Key Spec 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|
|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 specification and global reprogramming of histone epigenetic marks during gamete formation and early embryo development in C. elegans.|
Samson, M; Jow, MM; Wong, CC; Fitzpatrick, C; Aslanian, A; Saucedo, I; Estrada, R; Ito, T; Park, SK; Yates, JR; Chu, DS
PLoS genetics e1004588 2014
In addition to the DNA contributed by sperm and oocytes, embryos receive parent-specific epigenetic information that can include histone variants, histone post-translational modifications (PTMs), and DNA methylation. However, a global view of how such marks are erased or retained during gamete formation and reprogrammed after fertilization is lacking. To focus on features conveyed by histones, we conducted a large-scale proteomic identification of histone variants and PTMs in sperm and mixed-stage embryo chromatin from C. elegans, a species that lacks conserved DNA methylation pathways. The fate of these histone marks was then tracked using immunostaining. Proteomic analysis found that sperm harbor ∼2.4 fold lower levels of histone PTMs than embryos and revealed differences in classes of PTMs between sperm and embryos. Sperm chromatin repackaging involves the incorporation of the sperm-specific histone H2A variant HTAS-1, a widespread erasure of histone acetylation, and the retention of histone methylation at sites that mark the transcriptional history of chromatin domains during spermatogenesis. After fertilization, we show HTAS-1 and 6 histone PTM marks distinguish sperm and oocyte chromatin in the new embryo and characterize distinct paternal and maternal histone remodeling events during the oocyte-to-embryo transition. These include the exchange of histone H2A that is marked by ubiquitination, retention of HTAS-1, removal of the H2A variant HTZ-1, and differential reprogramming of histone PTMs. This work identifies novel and conserved features of paternal chromatin that are specified during spermatogenesis and processed in the embryo. Furthermore, our results show that different species, even those with diverged DNA packaging and imprinting strategies, use conserved histone modification and removal mechanisms to reprogram epigenetic information.
|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.
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