Dynamic Histone H2B Ubiquitination Regulates Co-transcriptional mRNA Processing of STAT1 inducible genes

The eukaryotic genome is packaged into chromatin. Chromatin is the ensemble of genomic DNA and a large number of proteins. Alteration of the chromatin structure by covalent modifications plays a key role in the regulation of gene expression. However, the molecular mechanism of chromatin modifications in inducible gene expression is poorly defined. Signal transducer and activator of transcription (STAT) activation of gene expression is both rapid and transient, and requires dynamic post-translational modification of the chromatin template. Using chromatin immunoprecipitation assays, I show that histone H2B monoubiquitination (ubH2B) is highly dynamic at interferon regulatory factor 1 (IRF1) gene during interferon gamma (IFN-) induction. RNF20, the major E3 ligase for histone H2B, is recruited to IRF1 during STAT1 signaling. shRNAmediated depletion of RNF20 reduces global and IRF1 specific ubH2B, but enhances IFN- induced IRF1 transcription. Furthermore, RNF20 ablation diminishes histone H3 methylation at lysine 4 (H3K4), providing support for a cross-talk mechanism in which ubH2B promotes H3K4 methylation. Importantly, loss of ubH2B in RNF20 depleted cells attenuates phosphorylation of the C-terminal domain (CTD) of RNA Polymerase II at both serine 2 (Ser2P) and 5 (Ser5P) residues. Ubiquitin-specific protease 22 (USP22) removes ubiquitin from monoubiquitinated H2B. Downregulation of USP22 by shRNA significantly increases global and IRF1 specific ubH2B, but reduces the expression of STAT1-target genes. Importantly, I demonstrate that increased ubH2B in USP22 depleted cells impairs 3-end processing of STAT1-governed genes. USP22 knockdown disrupts the recruitment of the cleavage and polyadenylation specificity factor (CPSF73) and decreases Ser2P in the CTD of RNA Polymerase II at IRF1. The catalytic activity of USP22 depends upon interactions with other members of the deubiquitination module (DUBm) of hSAGA. Accordingly, depletion of the DUBm component, ATXN7L3, also iv results in defective 3'-end RNA processing. Taken together, my findings imply that a dynamic equilibrium of H2B ubiquitination and deubiquitination regulates IRF1 transcription and 3-end processing by controlling the phosphorylation of the CTD of RNA Polymerase II. My results revealed a novel role of USP22 during 3-end processing of STAT1 genes and also help explain the molecular mechanism for transcriptional regulation by histone H2B ubiquitination. v I am indebted to my parents, for this work would not have been possible without their encouragement, love and support. My heartfelt thanks goes to my young brother, Admire, who passed before I could finish this thesis. I love you & may peace be with you until we meet again. We miss you everyday.

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