin state data, transcription of 11q TERRA but not 7q TERRA was detected. Our study provides a detailed description of human subtelomeric chromatin dynamics and shows distinct regulation of the TERRA promoters of 7q and 11q upon cellular senescence.
|Epigenetic expansion of VHL-HIF signal output drives multiorgan metastasis in renal cancer. |
Vanharanta, S; Shu, W; Brenet, F; Hakimi, AA; Heguy, A; Viale, A; Reuter, VE; Hsieh, JJ; Scandura, JM; Massagué, J
Nature medicine 19 50-6 2013
Inactivation of the von Hippel-Lindau tumor suppressor gene, VHL, is an archetypical tumor-initiating event in clear cell renal carcinoma (ccRCC) that leads to the activation of hypoxia-inducible transcription factors (HIFs). However, VHL mutation status in ccRCC is not correlated with clinical outcome. Here we show that during ccRCC progression, cancer cells exploit diverse epigenetic alterations to empower a branch of the VHL-HIF pathway for metastasis, and the strength of this activation is associated with poor clinical outcome. By analyzing metastatic subpopulations of VHL-deficient ccRCC cells, we discovered an epigenetically altered VHL-HIF response that is specific to metastatic ccRCC. Focusing on the two most prominent pro-metastatic VHL-HIF target genes, we show that loss of Polycomb repressive complex 2 (PRC2)-dependent histone H3 Lys27 trimethylation (H3K27me3) activates HIF-driven chemokine (C-X-C motif) receptor 4 (CXCR4) expression in support of chemotactic cell invasion, whereas loss of DNA methylation enables HIF-driven cytohesin 1 interacting protein (CYTIP) expression to protect cancer cells from death cytokine signals. Thus, metastasis in ccRCC is based on an epigenetically expanded output of the tumor-initiating pathway.
|Polycomb repressive complex 2 (PRC2) protein ESC regulates insect developmental timing by mediating H3K27me3 and activating prothoracicotropic hormone gene expression. |
Lu, YX; Denlinger, DL; Xu, WH
The Journal of biological chemistry 288 23554-64 2013
The decision made by insects to develop into adults or halt development (enter diapause and prolong lifespan) is commonly based on environmental signals that provide reliable predictors of future seasons of adversity. For example, the short day lengths of early autumn accurately foretell the advent of winter, but little is known about the molecular mechanisms that preside over the hormonal events dictating whether the insect proceeds with development or enters diapause. In Helicoverpa armigera we show that day length affects H3K27me3 by affecting polycomb repressive complex 2 (PRC2) protein extra sex comb (ESC) and regulates the prothoracicotropic hormone (PTTH) gene, thus directly influencing developmental timing. ESC expression in brains of developing (nondiapause) pupae is higher than in brains from diapausing pupae. High ESC expression is localized in two pairs of PTTH neurosecretory cells, and H3K27me3 recruits on the PTTH promoter. Double strand ESC and PRC2 inhibitor (DzNep) treatment in vitro show that ESC triggers PTTH promoter activity, which in turn depends on PRC2 methyltransferase activity. Injection of DzNep into pupae programmed for development reduces the H3K27me3 mark and PTTH gene expression, thereby delaying development. Although ESC is best known as a transcriptional repressor, our results show that ESC prompts development and metamorphosis. We believe this is the first report showing that the PRC2 complex functions as an activator and that a low level of H3K27me3 can prolong lifespan (i.e. induce diapause) by controlling PTTH gene expression in insects.
|Transgene- and locus-dependent imprinting reveals allele-specific chromosome conformations. |
Lonfat, N; Montavon, T; Jebb, D; Tschopp, P; Nguyen Huynh, TH; Zakany, J; Duboule, D
Proceedings of the National Academy of Sciences of the United States of America 110 11946-51 2013
When positioned into the integrin α-6 gene, an Hoxd9lacZ reporter transgene displayed parental imprinting in mouse embryos. While the expression from the paternal allele was comparable with patterns seen for the same transgene when present at the neighboring HoxD locus, almost no signal was scored at this integration site when the transgene was inherited from the mother, although the Itga6 locus itself is not imprinted. The transgene exhibited maternal allele-specific DNA hypermethylation acquired during oogenesis, and its expression silencing was reversible on passage through the male germ line. Histone modifications also corresponded to profiles described at known imprinted loci. Chromosome conformation analyses revealed distinct chromatin microarchitectures, with a more compact structure characterizing the maternally inherited repressed allele. Such genetic analyses of well-characterized transgene insertions associated with a de novo-induced parental imprint may help us understand the molecular determinants of imprinting.
|Cell cycle-dependent recruitment of polycomb proteins to the ASNS promoter counteracts C/ebp-mediated transcriptional activation in Bombyx mori. |
Li, Z; Cheng, D; Mon, H; Zhu, L; Xu, J; Tatsuke, T; Lee, JM; Xia, Q; Kusakabe, T
PloS one 8 e52320 2013
Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori.
|SINE retrotransposons cause epigenetic reprogramming of adjacent gene promoters. |
Estécio, MR; Gallegos, J; Dekmezian, M; Lu, Y; Liang, S; Issa, JP
Molecular cancer research : MCR 10 1332-42 2012
Almost half of the human genome and as much as 40% of the mouse genome is composed of repetitive DNA sequences. The majority of these repeats are retrotransposons of the SINE and LINE families, and such repeats are generally repressed by epigenetic mechanisms. It has been proposed that these elements can act as methylation centers from which DNA methylation spreads into gene promoters in cancer. Contradictory to a methylation center function, we have found that retrotransposons are enriched near promoter CpG islands that stay methylation-free in cancer. Clearly, it is important to determine which influence, if any, these repetitive elements have on nearby gene promoters. Using an in vitro system, we confirm here that SINE B1 elements can influence the activity of downstream gene promoters, with acquisition of DNA methylation and loss of activating histone marks, thus resulting in a repressed state. SINE sequences themselves did not immediately acquire DNA methylation but were marked by H3K9me2 and H3K27me3. Moreover, our bisulfite sequencing data did not support that gain of DNA methylation in gene promoters occurred by methylation spreading from SINE B1 repeats. Genome-wide analysis of SINE repeats distribution showed that their enrichment is directly correlated with the presence of USF1, USF2, and CTCF binding, proteins with insulator function. In summary, our work supports the concept that SINE repeats interfere negatively with gene expression and that their presence near gene promoters is counter-selected, except when the promoter is protected by an insulator element.
|Fibroblast growth factor receptor 2 homodimerization rapidly reduces transcription of the pluripotency gene Nanog without dissociation of activating transcription factors. |
Santostefano, KE; Hamazaki, T; Pardo, CE; Kladde, MP; Terada, N
The Journal of biological chemistry 287 30507-17 2012
Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.
|Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1. |
Hong, GM; Bain, LJ
Toxicology and applied pharmacology 260 250-9 2012
Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (~2-fold) to the myogenin promoter at the transcription start site (-40 to +42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (-40 to +42).
|Long noncoding RNA-mediated maintenance of DNA methylation and transcriptional gene silencing. |
Mohammad, F; Pandey, GK; Mondal, T; Enroth, S; Redrup, L; Gyllensten, U; Kanduri, C
Development (Cambridge, England) 139 2792-803 2012
Establishment of silencing by noncoding RNAs (ncRNAs) via targeting of chromatin remodelers is relatively well investigated; however, their role in the maintenance of silencing is poorly understood. Here, we explored the functional role of the long ncRNA Kcnq1ot1 in the maintenance of transcriptional gene silencing in the one mega-base Kcnq1 imprinted domain in a transgenic mouse model. By conditionally deleting the Kcnq1ot1 ncRNA at different stages of mouse development, we suggest that Kcnq1ot1 ncRNA is required for the maintenance of the silencing of ubiquitously imprinted genes (UIGs) at all developmental stages. In addition, Kcnq1ot1 ncRNA is also involved in guiding and maintaining the CpG methylation at somatic differentially methylated regions flanking the UIGs, which is a hitherto unknown role for a long ncRNA. On the other hand, silencing of some of the placental-specific imprinted genes (PIGs) is maintained independently of Kcnq1ot1 ncRNA. Interestingly, the non-imprinted genes (NIGs) that escape RNA-mediated silencing are enriched with enhancer-specific modifications. Taken together, this study illustrates the gene-specific maintenance mechanisms operational at the Kcnq1 locus for tissue-specific transcriptional gene silencing and activation.
|Maternal low-protein diet causes epigenetic deregulation of HMGCR and CYP7α1 in the liver of weaning piglets. |
Rihua Cong,Yimin Jia,Runsheng Li,Yingdong Ni,Xiaojing Yang,Qinwei Sun,Nahid Parvizi,Ruqian Zhao
The Journal of nutritional biochemistry 23 2012
To investigate the effect of maternal dietary protein on hepatic cholesterol metabolism in offspring pigs and to detect underlying epigenetic mechanisms, 14 primiparous purebred Meishan sows were fed standard-protein (SP, n=7) or low-protein (LP, 50% of SP, n=7) diets during pregnancy and lactation, respectively. LP piglets showed significantly lower body weight and liver weight at weaning, associated with decreased liver and serum cholesterol content. Hepatic SREBP2, HMGCR and CYP7α1 mRNA expressions were all up-regulated in LP piglets, as well as SREBP2 protein content and HMGCR enzyme activity, compared to SP piglets, while the mRNA expression of LDLR, FXR, LXR and CYP27α1 was not altered. Hepatic activation of HMGCR gene transcription in LP piglets was associated with promoter hypomethylation, together with decreased histone H3, H3 lysine 9 monomethylation (H3K9me1) and H3 lysine 27 trimethylation (H3K27me3) and increased H3 acetylation. No CpG islands were predicted in the CYP7α1 promoter, and the augmented CYP7α1 transcription in LP piglets was associated with decreased H3, H3K9me1 and H3K27me3. No alterations were detected for hepatic expression of microRNAs predicted to target 3'-UTR of HMGCR or CYP7α1 gene. These results indicate that maternal low-protein diet during gestation and lactation affects hepatic cholesterol metabolism in weaning piglets by modifying the epigenetic regulation of HMGCR and CYP7α1 genes, which implicates possible long-term consequences in cholesterol homeostasis later in adult life.
|Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47. |
Balog, J; Miller, D; Sanchez-Curtailles, E; Carbo-Marques, J; Block, G; Potman, M; de Knijff, P; Lemmers, RJ; Tapscott, SJ; van der Maarel, SM
European journal of human genetics : EJHG 20 185-91 2012
Macrosatellite repeats (MSRs) present an extreme example of copy number variation, yet their epigenetic regulation in normal and malignant cells is largely understudied. The CT47 cancer/testis antigen located on human Xq24 is organized as an array of 4.8 kb large units. CT47 is expressed in the testis and in certain types of cancer, but not in non-malignant somatic tissue. We used CT47 as a model to study a possible correlation between copy number variation, epigenetic regulation and transcription originating from MSRs in normal and malignant cells. In lymphoblastoid cell lines and primary fibroblasts, CT47 expression was absent, consistent with the observed heterochromatic structure and DNA hypermethylation of the CT47 promoter. Heterochromatinization of CT47 occurs early during development as human embryonic stem cells show high levels of DNA methylation and repressive chromatin modifications in the absence of CT47 expression. In small-cell lung carcinoma cell lines with low levels of CT47 transcripts, we observed reduced levels of histone 3 lysine 9 trimethylation (H3K9me3) and trimethylated lysine 27 of histone H3 (H3K27me3) without concomitant increase in euchromatic histone modifications. DNA methylation levels in the promoter region of CT47 are also significantly reduced in these cells. This supports a model in which during oncogenic transformation, there is a relative loss of repressive chromatin markers resulting in leaky expression of CT47. We conclude that some MSRs, like CT47 and the autosomal MSRs TAF11-Like, PRR20, ZAV and D4Z4, the latter being involved in facioscapulohumeral muscular dystrophy, seem to be governed by common regulatory mechanisms with their abundant expression mostly being restricted to the germ line.
|IDH mutation impairs histone demethylation and results in a block to cell differentiation. |
Lu, C; Ward, PS; Kapoor, GS; Rohle, D; Turcan, S; Abdel-Wahab, O; Edwards, CR; Khanin, R; Figueroa, ME; Melnick, A; Wellen, KE; O'Rourke, DM; Berger, SL; Chan, TA; Levine, RL; Mellinghoff, IK; Thompson, CB
Nature 483 474-8 2012
Recurrent mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 have been identified in gliomas, acute myeloid leukaemias (AML) and chondrosarcomas, and share a novel enzymatic property of producing 2-hydroxyglutarate (2HG) from α-ketoglutarate. Here we report that 2HG-producing IDH mutants can prevent the histone demethylation that is required for lineage-specific progenitor cells to differentiate into terminally differentiated cells. In tumour samples from glioma patients, IDH mutations were associated with a distinct gene expression profile enriched for genes expressed in neural progenitor cells, and this was associated with increased histone methylation. To test whether the ability of IDH mutants to promote histone methylation contributes to a block in cell differentiation in non-transformed cells, we tested the effect of neomorphic IDH mutants on adipocyte differentiation in vitro. Introduction of either mutant IDH or cell-permeable 2HG was associated with repression of the inducible expression of lineage-specific differentiation genes and a block to differentiation. This correlated with a significant increase in repressive histone methylation marks without observable changes in promoter DNA methylation. Gliomas were found to have elevated levels of similar histone repressive marks. Stable transfection of a 2HG-producing mutant IDH into immortalized astrocytes resulted in progressive accumulation of histone methylation. Of the marks examined, increased H3K9 methylation reproducibly preceded a rise in DNA methylation as cells were passaged in culture. Furthermore, we found that the 2HG-inhibitable H3K9 demethylase KDM4C was induced during adipocyte differentiation, and that RNA-interference suppression of KDM4C was sufficient to block differentiation. Together these data demonstrate that 2HG can inhibit histone demethylation and that inhibition of histone demethylation can be sufficient to block the differentiation of non-transformed cells.
|Downregulation of integrin receptor-signaling genes by Epstein-Barr virus EBNA 3C via promoter-proximal and -distal binding elements. |
McClellan, MJ; Khasnis, S; Wood, CD; Palermo, RD; Schlick, SN; Kanhere, AS; Jenner, RG; West, MJ
Journal of virology 86 5165-78 2012
Epstein-Barr virus (EBV) establishes a persistent latent infection in B lymphocytes and is associated with the development of numerous human tumors. Epstein-Barr nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization, has potent cell cycle deregulation capabilities, and functions as a regulator of both viral- and cellular-gene expression. We performed transcription profiling on EBNA 3C-expressing B cells and identified several chemokines and members of integrin receptor-signaling pathways, including CCL3, CCL4, CXCL10, CXCL11, ITGA4, ITGB1, ADAM28, and ADAMDEC1, as cellular target genes that could be repressed by the action of EBNA 3C alone. Chemotaxis assays demonstrated that downregulation of CXCL10 and -11 by EBNA 3C is sufficient to reduce the migration of cells expressing the CXCL10 and -11 receptor CXCR3. Gene repression by EBNA 3C was accompanied by decreased histone H3 lysine 9/14 acetylation and increased histone H3 lysine 27 trimethylation. In an EBV-positive cell line expressing all latent genes, we identified binding sites for EBNA 3C at ITGB1 and ITGA4 and in a distal regulatory region between ADAMDEC1 and ADAM28, providing the first demonstration of EBNA 3C association with cellular-gene control regions. Our data implicate indirect mechanisms in CXCL10 and CXCL11 repression by EBNA 3C. In summary, we have unveiled key cellular pathways repressed by EBNA 3C that are likely to contribute to the ability of EBV-immortalized cells to modulate immune responses, adhesion, and B-lymphocyte migration to facilitate persistence in the host.
|Telomere length regulates TERRA levels through increased trimethylation of telomeric H3K9 and HP1α. |
Arnoult, Nausica, et al.
Nat. Struct. Mol. Biol., 19: 948-56 (2012) 2012
Gene silencing by the repressive telomeric chromatin environment, referred to as telomere position effect (TPE), has been well characterized in yeast and depends on telomere length. However, proof of its existence at native human chromosome ends has remained elusive, mainly owing to the paucity of genes near telomeres. The discovery of TERRAs, the telomeric noncoding RNAs transcribed from subtelomeric promoters, paved the way to probing for telomere-length impact on physiological TPE. Using cell lines of various origins, we show that telomere elongation consistently represses TERRA expression. Repression is mediated by increased trimethylated H3K9 density at telomeres and by heterochromatin protein HP1α, with no detectable spreading of the marks beyond the telomeric tract, restricting human TPE to telomere transcription. Our data further support the existence of a negative-feedback mechanism in which longer TERRA molecules repress their own transcription upon telomere elongation.
|The 3D tissue microenvironment modulates DNA methylation and E-cadherin expression in squamous cell carcinoma. |
DesRochers, TM; Shamis, Y; Alt-Holland, A; Kudo, Y; Takata, T; Wang, G; Jackson-Grusby, L; Garlick, JA
Epigenetics 7 34-46 2012
The microenvironment plays a significant role in human cancer progression. However, the role of the tumor microenvironment in the epigenetic control of genes critical to cancer progression remains unclear. As transient E-cadherin expression is central to many stages of neoplasia and is sensitive to regulation by the microenvironment, we have studied if microenvironmental control of E-cadherin expression is linked to transient epigenetic regulation of its promoter, contributing to the unstable and reversible expression of E-cadherin seen during tumor progression. We used 3D, bioengineered human tissue constructs that mimic the complexity of their in vivo counterparts, to show that the tumor microenvironment can direct the re-expression of E-cadherin through the reversal of methylation-mediated silencing of its promoter. This loss of DNA methylation results from the induction of homotypic cell-cell interactions as cells undergo tissue organization. E-cadherin re-expression is associated with multiple epigenetic changes including altered methylation of a small number of CpGs, specific histone modifications, and control of miR-148a expression. These epigenetic changes may drive the plasticity of E-cadherin-mediated adhesion in different tissue microenvironments during tumor cell invasion and metastasis. Thus, we suggest that epigenetic regulation is a mechanism through which tumor cell colonization of metastatic sites occurs as E-cadherin-expressing cells arise from E-cadherin-deficient cells.
|Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1. |
Musselman, CA; Avvakumov, N; Watanabe, R; Abraham, CG; Lalonde, ME; Hong, Z; Allen, C; Roy, S; Nuñez, JK; Nickoloff, J; Kulesza, CA; Yasui, A; Côté, J; Kutateladze, TG
Nature structural & molecular biology 19 1266-72 2012
The PHD finger protein 1 (PHF1) is essential in epigenetic regulation and genome maintenance. Here we show that the Tudor domain of human PHF1 binds to histone H3 trimethylated at Lys36 (H3K36me3). We report a 1.9-Å resolution crystal structure of the Tudor domain in complex with H3K36me3 and describe the molecular mechanism of H3K36me3 recognition using NMR. Binding of PHF1 to H3K36me3 inhibits the ability of the Polycomb PRC2 complex to methylate Lys27 of histone H3 in vitro and in vivo. Laser microirradiation data show that PHF1 is transiently recruited to DNA double-strand breaks, and PHF1 mutants impaired in the H3K36me3 interaction exhibit reduced retention at double-strand break sites. Together, our findings suggest that PHF1 can mediate deposition of the repressive H3K27me3 mark and acts as a cofactor in early DNA-damage response.
|BZLF1 governs CpG-methylated chromatin of Epstein-Barr Virus reversing epigenetic repression. |
Woellmer, A; Arteaga-Salas, JM; Hammerschmidt, W
PLoS pathogens 8 e1002902 2012
Epigenetic mechanisms are essential for the regulation of all genes in mammalian cells but transcriptional repression including DNA methylation are also major epigenetic mechanisms of defense inactivating potentially harmful pathogens. Epstein-Barr Virus (EBV), however, has evolved to take advantage of CpG methylated DNA to regulate its own biphasic life cycle. We show here that latent EBV DNA has an extreme composition of methylated CpG dinucleotides with a bimodal distribution of unmethylated or fully methylated DNA at active latent genes or completely repressed lytic promoters, respectively. We find this scenario confirmed in primary EBV-infected memory B cells in vivo. Extensive CpG methylation of EBV's DNA argues for a very restricted gene expression during latency. Above-average nucleosomal occupancy, repressive histone marks, and Polycomb-mediated epigenetic silencing further shield early lytic promoters from activation during latency. The very tight repression of viral lytic genes must be overcome when latent EBV enters its lytic phase and supports de novo virus synthesis in infected cells. The EBV-encoded and AP-1 related transcription factor BZLF1 overturns latency and initiates virus synthesis in latently infected cells. Paradoxically, BZLF1 preferentially binds to CpG-methylated motifs in key viral promoters for their activation. Upon BZLF1 binding, we find nucleosomes removed, Polycomb repression lost, and RNA polymerase II recruited to the activated early promoters promoting efficient lytic viral gene expression. Surprisingly, DNA methylation is maintained throughout this phase of viral reactivation and is no hindrance to active transcription of extensively CpG methylated viral genes as thought previously. Thus, we identify BZLF1 as a pioneer factor that reverses epigenetic silencing of viral DNA to allow escape from latency and report on a new paradigm of gene regulation.
|Cancer-linked satellite 2 DNA hypomethylation does not regulate Sat2 non-coding RNA expression and is initiated by heat shock pathway activation. |
Tilman, G; Arnoult, N; Lenglez, S; Van Beneden, A; Loriot, A; De Smet, C; Decottignies, A
Epigenetics 7 903-13 2012
Epigenetic dysfunctions, including DNA methylation alterations, play major roles in cancer initiation and progression. Although it is well established that gene promoter demethylation activates transcription, it remains unclear whether hypomethylation of repetitive heterochromatin similarly affects expression of non-coding RNA from these loci. Understanding how repetitive non-coding RNAs are transcriptionally regulated is important given that their established upregulation by the heat shock (HS) pathway suggests important functions in cellular response to stress, possibly by promoting heterochromatin reconstruction. We found that, although pericentromeric satellite 2 (Sat2) DNA hypomethylation is detected in a majority of cancer cell lines of various origins, DNA methylation loss does not constitutively hyperactivate Sat2 expression, and also does not facilitate Sat2 transcriptional induction upon heat shock. In melanoma tumor samples, our analysis revealed that the HS response, frequently upregulated in tumors, is probably the main determinant of Sat2 RNA expression in vivo. Next, we tested whether HS pathway hyperactivation may drive Sat2 demethylation. Strikingly, we found that both hyperthermia and hyperactivated RasV12 oncogene, another potent inducer of the HS pathway, reduced Sat2 methylation levels by up to 27% in human fibroblasts recovering from stress. Demethylation occurred locally on Sat2 repeats, resulting in a demethylation signature that was also detected in cancer cell lines with moderate genome-wide hypomethylation. We therefore propose that upregulation of Sat2 transcription in response to HS pathway hyperactivation during tumorigenesis may promote localized demethylation of the locus. This, in turn, may contribute to tumorigenesis, as demethylation of Sat2 was previously reported to favor chromosomal rearrangements.
|Genome-wide bovine H3K27me3 modifications and the regulatory effects on genes expressions in peripheral blood lymphocytes. |
He, Y; Yu, Y; Zhang, Y; Song, J; Mitra, A; Zhang, Y; Wang, Y; Sun, D; Zhang, S
PloS one 7 e39094 2012
Gene expression of lymphocytes was found to be influenced by histone methylation in mammals and trimethylation of lysine 27 on histone H3 (H3K27me3) normally represses genes expressions. Peripheral blood lymphocytes are the main source of somatic cells in the milk of dairy cows that vary frequently in response to the infection or injury of mammary gland and number of parities.The genome-wide status of H3K27me3 modifications on blood lymphocytes in lactating Holsteins was performed via ChIP-Seq approach. Combined with digital gene expression (DGE) technique, the regulation effects of H3K27me3 on genes expressions were analyzed.The ChIP-seq results showed that the peaks of H3K27me3 in cows lymphocytes were mainly enriched in the regions of up20K (~50%), down20K (~30%) and intron (~28%) of the genes. Only ~3% peaks were enriched in exon regions. Moreover, the highest H3K27me3 modification levels were mainly around the 2 Kb upstream of transcriptional start sites (TSS) of the genes. Using conjoint analysis with DGE data, we found that H3K27me3 marks tended to repress target genes expressions throughout whole gene regions especially acting on the promoter region. A total of 53 differential expressed genes were detected in third parity cows compared to first parity, and the 25 down-regulated genes (PSEN2 etc.) were negatively correlated with H3K27me3 levels on up2Kb to up1Kb of the genes, while the up-regulated genes were not showed in this relationship.The first blueprint of bovine H3K27me3 marks that mediates gene silencing was generated. H3K27me3 plays its repressed role mainly in the regulatory region in bovine lymphocytes. The up2Kb to up1Kb region of the down-regulated genes in third parity cows could be potential target of H3K27me3 regulation. Further studies are warranted to understand the regulation mechanisms of H3K27me3 on somatic cell count increases and milk losses in latter parities of cows.
|Epigenetic regulation of the latency-associated region of Marek's disease virus in tumor-derived T-cell lines and primary lymphoma. |
Brown, AC; Nair, V; Allday, MJ
Journal of virology 86 1683-95 2012
Meq is the major Marek's disease virus (MDV)-encoded oncoprotein and is essential for T-cell lymphomagenesis. Meq and several noncoding RNAs, including three microRNA (MiR) clusters, are expressed from the repeats of the MDV genome during latent infection of T cells. To investigate the state of the chromatin in this and flanking regions, we carried out chromatin immunoprecipitation (ChIP) analysis of covalent histone modifications and associated bound proteins. T-cell lines and a lymphoma were compared. The chromatin around the promoters for Meq and the noncoding RNAs in both cell lines and the lymphoma were associated with H3K9 acetylation and H3K4 trimethylation, which are marks of transcriptionally active chromatin. These correlated with bound Meq-c-Jun heterodimers. The only binding site for Meq homodimers is located at the lytic origin of replication (OriLyt), next to the lytic gene pp38. This region lacked active marks and was associated with repressive histone modifications (H3K27 and H3K9 trimethylation). DNA CpG methylation was investigated using methylated DNA precipitation (MeDP). In cell lines, DNA methylation was abundant across the repeats but noticeably reduced or absent around the active promoters. In primary tumors, CpG methylation occurred less than 2 months after infection, focused within the ICP4 gene. These data suggest that nonrandom de novo DNA methylation occurs early in lymphomagenesis. In addition, the histone data indicate a role for Meq in the epigenetic regulation of the MDV genome repeats in transformed T cells and suggest that the OriLyt region and the Meq/MiR region might be separated by chromatin boundary elements, and preliminary data on CTCF binding are consistent with this.
|Three murine leukemia virus integration regions within 100 kilobases upstream of c-myb are proximal to the 5' regulatory region of the gene through DNA looping. |
Zhang, J; Markus, J; Bies, J; Paul, T; Wolff, L
Journal of virology 86 10524-32 2012
Retroviruses integrated into genomic DNA participate in long-range gene activation from as far away as several hundred kilobases. Hypotheses have been put forth to account for these phenomena, but data have not been provided to support a physical mechanism that explains long-range activation. In murine leukemia virus-induced myeloid leukemia in mice, integrated proviruses have been found upstream of c-myb in three regions, named Mml1, Mml2, and Mml3 (25, 50, and 70 kb upstream, respectively). The transcription factor c-Myb is an oncogene whose dysregulation and/or mutation can lead to human leukemia. We hypothesized that the murine c-myb upstream region contains regulatory elements accessed by the retrovirus. To identify regulatory sites in the murine c-myb upstream region, we looked by chromatin immunoprecipitation with microarray technology (ChIP-on-chip) for histone modifications implicating gene activation in normal cells. H3K4me3, H3K4me1, and H3K9/14ac were enriched at Mml1 and/or Mml2 in the myeloblastic cell line M1, which expresses c-myb. The enrichment of all of these histone marks decreased with differentiation-induced downregulation of the gene in M1 cells but increased and spread in tumor cells containing integrated provirus. Importantly, using chromosome conformation capture (3C)-quantitative PCR assays, interactions between the 5' region, including the promoter and all Mml sites (Mml1, Mml2, and Mml3), were detected due to DNA looping in M1 cells and tumor cells with provirus in Mml1, Mml2, or Mml3. Therefore, our study provides a new mechanism of retrovirus insertional mutagenesis whereby spatial chromatin organization allows distally located provirus, with its own enhancer elements, to access the 5' regulatory region of the gene.
|Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. |
Efe JA, Hilcove S, Kim J, Zhou H, Ouyang K, Wang G, Chen J, Ding S.
Nature cell biology 13 215-22 2011
Here we show that conventional reprogramming towards pluripotency through overexpression of Oct4, Sox2, Klf4 and c-Myc can be shortcut and directed towards cardiogenesis in a fast and efficient manner. With as little as 4 days of transgenic expression of these factors, mouse embryonic fibroblasts (MEFs) can be directly reprogrammed to spontaneously contracting patches of differentiated cardiomyocytes over a period of 11-12 days. Several lines of evidence suggest that a pluripotent intermediate is not involved. Our method represents a unique strategy that allows a transient, plastic developmental state established early in reprogramming to effectively function as a cellular transdifferentiation platform, the use of which could extend beyond cardiogenesis. Our study has potentially wide-ranging implications for induced pluripotent stem cell (iPSC)-factor-based reprogramming and broadens the existing paradigm.
|Ethanol and acetaldehyde exposure induces specific epigenetic modifications in the prodynorphin gene promoter in a human neuroblastoma cell line. |
D'Addario, C; Johansson, S; Candeletti, S; Romualdi, P; Ögren, SO; Terenius, L; Ekström, TJ
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 25 1069-75 2011
Ethanol alters neural activity through interaction with multiple neurotransmitters and neuromodulators. The endogenous opioid system seems to play a key role, since the opioid receptor antagonist naltrexone (ReVia®) attenuates craving for alcohol. We recently reported that ethanol and acetaldehyde, the first product of ethanol metabolism, affect transcription of opioid system genes in human SH-SY5Y neuroblastoma cells. In the current study, potential epigenetic mechanisms were investigated to clarify these effects on prodynorphin gene expression. DNA methylation was analyzed by bisulfite pyrosequencing, and chromatin immunoprecipitation was used to assess putative specific histone modifications at the prodynorphin gene promoter. The results demonstrated a temporal relationship between selective chromatin modifications induced by ethanol and acetaldehyde and changes in prodynorphin gene expression quantitated by real-time qPCR. DNA methylation was not altered in any of the experimental conditions used. The epigenetic changes may precede gene transcription, and histone modifications might keep the prodynorphin gene in a poised state for later reactivation. A link has been observed between gene expression alterations and selective epigenetic modulation in the prodynorphin promoter region, demonstrating a specificity of the changes induced by ethanol and acetaldehyde. The latter may be mediating ethanol effects at the genomic level.
|Epigenetic inactivation of the miR-124-1 in haematological malignancies. |
Wong, KY; So, CC; Loong, F; Chung, LP; Lam, WW; Liang, R; Li, GK; Jin, DY; Chim, CS
PloS one 6 e19027 2011
miR-124-1 is a tumour suppressor microRNA (miR). Epigenetic deregulation of miRs is implicated in carcinogenesis. Promoter DNA methylation and histone modification of miR-124-1 was studied in 5 normal marrow controls, 4 lymphoma, 8 multiple myeloma (MM) cell lines, 230 diagnostic primary samples of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), MM, and non-Hodgkin's lymphoma (NHL), and 53 MM samples at stable disease or relapse. Promoter of miR-124-1 was unmethylated in normal controls but homozygously methylated in 4 of 4 lymphoma and 4 of 8 myeloma cell lines. Treatment of 5-Aza-2'-deoxycytidine led to miR-124-1 demethylation and re-expression of mature miR-124, which also associated with emergence of euchromatic trimethyl H3K4 and consequent downregulation of CDK6 in myeloma cells harboring homozygous miR-124-1 methylation. In primary samples at diagnosis, miR-124-1 methylation was absent in CML but detected in 2% each of MM at diagnosis and relapse/progression, 5% ALL, 15% AML, 14% CLL and 58.1% of NHL (pless than 0.001). Amongst lymphoid malignancies, miR-124-1 was preferentially methylated in NHL than MM, CLL or ALL. In primary lymphoma samples, miR-124-1 was preferentially hypermethylated in B- or NK/T-cell lymphomas and associated with reduced miR-124 expression. In conclusion, miR-124-1 was hypermethylated in a tumour-specific manner, with a heterochromatic histone configuration. Hypomethylation led to partial restoration of euchromatic histone code and miR re-expression. Infrequent miR-124-1 methylation detected in diagnostic and relapse MM samples showed an unimportant role in MM pathogenesis, despite frequent methylation found in cell lines. Amongst haematological cancers, miR-124-1 was more frequently hypermethylated in NHL, and hence warrants further study.Texto completo do artigo
|Aberrantly silenced promoters retain a persistent memory of the silenced state after long-term reactivation. |
Oyer, JA; Yates, PA; Godsey, S; Turker, MS
Mutation research 706 21-7 2011
A hallmark of aberrant DNA methylation-associated silencing is reversibility. However, long-term stability of reactivated promoters has not been explored. To examine this issue, spontaneous reactivant clones were isolated from mouse embryonal carcinoma cells bearing aberrantly silenced Aprt alleles and re-silencing frequencies were determined as long as three months after reactivation occurred. Despite continuous selection for expression of the reactivated Aprt alleles, exceptionally high spontaneous re-silencing frequencies were observed. A DNA methylation analysis demonstrated retention of sporadic methylation of CpG sites in a protected region of the Aprt promoter in many reactivant alleles suggesting a role for these methylated sites in the re-silencing process. In contrast, a chromatin immunoprecipitation (ChIP) analysis for methyl-H3K4, acetyl-H3K9, and dimethyl-H3K9 levels failed to reveal a specific histone modification that could explain high frequency re-silencing. These results demonstrate that aberrantly silenced and reactivated promoters retain a persistent memory of having undergone the silencing process and suggest the failure to eliminate all CpG methylation as a potential contributing mechanism.Texto completo do artigo
|Specific histone lysine 4 methylation patterns define TR-binding capacity and differentiate direct T3 responses. |
Bilesimo, P; Jolivet, P; Alfama, G; Buisine, N; Le Mevel, S; Havis, E; Demeneix, BA; Sachs, LM
Molecular endocrinology (Baltimore, Md.) 25 225-37 2011
The diversity of thyroid hormone T(3) effects in vivo makes their molecular analysis particularly challenging. Indeed, the current model of the action of T(3) and its receptors on transcription does not reflect this diversity. Here, T(3)-dependent amphibian metamorphosis was exploited to investigate, in an in vivo developmental context, how T(3) directly regulates gene expression. Two, direct positively regulated T(3)-response genes encoding transcription factors were analyzed: thyroid hormone receptor β (TRβ) and TH/bZIP. Reverse transcription-real-time quantitative PCR analysis on Xenopus tropicalis tadpole brain and tail fin showed differences in expression levels in premetamorphic tadpoles (lower for TH/bZIP than for TRβ) and differences in induction after T(3) treatment (lower for TRβ than for TH/bZIP). To dissect the mechanisms underlying these differences, chromatin immunoprecipitation was used. T(3) differentially induced RNA polymerase II and histone tail acetylation as a function of transcriptional level. Gene-specific patterns of TR binding were found on the different T(3) -responsive elements (higher for TRβ than for TH/bZIP), correlated with gene-specific modifications of H3K4 methylation (higher for TRβ than for TH/bZIP). Moreover, tissue-specific modifications of H3K27 were found (lower in brain than in tail fin). This first in vivo analysis of the association of histone modifications and TR binding/gene activation during vertebrate development for any nuclear receptor indicate that chromatin context of thyroid-responsive elements loci controls the capacity to bind TR through variations in histone H3K4 methylation, and that the histone code, notably H3, contributes to the fine tuning of gene expression that underlies complex physiological T(3) responses.
|Chromatin Immunoprecipitation (ChIP)||Xenopus||21239616|
|Epstein-Barr virus nuclear antigens 3C and 3A maintain lymphoblastoid cell growth by repressing p16INK4A and p14ARF expression. |
Maruo, S; Zhao, B; Johannsen, E; Kieff, E; Zou, J; Takada, K
Proceedings of the National Academy of Sciences of the United States of America 108 1919-24 2011
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and EBNA3A are each essential for EBV conversion of primary human B lymphocytes into continuously proliferating lymphoblast cell lines (LCLs) and for maintaining LCL growth. We now find that EBNA3C and EBNA3A's essential roles are to repress p16(INK4A) and p14(ARF). In the absence of EBNA3C or EBNA3A, p16(INK4A) and p14(ARF) expression increased and cell growth ceased. EBNA3C inactivation did not alter p16(INK4A) promoter CpG methylation, but reduced already low H3K27me3, relative to resting B cells, and increased H3K4me3 and H3-acetylation, linking EBNA3C inactivation to histone modifications associated with increased transcription. Importantly, knockdown of p16(INK4A) or p14(ARF) partially rescued LCLs from EBNA3C or EBNA3A inactivation-induced growth arrest and knockdown of both rescued LCL growth, confirming central roles for p16(INK4A) and p14(ARF) in LCL growth arrest following EBNA3C or EBNA3A inactivation. Moreover, blockade of p16(INK4A) and p14(ARF) effects on pRb and p53 by human papilloma virus type 16 E7 and E6 expression, sustained LCL growth after EBNA3C or EBNA3A inactivation. These data indicate that EBNA3C and EBNA3A joint repression of CDKN2A p16(INK4A) and p14(ARF) is essential for LCL growth.Texto completo do artigo
|cAMP-responsive element modulator (CREM)α protein signaling mediates epigenetic remodeling of the human interleukin-2 gene: implications in systemic lupus erythematosus. |
Hedrich, Christian M, et al.
J. Biol. Chem., 286: 43429-36 (2011) 2011
IL-2 is a key cytokine during proliferation and activation of T lymphocytes and functions as an auto- and paracrine growth factor. Regardless of activating effects on T lymphocytes, the absence of IL-2 has been linked to the development of autoimmune pathology in mice and humans. Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease and characterized by dysregulation of lymphocyte function, transcription factor and cytokine expression, and antigen presentation. Reduced IL-2 expression is a hallmark of SLE T lymphocytes and results in decreased numbers of regulatory T lymphocytes which play an important role in preventing autoimmunity. Reduced IL-2 expression was linked to overproduction of the transcription regulatory factor cAMP-responsive element modulator (CREM)α in SLE T lymphocytes and subsequent CREMα binding to a CRE site within the IL2 promoter (-180 CRE). In this study, we demonstrate the involvement of CREMα-mediated IL2 silencing in T lymphocytes from SLE patients through a gene-wide histone deacetylase 1-directed deacetylation of histone H3K18 and DNA methyltransferase 3a-directed cytosine phosphate guanosine (CpG)-DNA hypermethylation. For the first time, we provide direct evidence that CREMα mediates silencing of the IL2 gene in SLE T cells though histone deacetylation and CpG-DNA methylation.
|Long term transcriptional reactivation of epigenetically silenced genes in colorectal cancer cells requires DNA hypomethylation and histone acetylation. |
Mossman, D; Scott, RJ
PloS one 6 e23127 2011
Epigenetic regulation of genes involves the coordination of DNA methylation and histone modifications to maintain transcriptional status. These two features are frequently disrupted in malignancy such that critical genes succumb to inactivation. 5-aza-2'-deoxycytidine (5-aza-dC) is an agent which inhibits DNA methyltransferase, and holds great potential as a treatment for cancer, yet the extent of its effectiveness varies greatly between tumour types. Previous evidence suggests expression status after 5-aza-dC exposure cannot be explained by the DNA methylation status alone.We sought to identify chromatin changes involved with short and long term gene reactivation following 5-aza-dC exposure. Two colorectal cancer cell lines, HCT116 and SW480, were treated with 5-aza-dC and then grown in drug-free media to allow DNA re-methylation. DNA methylation and chromatin modifications were assessed with bisulfite sequencing and Chromatin Immuno-Precipitation analysis.Increased H3 acetylation, H3K4 tri-methylation and loss of H3K27 tri-methylation were associated with reactivation. Hypermethylated genes that did not show increased acetylation were transiently expressed with 5-aza-dC treatment before reverting to an inactive state. Three reactivated genes, CDO1, HSPC105 and MAGEA3, were still expressed 10 days post 5-aza-dC treatment and displayed localised hypomethylation at the transcriptional start site, and also an increased enrichment of histone H3 acetylation.These observations suggest that hypomethylation alone is insufficient to reactivate silenced genes and that increased Histone H3 acetylation in unison with localised hypomethylation allows long term reversion of these epigenetically silenced genes. This study suggests that combined DNA methyltransferase and histone deacetylase inhibitors may aid long term reactivation of silenced genes.Texto completo do artigo
|cAMP response element modulator (CREM)α suppresses IL-17F expression in T lymphocytes from patients with systemic lupus erythematosus (SLE). |
Hedrich, Christian M, et al.
The Journal of biological chemistry, (2011) 2011
The pro-inflammatory cytokines IL-17A and IL-17F are primarily produced by Th17 lymphocytes. Both are involved in host defence mechanisms against bacterial and fungal pathogens, and contribute to the development of various autoimmune diseases. T lymphocytes from patients with systemic lupus erythematosus (SLE) display increased expression of transcription factor cAMP response element modulator (CREM)α which has been documented to account for aberrant T cell function and contributes to the pathogenesis of SLE. Here, we provide evidence that IL-17F expression is reduced in SLE T cells. We demonstrate that CREMα binds to a yet unidentified CRE site within the proximal promoter. This results in reduced IL-17F expression in SLE T lymphocytes and is independent of activating epigenetic patterns (increased H3K18 acetylation, reduced H3K27 tri-methylation and CpG-DNA demethylation). Forced CREMα expression in human T lymphocytes results in reduced IL-17F expression. Our findings demonstrate extended involvement of CREMα in cytokine dysregulation in SLE by contributing to a disrupted balance between IL-17A and IL-17F. An increased IL-17A/IL-17F ratio may aggravate the pro-inflammatory phenotype of SLE.
|cAMP-responsive element modulator (CREM)α protein induces interleukin 17A expression and mediates epigenetic alterations at the interleukin-17A gene locus in patients with systemic lupus erythematosus. |
Rauen, Thomas, et al.
J. Biol. Chem., 286: 43437-46 (2011) 2011
IL-17A is a proinflammatory cytokine that is produced by specialized T helper cells and contributes to the development of several autoimmune diseases such as systemic lupus erythematosus (SLE). Transcription factor cAMP-responsive element modulator (CREM)α displays increased expression levels in T cells from SLE patients and has been described to account for aberrant T cell function in SLE pathogenesis. In this report, we provide evidence that CREMα physically binds to a cAMP-responsive element, CRE (-111/-104), within the proximal human IL17A promoter and increases its activity. Chromatin immunoprecipitation assays reveal that activated naïve CD4(+) T cells as well as T cells from SLE patients display increased CREMα binding to this site compared with T cells from healthy controls. The histone H3 modification pattern at the CRE site (-111/-104) and neighboring conserved noncoding sequences within the human IL17A gene locus suggests an accessible chromatin structure (H3K27 hypomethylation/H3K18 hyperacetylation) in activated naïve CD4(+) T cells and SLE T cells. H3K27 hypomethylation is accompanied by decreased cytosine phosphate guanosine (CpG)-DNA methylation in these regions in SLE T cells. Decreased recruitment of histone deacetylase (HDAC)1 and DNA methyltransferase (DNMT)3a to the CRE site (-111/-104) probably accounts for the observed epigenetic alterations. Reporter studies confirmed that DNA methylation of the IL17A promoter indeed abrogates its inducibility. Our findings demonstrate an extended role for CREMα in the immunopathogenesis of SLE because it contributes to increased expression of IL-17A.
|Regulation of DCC localization by HTZ-1/H2A.Z and DPY-30 does not correlate with H3K4 methylation levels. |
Petty, E; Laughlin, E; Csankovszki, G
PloS one 6 e25973 2011
Dosage compensation is a specialized form of gene regulation that balances sex-chromosome linked gene expression between the sexes. In C. elegans, dosage compensation is achieved by the activity of the dosage compensation complex (DCC). The DCC binds along both X chromosomes in hermaphrodites to down-regulate gene expression by half, limiting X-linked gene products to levels produced in XO males. Sequence motifs enriched on the X chromosome play an important role in targeting the DCC to the X. However, these motifs are not strictly X-specific and therefore other factors, such as the chromatin environment of the X chromosome, are likely to aid in DCC targeting. Previously, we found that loss of HTZ-1 results in partial disruption of dosage compensation localization to the X chromosomes. We wanted to know whether other chromatin components coordinated with HTZ-1 to regulate DCC localization. One candidate is DPY-30, a protein known to play a role in DCC localization. DPY-30 homologs in yeast, flies, and mammals are highly conserved members of histone H3 lysine 4 (H3K4) methyltransferase Set1/MLL complexes. Therefore, we investigated the hypothesis that the dosage compensation function of DPY-30 involves H3K4 methylation. We found that in dpy-30 animals the DCC fails to stably bind chromatin. Interestingly, of all the C. elegans homologs of Set1/MLL complex subunits, only DPY-30 is required for stable DCC binding to chromatin. Additionally, loss of H3K4 methylation does not enhance DCC mislocalization in htz-1 animals. We conclude that DPY-30 and HTZ-1 have unique functions in DCC localization, both of which are largely independent of H3K4 methylation.
|A regulatory archipelago controls Hox genes transcription in digits. |
Montavon, T; Soshnikova, N; Mascrez, B; Joye, E; Thevenet, L; Splinter, E; de Laat, W; Spitz, F; Duboule, D
Cell 147 1132-45 2011
The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a "regulatory archipelago," provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.
|Epigenetic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of Zeste 2. |
Friedman, J; Cho, WK; Chu, CK; Keedy, KS; Archin, NM; Margolis, DM; Karn, J
Journal of virology 85 9078-89 2011
Latent HIV proviruses are silenced as the result of deacetylation and methylation of histones located at the viral long terminal repeat (LTR). Inhibition of histone deacetylases (HDACs) leads to the reemergence of HIV-1 from latency, but the contribution of histone lysine methyltransferases (HKMTs) to maintaining HIV latency remains uncertain. Chromatin immunoprecipitation experiments using latently infected Jurkat T-cell lines demonstrated that the HKMT enhancer of Zeste 2 (EZH2) was present at high levels at the LTR of silenced HIV proviruses and was rapidly displaced following proviral reactivation. Knockdown of EZH2, a key component of the Polycomb repressive complex 2 (PRC2) silencing machinery, and the enzyme which is required for trimethyl histone lysine 27 (H3K27me3) synthesis induced up to 40% of the latent HIV proviruses. In contrast, there was less than 5% induction of latent proviruses following knockdown of SUV39H1, which is required for H3K9me3 synthesis. Knockdown of EZH2 also sensitized latent proviruses to external stimuli, such as T-cell receptor stimulation, and slowed the reversion of reactivated proviruses to latency. Similarly, cell populations that responded poorly to external stimuli carried HIV proviruses that were enriched in H3K27me3 and relatively depleted in H3K9me3. Treating latently infected cells with the HKMT inhibitor 3-deazaneplanocin A, which targets EZH2, led to the reactivation of silenced proviruses, whereas chaetocin and BIX01294 showed only minimal reactivation activities. These findings suggest that PRC2-mediated silencing is an important feature of HIV latency and that inhibitors of histone methylation may play a useful role in induction strategies designed to eradicate latent HIV pools.
|Mutant p53(R175H) upregulates Twist1 expression and promotes epithelial-mesenchymal transition in immortalized prostate cells. |
Kogan-Sakin, I; Tabach, Y; Buganim, Y; Molchadsky, A; Solomon, H; Madar, S; Kamer, I; Stambolsky, P; Shelly, A; Goldfinger, N; Valsesia-Wittmann, S; Puisieux, A; Zundelevich, A; Gal-Yam, EN; Avivi, C; Barshack, I; Brait, M; Sidransky, D; Domany, E; Rotter, V
Cell death and differentiation 18 271-81 2011
A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 'gain of function' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53(R175H) mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53(R175H), was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial-mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53(R175H) mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53(R175H) mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.
|Structural and functional differences in the long non-coding RNA hotair in mouse and human. |
Schorderet, P; Duboule, D
PLoS genetics 7 e1002071 2011
Long non-coding RNAs regulate various biological processes such as dosage compensation, imprinting, and chromatin organization. HOTAIR, a paradigm of this new class of RNAs, is localized within the human HOXC gene cluster and was shown, in human cells, to regulate HOXD genes in trans via the recruitment of Polycomb Repressive Complex 2 (PRC2), followed by the trimethylation of lysine 27 of histone H3. We looked for the presence of Hotair in mice to assess whether this in trans mechanism was conserved, in particular at the developmental stages, when Hoxd genes must be tightly regulated. We show that the cognate mouse Hotair is poorly conserved in sequence; and its absence, along with the deletion of the HoxC cluster, has surprisingly little effect in vivo, neither on the expression pattern or transcription efficiency, nor on the amount of K27me3 coverage of different Hoxd target genes. We conclude that Hotair may have rapidly evolved within mammals and acquired a functional importance in humans that is not easily revealed in mice. Alternatively, redundant or compensatory mechanisms may mask its function when studied under physiological conditions.
|H3K27 trimethylation is an early epigenetic event of p16INK4a silencing for regaining tumorigenesis in fusion reprogrammed hepatoma cells. |
Yao, JY; Zhang, L; Zhang, X; He, ZY; Ma, Y; Hui, LJ; Wang, X; Hu, YP
The Journal of biological chemistry 285 18828-37 2010
Stable epigenetic silencing of p16(INK4a) is a common event in hepatocellular carcinoma (HCC) cells, which is associated with abnormal cell proliferation and liberation from cell cycle arrest. Understanding the early epigenetic events in silencing p16(INK4a) expression may illuminate a prognostic strategy to block HCC development. Toward this end, we created a reprogram cell model by the fusion mouse HCC cells with mouse embryonic stem cells, in which the ES-Hepa hybrids forfeited HCC cell characteristics along with reactivation of the silenced p16(INK4a). HCC characteristics, in terms of gene expression pattern and tumorigenic potential, was restored upon induced differentiation of these reprogrammed ES-Hepa hybrids. The histone methylation pattern relative to p16(INK4a) silencing during differentiation of the ES-Hepa hybrids was analyzed. H3K27 trimethylation at the p16(INK4a) promoter region, occurring in the early onset of p16(INK4a) silencing, was followed by H3K9 dimethylation at later stages. During the induced differentiation of the ES-Hepa hybrids, H3K4 di- and trimethylations were maintained at high levels during the silencing of p16(INK4a), strongly suggesting that H3K4 methylation events did not cause the silencing of p16(INK4a). Our results suggested that the enrichment of H3K27 trimethylation, independent of H3K9 dimethylation, trimethylation, and DNA methylation, was an early event in the silencing of p16(INK4a) during the tumor development. This unique chromatin pattern may be a heritable marker of epigenetic regulation for p16(INK4a) silencing during the developmental process of hepatocellular carcinogenesis.
|The latent origin of replication of Epstein-Barr virus directs viral genomes to active regions of the nucleus. |
Manuel J Deutsch,Elisabeth Ott,Peer Papior,Aloys Schepers
Journal of virology 84 2010
The Epstein-Barr virus efficiently infects human B cells. The EBV genome is maintained extrachromosomally and replicates synchronously with the host's chromosomes. The latent origin of replication (oriP) guarantees plasmid stability by mediating two basic functions: replication and segregation of the viral genome. While the segregation process of EBV genomes is well understood, little is known about its chromatin association and nuclear distribution during interphase. Here, we analyzed the nuclear localization of EBV genomes and the role of functional oriP domains FR and DS for basic functions such as the transformation of primary cells, their role in targeting EBV genomes to distinct nuclear regions, and their association with epigenetic domains. Fluorescence in situ hybridization visualized the localization of extrachromosomal EBV genomes in the regions adjacent to chromatin-dense territories called the perichromatin. Further, immunofluorescence experiments demonstrated a preference of the viral genome for histone 3 lysine 4-trimethylated (H3K4me3) and histone 3 lysine 9-acetylated (H3K9ac) nuclear regions. To determine the role of FR and DS for establishment and subnuclear localization of EBV genomes, we transformed primary human B lymphocytes with recombinant mini-EBV genomes containing different oriP mutants. The loss of DS results in a slightly increased association in H3K27me3 domains. This study demonstrates that EBV genomes or oriP-based extrachromosomal vector systems are integrated into the higher order nuclear organization. We found that viral genomes are not randomly distributed in the nucleus. FR but not DS is crucial for the localization of EBV in perichromatic regions that are enriched for H3K4me3 and H3K9ac, which are hallmarks of transcriptionally active regions.Texto completo do artigo
|H2A.Z maintenance during mitosis reveals nucleosome shifting on mitotically silenced genes. |
Kelly, TK; Miranda, TB; Liang, G; Berman, BP; Lin, JC; Tanay, A; Jones, PA
Molecular cell 39 901-11 2010
Profound chromatin changes occur during mitosis to allow for gene silencing and chromosome segregation followed by reactivation of memorized transcription states in daughter cells. Using genome-wide sequencing, we found H2A.Z-containing +1 nucleosomes of active genes shift upstream to occupy TSSs during mitosis, significantly reducing nucleosome-depleted regions. Single-molecule analysis confirmed nucleosome shifting and demonstrated that mitotic shifting is specific to active genes that are silenced during mitosis and, thus, is not seen on promoters, which are silenced by methylation or mitotically expressed genes. Using the GRP78 promoter as a model, we found H3K4 trimethylation is also maintained while other indicators of active chromatin are lost and expression is decreased. These key changes provide a potential mechanism for rapid silencing and reactivation of genes during the cell cycle.Texto completo do artigo
|The ubiquitin carboxyl hydrolase BAP1 forms a ternary complex with YY1 and HCF-1 and is a critical regulator of gene expression. |
Yu, H; Mashtalir, N; Daou, S; Hammond-Martel, I; Ross, J; Sui, G; Hart, GW; Rauscher, FJ; Drobetsky, E; Milot, E; Shi, Y; Affar, el B
Molecular and cellular biology 30 5071-85 2010
The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with and deubiquitinate the transcriptional regulator host cell factor 1 (HCF-1). Here we show that BAP1 assembles multiprotein complexes containing numerous transcription factors and cofactors, including HCF-1 and the transcription factor Yin Yang 1 (YY1). Through its coiled-coil motif, BAP1 directly interacts with the zinc fingers of YY1. Moreover, HCF-1 interacts with the middle region of YY1 encompassing the glycine-lysine-rich domain and is essential for the formation of a ternary complex with YY1 and BAP1 in vivo. BAP1 activates transcription in an enzymatic-activity-dependent manner and regulates the expression of a variety of genes involved in numerous cellular processes. We further show that BAP1 and HCF-1 are recruited by YY1 to the promoter of the cox7c gene, which encodes a mitochondrial protein used here as a model of BAP1-activated gene expression. Our findings (i) establish a direct link between BAP1 and the transcriptional control of genes regulating cell growth and proliferation and (ii) shed light on a novel mechanism of transcription regulation involving ubiquitin signaling.
|Extensive co-operation between the Epstein-Barr virus EBNA3 proteins in the manipulation of host gene expression and epigenetic chromatin modification. |
White, RE; Groves, IJ; Turro, E; Yee, J; Kremmer, E; Allday, MJ
PloS one 5 e13979 2010
Epstein-Barr virus (EBV) is able to drive the transformation of B-cells, resulting in the generation of lymphoblastoid cell lines (LCLs) in vitro. EBV nuclear proteins EBNA3A and EBNA3C are necessary for efficient transformation, while EBNA3B is dispensable. We describe a transcriptome analysis of BL31 cells infected with a series of EBNA3-knockout EBVs, including one deleted for all three EBNA3 genes. Using Affymetrix Exon 1.0 ST microarrays analysed with the MMBGX algorithm, we have identified over 1000 genes whose regulation by EBV requires one of the EBNA3s. Remarkably, a third of the genes identified require more than one EBNA3 for their regulation, predominantly EBNA3C co-operating with either EBNA3B, EBNA3A or both. The microarray was validated by real-time PCR, while ChIP analysis of a selection of co-operatively repressed promoters indicates a role for polycomb group complexes. Targets include genes involved in apoptosis, cell migration and B-cell differentiation, and show a highly significant but subtle alteration in genes involved in mitosis. In order to assess the relevance of the BL31 system to LCLs, we analysed the transcriptome of a set of EBNA3B knockout (3BKO) LCLs. Around a third of the genes whose expression level in LCLs was altered in the absence of EBNA3B were also altered in 3BKO-BL31 cell lines.Among these are TERT and TCL1A, implying that EBV-induced changes in the expression of these genes are not required for B-cell transformation. We also identify 26 genes that require both EBNA3A and EBNA3B for their regulation in LCLs. Together, this shows the complexity of the interaction between EBV and its host, whereby multiple EBNA3 proteins co-operate to modulate the behaviour of the host cell.
|Modular utilization of distal cis-regulatory elements controls Ifng gene expression in T cells activated by distinct stimuli. |
Balasubramani, A; Shibata, Y; Crawford, GE; Baldwin, AS; Hatton, RD; Weaver, CT
Immunity 33 35-47 2010
Distal cis-regulatory elements play essential roles in the T lineage-specific expression of cytokine genes. We have mapped interactions of three trans-acting factors-NF-kappaB, STAT4, and T-bet-with cis elements in the Ifng locus. We find that RelA is critical for optimal Ifng expression and is differentially recruited to multiple elements contingent upon T cell receptor (TCR) or interleukin-12 (IL-12) plus IL-18 signaling. RelA recruitment to at least four elements is dependent on T-bet-dependent remodeling of the Ifng locus and corecruitment of STAT4. STAT4 and NF-kappaB therefore cooperate at multiple cis elements to enable NF-kappaB-dependent enhancement of Ifng expression. RelA recruitment to distal elements was similar in T helper 1 (Th1) and effector CD8(+) T (Tc1) cells, although T-bet was dispensable in CD8 effectors. These results support a model of Ifng regulation in which distal cis-regulatory elements differentially recruit key transcription factors in a modular fashion to initiate gene transcription induced by distinct activation signals.
|Stwl modifies chromatin compaction and is required to maintain DNA integrity in the presence of perturbed DNA replication. |
Yi, X; de Vries, HI; Siudeja, K; Rana, A; Lemstra, W; Brunsting, JF; Kok, RM; Smulders, YM; Schaefer, M; Dijk, F; Shang, Y; Eggen, BJ; Kampinga, HH; Sibon, OC
Molecular biology of the cell 20 983-94 2009
Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall (stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 (two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress.Texto completo do artigo
|Analysis of intergenic transcription and histone modification across the human immunoglobulin heavy-chain locus. |
Chowdhury, M; Forouhi, O; Dayal, S; McCloskey, N; Gould, HJ; Felsenfeld, G; Fear, DJ
Proceedings of the National Academy of Sciences of the United States of America 105 15872-7 2008
Ig class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID) mediated deamination of the switch (S) regions; the resultant mismatch is processed to yield the DNA breaks required for recombination. Whereas many of the pathways involved in the mechanism of recombination have been identified, little is known about how CSR is regulated. AID action is known to require transcription of the Ig heavy-chain genes. However, it is not understood how AID is restricted to the Ig genes. Many aspects of gene expression are known to be regulated by modification of chromatin structure. In turn, chromatin is known to be regulated by several RNA-dependent activities. We have mapped the transcriptional and chromatin landscape of the human Ig heavy-chain locus to investigate the effect these activities have on CSR. We demonstrate that the Ig heavy-chain constant genes and 3'-regulatory regions are in an active chromatin conformation in unstimulated total human B cells: the locus undergoes both genic and intergenic transcription and possesses histone modifications associated with "active" chromatin (acetylated H3 and H4 and lysine 4 trimethylated H3). However, on cytokine stimulation, these modifications spread into the S regions, demonstrating a chromatin remodeling activity associated with switching. Surprisingly, after stimulation, the S regions also accumulate lysine 9 trimethylated H3, a modification previously associated with gene silencing. These data demonstrates that the Ig locus is maintained with a complex pattern of both positive and negative histone marks and suggest that some of these marks may have dual functions.
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