Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, R, M, Mk, Am||ICC, WB, ChIP||M||Purified||Monoclonal Antibody|
|Presentation||Purified mouse monoclonal IgM in buffer containing PBS with 0.05% sodium azide before the addition of glycerol to 30%. Liquid at -20ºC.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at -20°C from date of receipt. For maximum recovery of product, centrifuge the vial prior to removing the cap.|
|Material Size||200 µL|
References | 44 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|Epigenetic Regulation of Antibody Responses by the Histone H2A Deubiquitinase MYSM1. |
Jiang, XX; Chou, Y; Jones, L; Wang, T; Sanchez, S; Huang, XF; Zhang, L; Wang, C; Chen, SY
Scientific reports 5 13755 2015
B cell-mediated antibody response plays critical roles in protective immunity, as well as in the pathogenesis of allergic and autoimmune diseases. Epigenetic histone and DNA modifications regulate gene transcription and immunity; however, so far, little is known about the role of epigenetic regulation in antibody responses. In this study, we found that mice deficient in the histone H2A deubiquitinase MYSM1, despite their severe defect in B cell development, exhibit an enhanced antibody response against both T cell-dependent and independent antigens. We revealed that MYSM1 intrinsically represses plasma cell differentiation and antibody production. Mechanistic studies demonstrated that MYSM1 is a transcriptional activator of Pax5, the repressors of plasma cell differentiation, by facilitating key transcriptional factor recruitment and coordinating histone modifications at the Pax5 loci. Hence, this study uncovers a critical role for MYSM1 in epigenetically repressing plasma cell differentiation and antibody production, in addition to its opposing, active role in B cell development. Importantly, this study further provides a new target and strategy to modulate antibody production and responses with profound therapeutic implications.
|Impact of flanking chromosomal sequences on localization and silencing by the human non-coding RNA XIST. |
Kelsey, AD; Yang, C; Leung, D; Minks, J; Dixon-McDougall, T; Baldry, SE; Bogutz, AB; Lefebvre, L; Brown, CJ
Genome biology 16 208 2015
X-chromosome inactivation is a striking example of epigenetic silencing in which expression of the long non-coding RNA XIST initiates the heterochromatinization and silencing of one of the pair of X chromosomes in mammalian females. To understand how the RNA can establish silencing across millions of basepairs of DNA we have modelled the process by inducing expression of XIST from nine different locations in human HT1080 cells.Localization of XIST, depletion of Cot-1 RNA, perinuclear localization, and ubiquitination of H2A occurs at all sites examined, while recruitment of H3K9me3 was not observed. Recruitment of the heterochromatic features SMCHD1, macroH2A, H3K27me3, and H4K20me1 occurs independently of each other in an integration site-dependent manner. Silencing of flanking reporter genes occurs at all sites, but the spread of silencing to flanking endogenous human genes is variable in extent of silencing as well as extent of spread, with silencing able to skip regions. The spread of H3K27me3 and loss of H3K27ac correlates with the pre-existing levels of the modifications, and overall the extent of silencing correlates with the ability to recruit additional heterochromatic features.The non-coding RNA XIST functions as a cis-acting silencer when expressed from nine different locations throughout the genome. A hierarchy among the features of heterochromatin reveals the importance of interaction with the local chromatin neighborhood for optimal spread of silencing, as well as the independent yet cooperative nature of the establishment of heterochromatin by the non-coding XIST RNA.
|The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth. |
Ohashi, M; Holthaus, AM; Calderwood, MA; Lai, CY; Krastins, B; Sarracino, D; Johannsen, E
PLoS pathogens 11 e1004822 2015
The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16(INK4A) and p14(ARF) by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex. Our results demonstrated that each EBNA3 protein forms a distinct complex with RBPJ. Mass-spectrometry revealed that the EBNA3A and EBNA3B complexes also contained the deubquitylation complex consisting of WDR48, WDR20, and USP46 (or its paralog USP12) and that EBNA3C complexes contained WDR48. Immunoprecipitation confirmed that EBNA3A, EBNA3B, and EBNA3C association with the USP46 complex. Using chromatin immunoprecipitation, we demonstrate that WDR48 and USP46 are recruited to the p14(ARF) promoter in an EBNA3C dependent manner. Mapping studies were consistent with WDR48 being the primary mediator of EBNA3 association with the DUB complex. By ChIP assay, WDR48 was recruited to the p14(ARF) promoter in an EBNA3C dependent manner. Importantly, WDR48 associated with EBNA3A and EBNA3C domains that are critical for LCL growth, suggesting a role for USP46/USP12 in EBV induced growth transformation.
|The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment. |
Yang, W; Lee, YH; Jones, AE; Woolnough, JL; Zhou, D; Dai, Q; Wu, Q; Giles, KE; Townes, TM; Wang, H
Nature communications 5 3818 2014
Polycomb Repressive Complex 1 and histone H2A ubiquitination (ubH2A) contribute to embryonic stem cell (ESC) pluripotency by repressing lineage-specific gene expression. However, whether active deubiquitination co-regulates ubH2A levels in ESCs and during differentiation is not known. Here we report that Usp16, a histone H2A deubiquitinase, regulates H2A deubiquitination and gene expression in ESCs, and importantly, is required for ESC differentiation. Usp16 knockout is embryonic lethal in mice, but does not affect ESC viability or identity. Usp16 binds to the promoter regions of a large number of genes in ESCs, and Usp16 binding is inversely correlated with ubH2A levels, and positively correlates with gene expression levels. Intriguingly, Usp16(-/-) ESCs fail to differentiate due to ubH2A-mediated repression of lineage-specific genes. Finally, Usp16, but not a catalytically inactive mutant, rescues the differentiation defects of Usp16(-/-) ESCs. Therefore, this study identifies Usp16 and H2A deubiquitination as critical regulators of ESC gene expression and differentiation.
|USP3 counteracts RNF168 via deubiquitinating H2A and γH2AX at lysine 13 and 15. |
Sharma, N; Zhu, Q; Wani, G; He, J; Wang, QE; Wani, AA
Cell cycle (Georgetown, Tex.) 13 106-14 2014
Histone ubiquitination plays a vital role in DNA damage response (DDR), which is important for maintaining genomic integrity in eukaryotic cells. In DDR, ubiquitination of histone H2A and γH2AX by the concerted action of ubiquitin (Ub) ligases, RNF168 and RNF8, generates a cascade of ubiquitination signaling. However, little is known about deubiquitinating enzymes (DUBs) that may catalyze the removal of Ub from these histones. This study demonstrated that USP3, an apparent DUB for mono-ubiquitinated H2A, is indeed the enzyme for deubiquitinating Ub conjugates of γH2AX and H2A from lysine sites, where the ubiquitination is initiated by RNF168. Here, we showed that ectopic expression of USP3 led to the deubiquitination of both H2A and γH2AX in response to UV-induced DNA damage. Moreover, ectopic USP3 expression abrogated FK2 antibody-reactive Ub-conjugate foci, which co-localize with damage-induced γH2AX foci. In addition, USP3 overexpression impaired the accumulation of downstream repair factors BRCA1 and 53BP1 at the damage sites in response to both UV and γ-irradiation. We further identified that the USP3 removes Ub at lysine 13 and 15 of H2A and γH2AX, as well as lysine 118 and 119 of H2AX in response to DNA damage. Taken together, the results suggested that USP3 is a negative regulator of ubiquitination signaling, counteracting RNF168- and RNF8-mediated ubiquitination.
|Impact of human MLL/COMPASS and polycomb complexes on the DNA methylome. |
Putiri, EL; Tiedemann, RL; Liu, C; Choi, JH; Robertson, KD
Oncotarget 5 6338-52 2014
The correlation between DNA methylation and a subset of histone post-translational modifications (positive and negative) has hinted at an underlying regulatory crosstalk between histone marks and DNA methylation in patterning the human DNA methylome, an idea further supported by corresponding alterations to both histone marks and DNA methylation during malignant transformation. This study investigated the framework by which histone marks influence DNA methylation at a genome-wide level. Using RNAi in a pluripotent human embryonic carcinoma cell line we depleted essential components of the MLL/COMPASS, polycomb repressive complex 2 (PRC2), and PRC1 histone modifying complexes that establish, respectively, the post-translational modifications H3K4me3, H3K27me3, and H2AK119ub, and assayed the impact of the subsequent depletion of these marks on the DNA methylome. Absence of H2AK119ub resulted predominantly in hypomethylation across the genome. Depletion of H3K4me3 and, surprisingly, H3K27me3 caused CpG island hypermethylation at a subset of loci. Intriguingly, many promoters were co-regulated by all three histone marks, becoming hypermethylated with loss of H3K4me3 or H3K27me3 and hypomethylated with depletion of H2AK119ub, and many of these co-regulated loci were among those commonly targeted for aberrant hypermethylation in cancer. Taken together, our results elucidate novel roles for polycomb and MLL/COMPASS in regulating DNA methylation and define targets of this regulation.
|BMI1 reprogrammes histone acetylation and enhances c-fos pathway via directly binding to Zmym3 in malignant myeloid progression. |
Shen, H; Chen, Z; Ding, X; Qi, X; Cen, J; Wang, Y; Yao, L; Chen, Y
Journal of cellular and molecular medicine 18 1004-17 2014
The polycomb group BMI1 is proved to be crucial in malignant myeloid progression. However, the underlying mechanism of the action of BMI1 in myeloid malignant progression was not well characterized. In this study, we found that the patients of both myelodysplastic syndromes and chronic myeloid leukaemia with BMI1 overexpression had a higher risk in malignant myeloid progression. In vitro gene transfection studies showed that BMI1 inhibited cell myeloid and erythroid differentiation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and histone deacetylase inhibitor sodium butyrate respectively. BMI1 also resisted apoptosis induced by arsenic trioxide. Moreover, the transcript levels of Runx1 and Pten were down-regulated in Bmi1-transfected cells in company with histone deacetylation modification. By using chromatin immunoprecipitation (ChIP) collaborated with secondary generation sequencing and verified by ChIP-PCR, we found that BMI1 directly bound to the promoter region of Zmym3, which encodes a component of histone deacetylase-containing complexes. In addition, as one of the downstream target genes of this complex, c-fos was activated with increasing histone acetylation when ZMYM3 was suppressed in the Bmi1-transfected cells. These results suggested that BMI1 may reprogramme the histone acetylation profile in multiple genes through either indirect or direct binding effects which probably contributes to the malignant progression of myeloid progenitor cells.
|Ubiquitin-specific peptidase 42 (USP42) functions to deubiquitylate histones and regulate transcriptional activity. |
Hock, AK; Vigneron, AM; Vousden, KH
The Journal of biological chemistry 289 34862-70 2014
Ubiquitin-specific peptidase 42 (USP42) is a deubiquitylating enzyme that can target p53 and contribute to the stabilization of p53 in response to stress. We now show that USP42 can also regulate transcription independently of p53. USP42 co-localized with RNA polymerase II (RNA Pol II) in nuclear foci, bound to histone H2B, and deubiquitylated H2B. Depletion of USP42 increased H2B ubiquitylation at a model promoter and decreased both basal and induced transcription from a number of promoters. These results are consistent with a role for USP42 in regulating transcription by deubiquitylating histones.
|USP49 deubiquitinates histone H2B and regulates cotranscriptional pre-mRNA splicing. |
Zhang, Z; Jones, A; Joo, HY; Zhou, D; Cao, Y; Chen, S; Erdjument-Bromage, H; Renfrow, M; He, H; Tempst, P; Townes, TM; Giles, KE; Ma, L; Wang, H
Genes & development 27 1581-95 2013
Post-translational histone modifications play important roles in regulating chromatin structure and function. Histone H2B ubiquitination and deubiquitination have been implicated in transcriptional regulation, but the function of H2B deubiquitination is not well defined, particularly in higher eukaryotes. Here we report the purification of ubiquitin-specific peptidase 49 (USP49) as a histone H2B-specific deubiquitinase and demonstrate that H2B deubiquitination by USP49 is required for efficient cotranscriptional splicing of a large set of exons. USP49 forms a complex with RuvB-like1 (RVB1) and SUG1 and specifically deubiquitinates histone H2B in vitro and in vivo. USP49 knockdown results in small changes in gene expression but affects the abundance of greater than 9000 isoforms. Exons down-regulated in USP49 knockdown cells show both elevated levels of alternative splicing and a general decrease in splicing efficiency. Importantly, USP49 is relatively enriched at this set of exons. USP49 knockdown increased H2B ubiquitination (uH2B) levels at these exons as well as upstream 3' and downstream 5' intronic splicing elements. Change in H2B ubiquitination level, as modulated by USP49, regulates U1A and U2B association with chromatin and binding to nascent pre-mRNA. Although H3 levels are relatively stable after USP49 depletion, H2B levels at these exons are dramatically increased, suggesting that uH2B may enhance nucleosome stability. Therefore, this study identifies USP49 as a histone H2B-specific deubiquitinase and uncovers a critical role for H2B deubiquitination in cotranscriptional pre-mRNA processing events.
|Usp16 contributes to somatic stem-cell defects in Down's syndrome. |
Adorno, M; Sikandar, S; Mitra, SS; Kuo, A; Nicolis Di Robilant, B; Haro-Acosta, V; Ouadah, Y; Quarta, M; Rodriguez, J; Qian, D; Reddy, VM; Cheshier, S; Garner, CC; Clarke, MF
Nature 501 380-4 2013
Down's syndrome results from full or partial trisomy of chromosome 21. However, the consequences of the underlying gene-dosage imbalance on adult tissues remain poorly understood. Here we show that in Ts65Dn mice, which are trisomic for 132 genes homologous to genes on human chromosome 21, triplication of Usp16 reduces the self-renewal of haematopoietic stem cells and the expansion of mammary epithelial cells, neural progenitors and fibroblasts. In addition, Usp16 is associated with decreased ubiquitination of Cdkn2a and accelerated senescence in Ts65Dn fibroblasts. Usp16 can remove ubiquitin from histone H2A on lysine 119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal Usp16 allele or by short interfering RNAs, largely rescues all of these defects. Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and postnatal neural progenitors, whereas downregulation of USP16 partially rescues the proliferation defects of Down's syndrome fibroblasts. Taken together, these results suggest that USP16 has an important role in antagonizing the self-renewal and/or senescence pathways in Down's syndrome and could serve as an attractive target to ameliorate some of the associated pathologies.
|Combined modulation of polycomb and trithorax genes rejuvenates β cell replication. |
Zhou, JX; Dhawan, S; Fu, H; Snyder, E; Bottino, R; Kundu, S; Kim, SK; Bhushan, A
The Journal of clinical investigation 123 4849-58 2013
Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and regeneration also decrease with age through mechanisms that are not fully understood. Age-dependent loss of enhancer of zeste homolog 2 (EZH2) prevents adult β cell replication through derepression of the gene encoding cyclin-dependent kinase inhibitor 2a (INK4a). We investigated whether replenishing EZH2 could reverse the age-dependent increase of Ink4a transcription. We generated an inducible pancreatic β cell-specific Ezh2 transgenic mouse model and showed that transgene expression of Ezh2 was sufficient to increase β cell replication and regeneration in young adult mice. In mice older than 8 months, induction of Ezh2 was unable to repress Ink4a. Older mice had an enrichment of a trithorax group (TrxG) protein complex at the Ink4a locus. Knockdown of TrxG complex components, in conjunction with expression of Ezh2, resulted in Ink4a repression and increased replication of β cells in aged mice. These results indicate that combined modulation of polycomb group proteins, such as EZH2, along with TrxG proteins to repress Ink4a can rejuvenate the replication capacity of aged β cells. This study provides potential therapeutic targets for expansion of adult β cell mass.
|Histone H2A mono-ubiquitination is a crucial step to mediate PRC1-dependent repression of developmental genes to maintain ES cell identity. |
Endoh, M; Endo, TA; Endoh, T; Isono, K; Sharif, J; Ohara, O; Toyoda, T; Ito, T; Eskeland, R; Bickmore, WA; Vidal, M; Bernstein, BE; Koseki, H
PLoS genetics 8 e1002774 2012
Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation.
|Regulation of the potential marker for intestinal cells, Bmi1, by β-catenin and the zinc finger protein KLF4: implications for colon cancer. |
Yu, T; Chen, X; Zhang, W; Colon, D; Shi, J; Napier, D; Rychahou, P; Lu, W; Lee, EY; Weiss, HL; Evers, BM; Liu, C
The Journal of biological chemistry 287 3760-8 2012
B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear β-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/β-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.
|The interaction of the von Hippel-Lindau tumor suppressor and heterochromatin protein 1. |
Lai, Y; Song, M; Hakala, K; Weintraub, ST; Shiio, Y
Archives of biochemistry and biophysics 518 103-10 2012
Inactivation of the von Hippel-Lindau (VHL) tumor suppressor is associated with renal carcinoma, hemangioblastoma and pheochromocytoma. The VHL protein is a component of a ubiquitin ligase complex that ubiquitinates and degrades hypoxia inducible factor-α (HIF-α). Degradation of HIF-α by VHL is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in tumor suppression and other biological processes. Using GST-VHL pull-down experiment and mass spectrometry, we detected an interaction between VHL and heterochromatin protein 1 (HP1). We identified a conserved HP1-binding motif (PXVXL) in the β domain of VHL, which is disrupted in a renal carcinoma-associated P81S mutant. We show that the VHL P81S mutant displays reduced binding to HP1, yet retains the ability to interact with elongin B, elongin C, and cullin 2 and is fully capable of degrading HIF-α. We also demonstrate that HP1 increases the chromatin association of VHL. These results suggest a role for the VHL-HP1 interaction in VHL chromatin targeting.
|The H3K4me3 histone demethylase Fbxl10 is a regulator of chemokine expression, cellular morphology, and the metabolome of fibroblasts. |
Janzer, A; Stamm, K; Becker, A; Zimmer, A; Buettner, R; Kirfel, J
The Journal of biological chemistry 287 30984-92 2012
Fbxl10 (Jhdm1b/Kdm2b) is a conserved and ubiquitously expressed member of the JHDM (JmjC domain-containing histone demethylase) family. Fbxl10 was implicated in the demethylation of H3K4me3 or H3K36me2 thereby removing active chromatin marks and inhibiting gene transcription. Apart from the JmjC domain, Fbxl10 consists of a CxxC domain, a PHD domain, and an Fbox domain. By purifying the JmjC and the PHD domain of Fbxl10 and using different approaches we were able to characterize the properties of these domains in vitro. Our results suggest that Fbxl10 is rather a H3K4me3 than a H3K36me2 histone demethylase. The PHD domain exerts a dual function in binding H3K4me3 and H3K36me2 and exhibiting E3 ubiquitin ligase activity. We generated mouse embryonic fibroblasts stably overexpressing Fbxl10. These cells reveal an increase in cell size but no changes in proliferation, mitosis, or apoptosis. Using a microarray approach we were able to identify potentially new target genes for Fbxl10 including chemokines, the noncoding RNA Xist, and proteins involved in metabolic processes. Additionally, we found that Fbxl10 is recruited to the promoters of Ccl7, Xist, Crabp2, and RipK3. Promoter occupancy by Fbxl10 was accompanied by reduced levels of H3K4me3 but unchanged levels of H3K36me2. Furthermore, knockdown of Fbxl10 using small interfering RNA approaches showed inverse regulation of Fbxl10 target genes. In summary, our data reveal a regulatory role of Fbxl10 in cell morphology, chemokine expression, and the metabolic control of fibroblasts.
|The critical role of histone H2A-deubiquitinase Mysm1 in hematopoiesis and lymphocyte differentiation. |
Nijnik, Anastasia, et al.
Blood, 119: 1370-9 (2012) 2012
Stem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin-modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and hematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1(tm1a/tm1a) and demonstrated defects in BM hematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation, and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes resulted from a cell-intrinsic requirement for Mysm1 in the BM. Importantly, Mysm1(tm1a/tm1a) HSCs were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the hematopoietic progenitors. Overall, these data establish a role for Mysm1 in the maintenance of BM stem cell function, in the control of oxidative stress and genetic stability in hematopoietic progenitors, and in the development of lymphoid and erythroid lineages.
|Lethal myelofibrosis induced by Bmi1-deficient hematopoietic cells unveils a tumor suppressor function of the polycomb group genes. |
Oguro, H; Yuan, J; Tanaka, S; Miyagi, S; Mochizuki-Kashio, M; Ichikawa, H; Yamazaki, S; Koseki, H; Nakauchi, H; Iwama, A
The Journal of experimental medicine 209 445-54 2012
Polycomb-group (PcG) proteins form the multiprotein polycomb repressive complexes (PRC) 1 and 2, and function as transcriptional repressors through histone modifications. They maintain the proliferative capacity of hematopoietic stem and progenitor cells by repressing the transcription of tumor suppressor genes, namely Ink4a and Arf, and thus have been characterized as oncogenes. However, the identification of inactivating mutations in the PcG gene, EZH2, unveiled a tumor suppressor function in myeloid malignancies, including primary myelofibrosis (PMF). Here, we show that loss of another PcG gene, Bmi1, causes pathological hematopoiesis similar to PMF. In a mouse model, loss of Bmi1 in Ink4a-Arf(-/-) hematopoietic cells induced abnormal megakaryocytopoiesis accompanied by marked extramedullary hematopoiesis, which eventually resulted in lethal myelofibrosis. Absence of Bmi1 caused derepression of a cohort of genes, including Hmga2, which is an oncogene overexpressed in PMF. Chromatin immunoprecipitation assays revealed that Bmi1 directly represses the transcription of Hmga2. Overexpression of Hmga2 in hematopoietic stem cells induced a myeloproliferative state with enhanced megakaryocytopoiesis in mice, implicating Hmga2 in the development of pathological hematopoiesis in the absence of Bmi1. Our findings provide the first genetic evidence of a tumor suppressor function of Bmi1 and uncover the role of PcG proteins in restricting growth by silencing oncogenes.
|Putative molecular mechanism underlying sperm chromatin remodelling is regulated by reproductive hormones. |
Gill-Sharma, MK; Choudhuri, J; Ansari, MA; D'Souza, S
Clinical epigenetics 4 23 2012
The putative regulatory role of the male reproductive hormones in the molecular mechanism underlying chromatin condensation remains poorly understood. In the past decade, we developed two adult male rat models wherein functional deficits of testosterone or FSH, produced after treatments with 20 mg/Kg/d of cyproterone acetate (CPA) per os, for a period of 15 days or 3 mg/Kg/d of fluphenazine decanoate (FD) subcutaneously, for a period of 60 days, respectively, affected the rate of sperm chromatin decondensation in vitro. These rat models have been used in the current study in order to delineate the putative roles of testosterone and FSH in the molecular mechanism underlying remodelling of sperm chromatin.We report that deficits of both testosterone and FSH affected the turnover of polyubiquitylated histones and led to their accumulation in the testis. Functional deficits of testosterone reduced expression of MIWI, the 5-methyl cap binding RNA-binding protein (PIWIlike murine homologue of the Drosophila protein PIWI/P-element induced wimpy testis) containing a PAZ/Piwi-Argonaut-Zwille domain and levels of histone deacetylase1 (HDAC1), ubiquitin ligating enzyme (URE-B1/E3), 20S proteasome α1 concomitant with reduced expression of ubiquitin activating enzyme (ube1), conjugating enzyme (ube2d2), chromodomain Y like protein (cdyl), bromodomain testis specific protein (brdt), hdac6 (histone deacetylase6), androgen-dependent homeobox placentae embryonic protein (pem/RhoX5), histones h2b and th3 (testis-specific h3). Functional deficits of FSH reduced the expression of cdyl and brdt genes in the testis, affected turnover of ubiquitylated histones, stalled the physiological DNA repair mechanism and culminated in spermiation of DNA damaged sperm.We aver that deficits of both testosterone and FSH differentially affected the process of sperm chromatin remodelling through subtle changes in the 'chromatin condensation transcriptome and proteome', thereby stalling the replacement of 'dynamic' histones with 'inert' protamines, and altering the epigenetic state of condensed sperm chromatin. The inappropriately condensed chromatin affected the sperm chromatin cytoarchitecture, evident from subtle ultrastructural changes in the nuclei of immature caput epididymal sperm of CPA- or FD-treated rats, incubated in vitro with dithiothreitol.
|Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery. |
Chakravarthy, H; Ormsbee, BD; Mallanna, SK; Rizzino, A
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 25 206-18 2011
The rapid formation of numerous tissues during development is highly dependent on the swift activation of key developmental regulators. Recent studies indicate that many key regulatory genes are repressed in embryonic stem cells (ESCs), yet poised for rapid activation due to the presence of both activating (H3K4 trimethylation) and repressive (H3K27 trimethylation) histone modifications (bivalent genes). However, little is known about bivalent gene regulation. In this study, we investigated the regulation of the bivalent gene Sox21, which is activated rapidly when ESCs differentiate in response to increases in Sox2. Chromatin immunoprecipitation demonstrated that prior to differentiation, the Sox21 gene is bound by a complex array of repressive and activating transcriptional machinery. Upon activation, all identified repressive machinery and histone modifications associated with the gene are lost, but the activating modifications and transcriptional machinery are retained. Notably, these changes do not occur when ESCs differentiate in response to retinoic acid. Moreover, ESCs lacking a functional PRC2 complex fail to activate this gene, apparently due to its association with other repressive complexes. Together, these findings suggest that bivalent genes, such as Sox21, are silenced by a complex set of redundant repressive machinery, which exit rapidly in response to appropriate differentiation signals.
|Regulation of histone H2A and H2B deubiquitination and Xenopus development by USP12 and USP46. |
Joo, HY; Jones, A; Yang, C; Zhai, L; Smith, AD; Zhang, Z; Chandrasekharan, MB; Sun, ZW; Renfrow, MB; Wang, Y; Chang, C; Wang, H
The Journal of biological chemistry 286 7190-201 2011
Post-translational histone modifications play important roles in regulating gene expression programs, which in turn determine cell fate and lineage commitment during development. One such modification is histone ubiquitination, which primarily targets histone H2A and H2B. Although ubiquitination of H2A and H2B has been generally linked to gene silencing and gene activation, respectively, the functions of histone ubiquitination during eukaryote development are not well understood. Here, we identified USP12 and USP46 as histone H2A and H2B deubiquitinases that regulate Xenopus development. USP12 and USP46 prefer nucleosomal substrates and deubiquitinate both histone H2A and H2B in vitro and in vivo. WDR48, a WD40 repeat-containing protein, interacts with USP12 and USP46 and is required for the histone deubiquitination activity. Overexpression of either gene leads to gastrulation defects without affecting mesodermal cell fate, whereas knockdown of USP12 in Xenopus embryos results in reduction of a subset of mesodermal genes at gastrula stages. Immunohistochemical staining and chromatin immunoprecipitation assays revealed that USP12 regulates histone deubiquitination in the mesoderm and at specific gene promoters during Xenopus development. Taken together, this study identifies USP12 and USP46 as histone deubiquitinases for H2A and H2B and reveals that USP12 regulates Xenopus development during gastrula stages.
|Meiotic functions of RAD18. |
Inagaki, A; Sleddens-Linkels, E; Wassenaar, E; Ooms, M; van Cappellen, WA; Hoeijmakers, JH; Seibler, J; Vogt, TF; Shin, MK; Grootegoed, JA; Baarends, WM
Journal of cell science 124 2837-50 2011
RAD18 is an ubiquitin ligase that is involved in replication damage bypass and DNA double-strand break (DSB) repair processes in mitotic cells. Here, we investigated the testicular phenotype of Rad18-knockdown mice to determine the function of RAD18 in meiosis, and in particular, in the repair of meiotic DSBs induced by the meiosis-specific topoisomerase-like enzyme SPO11. We found that RAD18 is recruited to a specific subfraction of persistent meiotic DSBs. In addition, RAD18 is recruited to the chromatin of the XY chromosome pair, which forms the transcriptionally silent XY body. At the XY body, RAD18 mediates the chromatin association of its interaction partners, the ubiquitin-conjugating enzymes HR6A and HR6B. Moreover, RAD18 was found to regulate the level of dimethylation of histone H3 at Lys4 and maintain meiotic sex chromosome inactivation, in a manner similar to that previously observed for HR6B. Finally, we show that RAD18 and HR6B have a role in the efficient repair of a small subset of meiotic DSBs.
|Histone crosstalk directed by H2B ubiquitination is required for chromatin boundary integrity. |
Ma, MK; Heath, C; Hair, A; West, AG
PLoS genetics 7 e1002175 2011
Genomic maps of chromatin modifications have provided evidence for the partitioning of genomes into domains of distinct chromatin states, which assist coordinated gene regulation. The maintenance of chromatin domain integrity can require the setting of boundaries. The HS4 insulator element marks the 3' boundary of a heterochromatin region located upstream of the chicken β-globin gene cluster. Here we show that HS4 recruits the E3 ligase RNF20/BRE1A to mediate H2B mono-ubiquitination (H2Bub1) at this insulator. Knockdown experiments show that RNF20 is required for H2Bub1 and processive H3K4 methylation. Depletion of RNF20 results in a collapse of the active histone modification signature at the HS4 chromatin boundary, where H2Bub1, H3K4 methylation, and hyperacetylation of H3, H4, and H2A.Z are rapidly lost. A remarkably similar set of events occurs at the HSA/HSB regulatory elements of the FOLR1 gene, which mark the 5' boundary of the same heterochromatin region. We find that persistent H2Bub1 at the HSA/HSB and HS4 elements is required for chromatin boundary integrity. The loss of boundary function leads to the sequential spreading of H3K9me2, H3K9me3, and H4K20me3 over the entire 50 kb FOLR1 and β-globin region and silencing of FOLR1 expression. These findings show that the HSA/HSB and HS4 boundary elements direct a cascade of active histone modifications that defend the FOLR1 and β-globin gene loci from the pervasive encroachment of an adjacent heterochromatin domain. We propose that many gene loci employ H2Bub1-dependent boundaries to prevent heterochromatin spreading.
|Human RAD18 interacts with ubiquitylated chromatin components and facilitates RAD9 recruitment to DNA double strand breaks. |
Inagaki, A; Sleddens-Linkels, E; van Cappellen, WA; Hibbert, RG; Sixma, TK; Hoeijmakers, JH; Grootegoed, JA; Baarends, WM
PloS one 6 e23155 2011
RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2.
|Polycomb group-dependent, heterochromatin protein 1-independent, chromatin structures silence retrotransposons in somatic tissues outside ovaries. |
Dufourt, J; Brasset, E; Desset, S; Pouchin, P; Vaury, C
DNA research : an international journal for rapid publication of reports on genes and genomes 18 451-61 2011
Somatic cells are equipped with different silencing mechanisms that protect the genome against retrotransposons. In Drosophila melanogaster, a silencing pathway implicating the argonaute protein PIWI represses retrotransposons in cells surrounding the oocyte, whereas a PIWI-independent pathway is involved in other somatic tissues. Here, we show that these two silencing mechanisms result in distinct chromatin structures. Using sensor transgenes, we found that, in somatic tissues outside of the ovaries, these transgenes adopt a heterochromatic configuration implicating hypermethylation of H3K9 and K27. We identified the Polycomb repressive complexes (PRC1 and 2), but not heterochromatin protein 1 to be necessary factors for silencing. Once established, the compact structure is stably maintained through cell divisions. By contrast, in cells where the silencing is PIWI-dependent, the transgenes display an open and labile chromatin structure. Our data suggest that a post-transcriptional gene silencing (PTGS) mechanism is responsible for the repression in the ovarian somatic cells, whereas a mechanism that couples PTGS to transcriptional gene silencing operates to silence retrotransposons in the other somatic tissues.
|Involvement of KDM1C histone demethylase-OTLD1 otubain-like histone deubiquitinase complexes in plant gene repression. |
Krichevsky, A; Zaltsman, A; Lacroix, B; Citovsky, V
Proceedings of the National Academy of Sciences of the United States of America 108 11157-62 2011
Covalent modifications of histones, such as acetylation, methylation and ubiquitination, are central for regulation of gene expression. Heterochromatic gene silencing, for example, is associated with hypoacetylation, methylation and demethylation, and deubiquitination of specific amino acid residues in histone molecules. Many of these changes can be effected by histone-modifying repressor complexes that include histone lysine demethylases, such as KDM1 in animals and KDM1C in plants. However, whereas KDM1-containing repressor complexes have been implicated in histone demethylation, methylation and deacetylation, whether or not they can also mediate histone deubiquitination remains unknown. We identify an Arabidopsis otubain-like deubiquitinase OTLD1 which directly interacts with the Arabidopsis KDM1C in planta, and use one target gene to exemplify that both OTLD1 and KDM1C are involved in transcriptional gene repression via histone deubiquitination and demethylation. We also show that OTLD1 binds plant chromatin and has enzymatic histone deubiquitinase activity, specific for the H2B histone. Thus, we suggest that, during gene repression, lysine demethylases can directly interact and function in a protein complex with histone deubiquitinases.
|A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells. |
Ng, K; Daigle, N; Bancaud, A; Ohhata, T; Humphreys, P; Walker, R; Ellenberg, J; Wutz, A
Molecular biology of the cell 22 2634-45 2011
In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2-tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome-bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement.
|UMI, a novel RNF168 ubiquitin binding domain involved in the DNA damage signaling pathway. |
Pinato, Sabrina, et al.
Mol. Cell. Biol., 31: 118-26 (2011) 2011
Ubiquitination regulates important cellular processes, including the DNA damage response (DDR) and DNA repair. The complexity of the ubiquitin-mediated signals is decoded by ubiquitin receptors, which contain protein modules named ubiquitin binding domains (UBDs). We previously identified a new ubiquitin ligase, RNF168, involved in DDR and endowed with two UBDs named MIU (motif interacting with ubiquitin). Here we have provided the identification of a novel UBD, the UMI (UIM- and MIU-related UBD), present in RNF168, and characterized the interaction surface with ubiquitin, centered on two Leu residues. We have demonstrated that integrity of the UMI, in addition to the MIUs, is necessary for the proper localization of RNF168 and for ubiquitination of nuclear proteins, including histone H2A. Finally, we have shown that simultaneous inactivation of UMI and MIUs prevents the recruitment to DDR foci of the crucial downstream mediator 53BP1.
|HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair. |
Baldeyron, C; Soria, G; Roche, D; Cook, AJ; Almouzni, G
The Journal of cell biology 193 81-95 2011
Heterochromatin protein 1 (HP1), a major component of constitutive heterochromatin, is recruited to DNA damage sites. However, the mechanism involved in this recruitment and its functional importance during DNA repair remain major unresolved issues. Here, by characterizing HP1α dynamics at laser-induced damage sites in mammalian cells, we show that the de novo accumulation of HP1α occurs within both euchromatin and heterochromatin as a rapid and transient event after DNA damage. This recruitment is strictly dependent on p150CAF-1, the largest subunit of chromatin assembly factor 1 (CAF-1), and its ability to interact with HP1α. We find that HP1α depletion severely compromises the recruitment of the DNA damage response (DDR) proteins 53BP1 and RAD51. Moreover, HP1α depletion leads to defects in homologous recombination-mediated repair and reduces cell survival after DNA damage. Collectively, our data reveal that HP1α recruitment at early stages of the DDR involves p150CAF-1 and is critical for proper DNA damage signaling and repair.
|Histone H2A and H2B are monoubiquitinated at AID-targeted loci. |
Borchert, GM; Holton, NW; Edwards, KA; Vogel, LA; Larson, ED
PloS one 5 e11641 2010
Somatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined.Here, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt's B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed V(H) and Sgamma3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci.Our results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation.Full Text Article
|MOF and H4 K16 acetylation play important roles in DNA damage repair by modulating recruitment of DNA damage repair protein Mdc1. |
Li, X; Corsa, CA; Pan, PW; Wu, L; Ferguson, D; Yu, X; Min, J; Dou, Y
Molecular and cellular biology 30 5335-47 2010
MOF (MYST1) is the major enzyme to catalyze acetylation of histone H4 lysine 16 (K16) and is highly conserved through evolution. Using a conditional knockout mouse model and the derived mouse embryonic fibroblast cell lines, we showed that loss of Mof led to a global reduction of H4 K16 acetylation, severe G(2)/M cell cycle arrest, massive chromosome aberration, and defects in ionizing radiation-induced DNA damage repair. We further showed that although early DNA damage sensing and signaling by ATM were normal in Mof-null cells, the recruitment of repair mediator protein Mdc1 and its downstream signaling proteins 53bp1 and Brca1 to DNA damage foci was completely abolished. Mechanistic studies suggested that Mof-mediated H4 K16 acetylation and an intact acidic pocket on H2A.X were essential for the recruitment of Mdc1. Removal of Mof and its associated proteins phenocopied a charge-neutralizing mutant of H2A.X. Given the well-characterized H4-H2A trans interactions in regulating higher-order chromatin structure, our study revealed a novel chromatin-based mechanism that regulates the DNA damage repair process.
|Alterations of histone modifications by cobalt compounds. |
Li, Q; Ke, Q; Costa, M
Carcinogenesis 30 1243-51 2009
In the present study, we examined the effects of CoCl(2) on multiple histone modifications at the global level. We found that in both human lung carcinoma A549 cells and human bronchial epithelial Beas-2B cells, exposure to CoCl(2) (greater than /=200 muM) for 24 h increased H3K4me3, H3K9me2, H3K9me3, H3K27me3, H3K36me3, uH2A and uH2B but decreased acetylation at histone H4 (AcH4). Further investigation demonstrated that in A549 cells, the increase in H3K4me3 and H3K27me3 by cobalt ions exposure was probably through enhancing histone methylation processes, as methionine-deficient medium blocked the induction of H3K4me3 and H3K27me3 by cobalt ions, whereas cobalt ions increased H3K9me3 and H3K36me3 by directly inhibiting JMJD2A demethylase activity in vitro, which was probably due to the competition of cobalt ions with iron for binding to the active site of JMJD2A. Furthermore, in vitro ubiquitination and deubiquitination assays revealed that the cobalt-induced histone H2A and H2B ubiquitination is the result of inhibition of deubiquitinating enzyme activity. Microarray data showed that exposed to 200 microM of CoCl(2) for 24 h, A549 cells not only increased but also decreased expression of hundreds of genes involved in different cellular functions, including tumorigenesis. This study is the first to demonstrate that cobalt ions altered epigenetic homeostasis in cells. It also sheds light on the possible mechanisms involved in cobalt-induced alteration of histone modifications, which may lead to altered programs of gene expression and carcinogenesis since cobalt at higher concentrations is a known carcinogen.Full Text Article
|A high incidence of meiotic silencing of unsynapsed chromatin is not associated with substantial pachytene loss in heterozygous male mice carrying multiple simple robertsonian translocations. |
Manterola, M; Page, J; Vasco, C; Berríos, S; Parra, MT; Viera, A; Rufas, JS; Zuccotti, M; Garagna, S; Fernández-Donoso, R
PLoS genetics 5 e1000625 2009
Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC). Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., gammaH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR). These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading of Robertsonian translocations, explaining the multitude of natural Robertsonian populations described in the mouse.
|Differential effects of arsenic on cutaneous and systemic immunity: focusing on CD4+ cell apoptosis in patients with arsenic-induced Bowen's disease. |
WT Liao, CL Yu, CC Lan, CH Lee, CH Chang, LW Chang, HL You, HS Yu
Carcinogenesis 30 1064-1072 2009
Bowen's disease (BD), a carcinoma in situ of the skin, has been identified as an early lesion in arsenic carcinogenesis. Patients with arsenic-induced Bowen's disease (As-BD) showed both cutaneous and systemic immune dysfunctions. We set out to evaluate the interactions between keratinocytes and lymphocytes in the context of As-BD carcinogenesis. Our results showed that As-BD lesions demonstrated a significant dermal CD4+ cell, an essential regulator of proper tumor immunity, undergoing apoptosis. In addition, it was found that the As-BD patients have lower percentage of peripheral CD4+ cells as compared with control subjects. However, the CD4+ cells from As-BD patients were less susceptible to arsenic-induced apoptosis, due to reduced tumor necrosis factor receptor 1 expression. Interestingly, arsenic was found to induce Fas expression on CD4+ cells and increase the soluble Fas ligand (sFasL) production from keratinocytes. This sFasL-containing keratinocyte supernatant was able to induce comparable CD4+ cell apoptosis for both patients and controls. Using immunofluorescent staining, increased FasL was observed in keratinocytes of As-BD lesions and Fas was expressed among infiltrating CD4+ cells. Our findings suggested that systemically, the percentage of CD4+ cells was decreased in the peripheral blood of As-BD patients. These residual CD4+ cells were less susceptible to arsenic-induced apoptosis. However, once infiltrated into the As-BD lesions, the selective CD4+ cell apoptosis might be mediated by FasL from keratinocytes. This additional tumor-anti-immune phenomenon present in the cutaneous environment provides a reasonable explanation for frequent occurrence of arsenic cancers in the skin.,
|RNF168, a new RING finger, MIU-containing protein that modifies chromatin by ubiquitination of histones H2A and H2AX. |
Pinato, Sabrina, et al.
BMC Mol. Biol., 10: 55 (2009) 2009
BACKGROUND: Modulation of chromatin structure has emerged as a critical molecular device to control gene expression. Histones undergo different post-translational modifications that increase chromatin accessibility to a number of regulatory factors. Among them, histone ubiquitination appears relevant in nuclear processes that govern gene silencing, either by inhibiting or activating transcription, and maintain genome stability, acting as scaffold to properly organize the DNA damage response. Thus, it is of paramount importance the identification and the characterization of new ubiquitin ligases that address histones. RESULTS: We identified and characterized RNF168, a new chromatin-associated RING finger protein. We demonstrated that RNF168 is endowed with ubiquitin ligase activity both in vitro and in vivo, which targets histones H2A and H2AX, but not H2B, forming K63 polyubiquitin chains. We previously described the presence within RNF168 sequence of two MIU domains, responsible for the binding to ubiquitinated proteins. Here we showed that inactivation of the MIUs impairs ubiquitin binding ability in vitro and reduces chromatin association of RNF168 in vivo. Moreover, upon formation of DNA double strand breaks induced by chemical and physical agents, RNF168 is recruited to the DNA damage foci, where it co-localizes with gammaH2AX and 53BP1. The localization of RNF168 at the site of damage highly increases the local concentration of ubiquitinated proteins and determines the prolonged ubiquitination signal. CONCLUSION: The RING finger protein RNF168 is a new ubiquitin ligase that functions as chromatin modifier, through histone ubiquitination. We hypothesize a dual function for RNF168. In normal condition RNF168 modifies chromatin structure by modulating ubiquitination of histone H2A. Upon DNA lesions, RNF168 is recruited to DNA damage response foci where it contributes to increase the amount of ubiquitinated proteins, thereby facilitating the downstream signalling cascade.
|Genome-wide uH2A localization analysis highlights Bmi1-dependent deposition of the mark at repressed genes. |
Kallin, EM; Cao, R; Jothi, R; Xia, K; Cui, K; Zhao, K; Zhang, Y
PLoS genetics 5 e1000506 2009
Polycomb group (PcG) proteins control organism development by regulating the expression of developmental genes. Transcriptional regulation by PcG proteins is achieved, at least partly, through the PRC2-mediated methylation on lysine 27 of histone H3 (H3K27) and PRC1-mediated ubiquitylation on lysine 119 of histone H2A (uH2A). As an integral component of PRC1, Bmi1 has been demonstrated to be critical for H2A ubiquitylation. Although recent studies have revealed the genome-wide binding patterns of some of the PRC1 and PRC2 components, as well as the H3K27me3 mark, there have been no reports describing genome-wide localization of uH2A. Using the recently developed ChIP-Seq technology, here, we report genome-wide localization of the Bmi1-dependent uH2A mark in MEF cells. Gene promoter averaging analysis indicates a peak of uH2A just inside the transcription start site (TSS) of well-annotated genes. This peak is enriched at promoters containing the H3K27me3 mark and represents the least expressed genes in WT MEF cells. In addition, peak finding reveals regions of local uH2A enrichment throughout the mouse genome, including almost 700 gene promoters. Genes with promoter peaks of uH2A exhibit lower-level expression when compared to genes that do not contain promoter peaks of uH2A. Moreover, we demonstrate that genes with uH2A peaks have increased expression upon Bmi1 knockout. Importantly, local enrichment of uH2A is not limited to regions containing the H3K27me3 mark. We describe the enrichment of H2A ubiquitylation at high-density CpG promoters and provide evidence to suggest that DNA methylation may be linked to uH2A at these regions. Thus, our work not only reveals Bmi1-dependent H2A ubiquitylation, but also suggests that uH2A targeting in differentiated cells may employ a different mechanism from that in ES cells.Full Text Article
|dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. |
Lagarou, A; Mohd-Sarip, A; Moshkin, YM; Chalkley, GE; Bezstarosti, K; Demmers, JA; Verrijzer, CP
Genes & development 22 2799-810 2008
Transcription regulation involves enzyme-mediated changes in chromatin structure. Here, we describe a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase dKDM2. In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression.Full Text Article
|Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity. |
Endoh, M; Endo, TA; Endoh, T; Fujimura, Y; Ohara, O; Toyoda, T; Otte, AP; Okano, M; Brockdorff, N; Vidal, M; Koseki, H
Development (Cambridge, England) 135 1513-24 2008
The Polycomb group (PcG) proteins mediate heritable silencing of developmental regulators in metazoans, participating in one of two distinct multimeric protein complexes, the Polycomb repressive complexes 1 (PRC1) and 2 (PRC2). Although PRC2 has been shown to share target genes with the core transcription network, including Oct3/4, to maintain embryonic stem (ES) cells, it is still unclear whether PcG proteins and the core transcription network are functionally linked. Here, we identify an essential role for the core PRC1 components Ring1A/B in repressing developmental regulators in mouse ES cells and, thereby, in maintaining ES cell identity. A significant proportion of the PRC1 target genes are also repressed by Oct3/4. We demonstrate that engagement of PRC1 at target genes is Oct3/4-dependent, whereas engagement of Oct3/4 is PRC1-independent. Moreover, upon differentiation induced by Gata6 expression, most of the Ring1A/B target genes are derepressed and the binding of Ring1A/B to their target loci is also decreased. Collectively, these results indicate that Ring1A/B-mediated Polycomb silencing functions downstream of the core transcriptional regulatory circuitry to maintain ES cell identity.
|Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity. |
Qingdong Ke, Thomas P Ellen, Max Costa, Qingdong Ke, Thomas P Ellen, Max Costa
Toxicology and applied pharmacology 228 190-9 2008
Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis.Full Text Article
|DNA damage triggers nucleotide excision repair-dependent monoubiquitylation of histone H2A. |
Bergink, S; Salomons, FA; Hoogstraten, D; Groothuis, TA; de Waard, H; Wu, J; Yuan, L; Citterio, E; Houtsmuller, AB; Neefjes, J; Hoeijmakers, JH; Vermeulen, W; Dantuma, NP
Genes & development 20 1343-52 2006
Chromatin changes within the context of DNA repair remain largely obscure. Here we show that DNA damage induces monoubiquitylation of histone H2A in the vicinity of DNA lesions. Ultraviolet (UV)-induced monoubiquitylation of H2A is dependent on functional nucleotide excision repair and occurs after incision of the damaged strand. The ubiquitin ligase Ring2 is required for the DNA damage-induced H2A ubiquitylation. UV-induced ubiquitylation of H2A is dependent on the DNA damage signaling kinase ATR (ATM- and Rad3-related) but not the related kinase ATM (ataxia telangiectasia-mutated). Although the response coincides with phosphorylation of variant histone H2AX, H2AX was not required for H2A ubiquitylation. Together our data show that monoubiquitylation of H2A forms part of the cellular response to UV damage and suggest a role of this modification in DNA repair-induced chromatin remodeling.Full Text Article
|Recruitment of PRC1 function at the initiation of X inactivation independent of PRC2 and silencing. |
Stefan Schoeftner, Aditya K Sengupta, Stefan Kubicek, Karl Mechtler, Laura Spahn, Haruhiko Koseki, Thomas Jenuwein, Anton Wutz, Stefan Schoeftner, Aditya K Sengupta, Stefan Kubicek, Karl Mechtler, Laura Spahn, Haruhiko Koseki, Thomas Jenuwein, Anton Wutz, Stefan Schoeftner, Aditya K Sengupta, Stefan Kubicek, Karl Mechtler, Laura Spahn, Haruhiko Koseki, Thomas Jenuwein, Anton Wutz
The EMBO journal 25 3110-22 2006
In mammals X inactivation is initiated by expression of Xist RNA and involves the recruitment of Polycomb repressive complex 1 (PRC1) and 2 (PRC2), which mediate chromosome-wide ubiquitination of histone H2A and methylation of histone H3, respectively. Here, we show that PRC1 recruitment by Xist RNA is independent of gene silencing. We find that Eed is required for the recruitment of the canonical PRC1 proteins Mph1 and Mph2 by Xist. However, functional Ring1b is recruited by Xist and mediates ubiquitination of histone H2A in Eed deficient embryonic stem (ES) cells, which lack histone H3 lysine 27 tri-methylation. Xist expression early in ES cell differentiation establishes a chromosomal memory, which allows efficient H2A ubiquitination in differentiated cells and is independent of silencing and PRC2. Our data show that Xist recruits PRC1 components by both PRC2 dependent and independent modes and in the absence of PRC2 function is sufficient for the establishment of Polycomb-based memory systems in X inactivation.Full Text Article
|Role of histone H2A ubiquitination in Polycomb silencing. |
Wang, Hengbin, et al.
Nature, 431: 873-8 (2004) 2004
Covalent modification of histones is important in regulating chromatin dynamics and transcription. One example of such modification is ubiquitination, which mainly occurs on histones H2A and H2B. Although recent studies have uncovered the enzymes involved in histone H2B ubiquitination and a 'cross-talk' between H2B ubiquitination and histone methylation, the responsible enzymes and the functions of H2A ubiquitination are unknown. Here we report the purification and functional characterization of an E3 ubiquitin ligase complex that is specific for histone H2A. The complex, termed hPRC1L (human Polycomb repressive complex 1-like), is composed of several Polycomb-group proteins including Ring1, Ring2, Bmi1 and HPH2. hPRC1L monoubiquitinates nucleosomal histone H2A at lysine 119. Reducing the expression of Ring2 results in a dramatic decrease in the level of ubiquitinated H2A in HeLa cells. Chromatin immunoprecipitation analysis demonstrated colocalization of dRing with ubiquitinated H2A at the PRE and promoter regions of the Drosophila Ubx gene in wing imaginal discs. Removal of dRing in SL2 tissue culture cells by RNA interference resulted in loss of H2A ubiquitination concomitant with derepression of Ubx. Thus, our studies identify the H2A ubiquitin ligase, and link H2A ubiquitination to Polycomb silencing.
|Chromatin Immunoprecipitation (ChIP)||15386022|
|Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase. |
Baarends, Willy M, et al.
Mol. Cell. Biol., 23: 1151-62 (2003) 2003
The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.
|Histone ubiquitination and chromatin remodeling in mouse spermatogenesis. |
Baarends, W M, et al.
Dev. Biol., 207: 322-33 (1999) 1999
Male infertility in HR6B knockout mice is associated with impairment of spermatogenesis. The HR6B gene is a mammalian, autosomal homolog of the Saccharomyces cerevisiae gene Rad6 encoding a ubiquitin-conjugating enzyme. In addition, X-chromosomal HR6A has been identified, in human and mouse. RAD6 in yeast is required for a variety of cellular functions, including sporulation, DNA repair, and mutagenesis. Since RAD6 and its mammalian homologs can ubiquitinate histones in vitro, we have investigated the pattern of histone ubiquitination in mouse testis. By immunoblot and immunohistochemical analysis of wild-type mouse testis, a high amount of ubiquitinated H2A (uH2A) was detected in pachytene spermatocytes. This signal became undetectable in round spermatids, but then increased again during a relatively short developmental period, in elongating spermatids. No other ubiquitinated histones were observed. In the HR6B knockout mice, we failed to detect an overt defect in the overall pattern of histone ubiquitination. For somatic cell types, it has been shown that histone ubiquitination is associated with destabilization of nucleosomes, in relation to active gene transcription. Unexpectedly, the most intense uH2A signal in pachytene spermatocytes was detected in the sex body, an inactive nuclear structure that contains the heterochromatic X and Y chromosomes. The postmeiotic uH2A immunoexpression in elongating spermatids indicates that nucleosome destabilization induced by histone ubiquitination may play a facilitating role during histone-to-protamine replacement.
|The levels of ubiquitinated histone H2A are highly upregulated in transformed human cells: partial colocalization of uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase. |
Vassilev, A P, et al.
J. Cell. Sci., 108 ( Pt 3): 1205-15 (1995) 1995
The levels and distribution of ubiquitinated histone H2A (uH2A) have been studied in normal and transformed human cells using a monoclonal antibody (mAb E6C5) that reacts specifically with this ubiquitin conjugate as determined by two-dimensional gel western blotting and microsequencing. Immunoblotting experiments demonstrated that the levels of the protein are highly upregulated in SV40-transformed human fibroblasts (WI-38 SV40) and keratinocytes (K14) relative to their normal counterparts, a finding that was further confirmed by indirect immunofluorescence studies of formaldehyde/Triton X-100-treated cells, which showed that about 97% of the transformed cells and 26% of the normal populations reacted with the antibody to yield a fine granular nuclear staining associated with the chromatin. Transformed cells contained in addition clusters of uH2A that were quite abundant and that showed variable size, shape and distribution even within a single cell line. The clusters, which were rare in normal cells, did not colocalize with other known nuclear antigens and may correspond to novel nuclear domains where ubiquitination/deubiquitination takes place. Electron microscopic immunocytochemistry of K14 cells confirmed the existence of the clusters. Double immunofluorescence studies of K14 keratinocytes with proliferating cell nuclear antigen (PCNA)/cyclin antibodies, which react with the nuclei of cells engaged in DNA replication, showed partial colocalization of PCNA/cyclin foci and large uH2A clusters in about 14% of the S-phase cells, and these corresponded mainly to late S-phase cells. Inhibition of DNA replication with hydroxyurea resulted in an overall increase in the intensity of the uH2A staining as well as in a more clear colocalization of uH2A clusters and PCNA/cyclin foci. Taken together, the results support the contention that uH2A plays a role at some stage of DNA replication.
|Product Selection Guide - Antibodies, small molecule inhibitors, kits, assays and proteins for signaling research.|