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.
Project description:In Penicillium oxalicum, histone H2B (PDE_01489) was one of the interacting proteins with the methyltransferase LaeA. It suggests that histone H2B may be a direct target of LaeA/Lae1. To verify whether histone H2B is one of the LaeA targets, an in vitro catalytic experiment was performed using Homo sapiens recombinant histone H2B (HsH2B, expressed in Escherichia coli, without histone modification), SAM as substrates, and LaeA. The products were analyzed by LC-MS/MS for histone modifications. Results showed that the mono and di-methylation of lysine 108 and the mono-methylation of lysine 116 were detected.
Project description:Rnf20 catalyzes lysine 120 mono-ubiquitination of histone H2B (H2Bub1) that has been previously involved in normal differentiation of embryonic stem (ES) and adult stem cells. However, the mechanisms underlying by which Rnf20 is recruited to its target chromosomal loci to generate H2Bub1 are still elusive. Here, we reveal that Fbxl19, a CxxC domain-containing protein, physically interacts with Rnf20, guides it preferentially to CpG island-containing target promoters, and thereby promotes mono-ubiqutination of H2B. We first show that up-regulation of Fbxl19 induces the level of global H2Bub1, while down-regulation of Fbxl19 reduces the level of H2Bub1 in mouse ES cells. Our genome-wide target mapping unveils the preferential occupancy of Fbxl19 on CpG island-containing promoters, and we further show that the binding of Fbxl19 is essential for the recruitment of Rnf20 to its target genes and subsequent H2Bub1. Altogether, our results demonstrate that Fbxl19 plays critical roles in the H2Bub1 pathway by recruiting Rnf20 to CGI target genes specifically and selectively.
Project description:Rnf20 catalyzes lysine 120 mono-ubiquitination of histone H2B (H2Bub1) that has been previously invloved in normal differentiation of embryonic stem (ES) and adult stem cells. However,the mechanims underlying by which Rnf20 is recruited to its target chromosomal loci to generate H2Bub1 is still elusive. Here, we reveal that Fbxl19, a CxxC domain-containing protein, physically interacts with Rnf20, guides it preferentially to CpG island-containing target promoters, and thereby promotes mono-ubiqutination of H2B. We first show that up-regulation of Fbxl19 induces the level of global H2Bub1, while down-regulation of Fbxl19 reduces the level of H2Bub1 in mouse ES cells. Our genome-wide target mapping unveils the preferential occupancy of Fbxl19 on CpG island-containing promoters, and we further show that the binding of Fbxl19 is essential for the recruitment of Rnf20 to its target genes and subsequent H2Bub1. Altogether, our results demonstrate that Fbxl19 plays critical roles in the H2Bub1 pathway by recruiting Rnf20 to CGI target genes specifically and selectively.
Project description:Mechanotransduction plays a critical role in regulating cell growth, proliferation, and metabolism. Cells respond to mechanical signals, which ultimately induces gene expression. However, the underlying mechanisms of this gene regulation remain largely unclear. In previous research, we discovered that the ubiquitin-conjugating enzymes UBE2A and UBE2B translocate between the cytoplasm and nucleus in a force-dependent manner. Additionally, we confirmed that UBE2A and UBE2B ubiquitinate histone H2B at lysine 120 (K120) when cells are cultured on a stiff substrate. In this study, we employed Chromatin Immunoprecipitation (ChIP) to investigate the distribution of H2B mono-ubiquitination in human skeletal muscle (hsSKM) cells cultured on substrates of varying stiffness—soft (0.2 kPa) and stiff (64 kPa).
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 histone H2B ubiquitination in wild type and USP49 knockdown cells [ChIP-Seq]
Project description:We have found that histone H2B is GlcNAcylated at residue S112 by O-GlcNAc transferase and that H2B S112 GlcNAcylation fluctuates in response to extracellular glucose level. We have also found that H2B S112 GlcAcylation promotes H2B K120 ubiquitination. To investigate whether these histone modification correlate to transcriptional activation, we performed comprehensive gene expression analysis using Affymetrix GeneChip in HeLa cell cultured with different conditions, i.e. without glucose, with glucose and with FBS.