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Examination of histone H2B ubiquitination in wild type and USP49 knockdown cells [ChIP-Seq]

ABSTRACT: Posttranslational 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 USP49 as a histone H2B specific deubiquitinase and demonstrate that H2B deubiquitination by USP49 is required for efficient co-transcriptional splicing of a large set of exons. USP49 forms a complex with 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 over 9,000 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 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 co-transcriptional pre-mRNA processing events. Examination of histone H2B ubiquitination in wild type and USP49 knockdown cells [ChIP-Seq]

ORGANISM(S): Homo sapiens

SUBMITTER: Hengbin Wang

PROVIDER: E-GEOD-38099 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Examination of gene expression in wild type and USP49 knockdown cells [RNA-Seq]

Project description:Posttranslational 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 USP49 as a histone H2B specific deubiquitinase and demonstrate that H2B deubiquitination by USP49 is required for efficient co-transcriptional splicing of a large set of exons. USP49 forms a complex with 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 over 9,000 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 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 co-transcriptional pre-mRNA processing events. Examination of gene expression in wild type and USP49 knockdown cells [RNA-Seq]

2013-07-28 | E-GEOD-38100 | biostudies-arrayexpress

Examination of gene expression in wild type and USP49 knockdown cells [RNA-Seq]

2013-07-28 | GSE38100 | GEO

Examination of histone H2B ubiquitination in wild type and USP49 knockdown cells [ChIP-Seq]

2013-07-28 | GSE38099 | GEO

Co-transcriptional histone H2B monoubiquitylation is tightly coupled with RNA polymerase II elongation rate

Project description:Various histone modifications decorate nucleosomes within transcribed genes. Among these, monoubiquitylation of histone H2B (H2Bub1) and methylation of histone H3 on lysines 36 (H3K36me2/3) and 79 (H3K79me2/3) correlate positively with gene expression. By measuring the progression of the transcriptional machinery along genes within live cells, we now report that H2B monoubiquitylation occurs co-transcriptionally and accurately reflects the advance of RNA polymerase II (Pol II). In contrast, H3K36me3 and H3K79me2 are less dynamic and represent Pol II movement less faithfully. High resolution ChIP-seq reveals that H2Bub1 levels are selectively reduced at exons, and decrease in an exon-dependent stepwise manner towards the 3' end of genes. Exonic depletion of H2Bub1 in gene bodies is highly correlated with Pol II pausing at exons, suggesting elongation rate changes associated with intron-exon structure. Overall, our data shed light on the organization of H2Bub1 within transcribed genes, and single out H2Bub1 as a reliable marker for ongoing transcription elongation. H2Bub1 and H2B ChIP-seq in NT2 cells

2014-07-15 | E-GEOD-56279 | biostudies-arrayexpress

Genome-wide maps of chromatin state in NCCIT cells

Project description:H2B mono-ubiquitylation is required for multiple methylations of both H3K4 and H3K79 and has been implicated in gene expression from yeast to human. However, molecular crosstalk between H2BUb1 and other modifications, especially H3K4 and H3K79 methylations, remains unclear in vertebrates. To understand the functional role of H2BUb1, genome-wide histone modification patterns were measured in human cells. This study proposes dual roles of H2BUb1 that are both H3 methylation dependent and independent. First, H2BUb1 is a 5'-enriched active transcription mark and is co-occupied with H3K79 methylations in actively transcribed regions. Importantly, this study found a unique role of H2BUb1 in chromatin architecture independent of histone H3 methylations. H2BUb1 is well positioned in exon-intron boundaries of highly expressed exons and is specifically enriched in 5'-biased exons. Furthermore, H2BUb1 demonstrates increased occupancy in skipped exons compared to flanking exons for the human and mouse genome. Our findings suggest that a potentiating mechanism links H2BUb1 to both H3K79 methylations in actively transcribed regions and the exon-intron structure of highly expressed exons through the regulation of nucleosome dynamics during transcription elongation. We generated high-throughput sequencing (ChIP-seq) data for genome-wide occupancy of H2BUb1, nucleosome, H3K4me3, H3K36me3, H379me1/2/3, H3Ac, and mRNA in human embryonic carcinoma cells. We performed ChIP-seq for seven different histone modifications, MNase-seq, mRNA-seq (two replications), and inputDNA-seq in NCCIT cell lines (human embryonic carcinoma cell lines). We also performed mRNA-seq for RNF20-siRNA transfected NCCIT cells, where H2BUb1 signals decreased.

2012-03-07 | E-GEOD-25882 | biostudies-arrayexpress

Genome-wide maps of Usp16, ubH2A and gene expression profiles in wild type and Usp16 knock out mouse embryonic stem cells (ESCs) and embryoid bodies (EBs)

Project description: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 the histone H2A deubiquitinase Usp16 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 correlated with gene expression levels. Intriguingly, Usp16-/- ESCs fail to differentiate due to ubH2A-mediated repression of lineage-specific genes. Finally, Usp16, but not the enzymatically 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. Examination of binding pattern of H2A deubiquitinase Usp16 and ubH2A in mouse embryonic stem cells and embroid bodies

2014-05-02 | E-GEOD-52899 | biostudies-arrayexpress

The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription [Yeast cells]

Project description:The SAGA co-activator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide co-factor for all RNA Polymerase II transcription. Comparison of H3K9ac, H2Bub and RNA Pol II distributions in WT yeast cells and upon the loss of SAGA activities.

2014-08-29 | E-GEOD-52997 | biostudies-arrayexpress

Genomic expression comparison between WT and H2B(K123R)

Project description:The study aims to elucidate the effect of mono-ubiquitination of histone H2B at K123. The WT and H2B(K123R) yeast in early stationary phase were inoculated in fresh YPD rich media at OD 0.3. The cells were then grown to OD2.0 and their total RNA was isolated for genomic expression profiling with S98 arrays (Affymetrix). The experiments were performed in triplicate.

2014-03-17 | E-GEOD-17527 | biostudies-arrayexpress

Intragenic DNA methylation modulates alternative splicing by recruiting MeCP2 to promote exon recognition [RNA-Seq]

Project description:Although the function of DNA methylation in gene promoter regions is well established in transcriptional repression, the function of the evolutionarily conserved widespread distribution of DNA methylation in gene body regions remains incompletely understood. Here, we show that DNA methylation is enriched in included alternatively spliced exons (ASEs) and inhibiting DNA methylation results in aberrant splicing of ASEs. The methyl-CpG binding protein MeCP2 is enriched in included ASEs, particularly those that are also highly DNA methylated, and inhibition of DNA methylation disrupts specific targeting of MeCP2 to exons. Interestingly, ablation of MeCP2 results in increased nucleosome acetylation and aberrant skipping events of ASEs. We further show that inhibition of histone deacetylases leads to a highly significant overlap of exon skipping events caused by knocking-down MeCP2. Together, our data indicate that intragenic DNA methylation operates in exon definition to modulate alternative splicing and can enhance exon recognition via recruitment of the multifunctional protein MeCP2, which thereby maintains local histone hypoacetylation through its established interaction with HDACs. RNA-Seq in IMR90 and HCT116

2013-08-13 | E-GEOD-47488 | biostudies-arrayexpress

The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription [HeLa cells]

Project description:The SAGA co-activator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide co-factor for all RNA Polymerase II transcription. Comparison of H3K9ac and H2Bub distributions in control HeLa cells and upon the inactivation of SAGA enzymatic activities

2014-08-29 | E-GEOD-52860 | biostudies-arrayexpress

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