GlcNAcylation of histone H2B facilitates its monoubiquitination
Ontology highlight
ABSTRACT: This SuperSeries is composed of the following subset Series: GSE33049: GlcNAcylation of histone H2B facilitates its monoubiquitination [Illumina Genome Analyzer data] GSE33050: GlcNAcylation of histone H2B facilitates its monoubiquitination [Affymetrix data] Refer to individual Series
Project description:We report that histone GlcNAcylation of H2B S112 is a vital histone modification which facilitates histone monoubiquitination (ub). In a genome-wide analysis, H2B S112 GlcNAcylation sites were observed widely distributed over entire chromosomes including transcribed gene loci, together with co-localization of H2B S112 GlcNAcylation and K120 ub. Examination of H2B S112 GlcNAc and H2B K120 ub in HeLa S3 cells
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. HeLa cells were cultured in DMEM with the following three conditions, 1) DMEM without glucose for 24 hours, 2) DMEM without glucose for 24 hours and followed by treatment with 4.5 g/L glucose for another 24 hours, 3) normal culture condition (DMEM with FBS). Total RNA was purified from these cells and each RNA was linearly amplified and hybridized to Affymetrix GeneChip.
Project description:We report that histone GlcNAcylation of H2B S112 is a vital histone modification which facilitates histone monoubiquitination (ub). In a genome-wide analysis, H2B S112 GlcNAcylation sites were observed widely distributed over entire chromosomes including transcribed gene loci, together with co-localization of H2B S112 GlcNAcylation and K120 ub.
2011-10-27 | GSE33049 | GEO
Project description:GlcNAcylation of histone H2B facilitates its monoubiquitination
Project description:Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing 13C-labeled UDP-GlcNAc from 13C6-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine site-specific protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 159 O-GlcNAc sites on 74 proteins and determine turnover rates of 24 O-GlcNAc peptides from 21 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein O-GlcNAcylation from different biological states and will provide more information on the significance of site-specific O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification in a site-specific manner for the first time.
Project description:OGT (O-GlcNAc transferase) is the distinctive enzyme responsible for catalyzing O-GlcNAc to the serine or threonine residues of thousands of cytoplasm and nuclear proteins that are involved in DNA damage, RNA splicing, and transcription preinitiation and initiation complex assembly. However, the molecular mechanism by OGT regulating gene transcription remains elusive. Using proximity labeling based mass spectrometry, we searched for functional partners of OGT and found that Dot1L, the conserved and unique histone methyltransferase mediated histone H3 lys79 methylation required for gene transcription, DNA damage repair, cell proliferation, and embryo development, interacts with OGT. Although this specific interaction does not regulate the enzymatic activity of Dot1L, it facilitates OGT-dependent histones O-GlcNAcylation. Moreover, OGT associates with Dot1L at transcription start sites, and depleting Dot1L decreased OGT associated with chromatin globally. Notably, downregulation of Dot1L reduces the levels of histone H2B S112 O-GlcNAcylation and histone H2B K120 ubiquitination in vivo, which are associated with gene transcription regulation. Taken together, these results reveal a Dot1L-dependent O-GlcNAcylation of chromatin.
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.