title: Systematic identification of functional residues in mammalian histone H2AX.
abstract: The histone variant H2AX is a principal component of chromatin involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). H2AX is thought to operate primarily through its C-terminal S139 phosphorylation, which mediates the recruitment of DNA damage response (DDR) factors to chromatin at DSB sites. Here, we describe a comprehensive screen of 67 residues in H2AX to determine their contributions to H2AX functions. Our analysis revealed that H2AX is both sumoylated and ubiquitylated. Individual residues defective for sumoylation, ubiquitylation, and S139 phosphorylation in untreated and damaged cells were identified. Specifically, we identified an acidic triad region in both H2A and H2AX that is required in cis for their ubiquitylation. We also report the characterization of a human H2AX knockout cell line, which exhibits DDR defects, including p53 activation, following DNA damage. Collectively, this work constitutes the first genetic complementation system for a histone in human cells. Finally, our data reveal new roles for several residues in H2AX and define distinct functions for H2AX in human cells.
authors.affiliation: Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas, USA.
authors.forename: Wei-Ta
authors.initials: WT
authors.lastname: Chen
authors.forename: Amir
authors.initials: A
authors.lastname: Alpert
authors.forename: Courtney
authors.initials: C
authors.lastname: Leiter
authors.forename: Fade
authors.initials: F
authors.lastname: Gong
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Jackson
authors.forename: Kyle M
authors.initials: KM
authors.lastname: Miller
mesh_terms: D000409:Alanine; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics; D055786:Gene Knockout Techniques; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000378:metabolism; D058207:Sumoylation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D000071857:Tumor Suppressor p53-Binding Protein 1; D054875:Ubiquitination
chemical_list: C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C498301:TP53BP1 protein, human; D016159:Tumor Suppressor Protein p53; D000071857:Tumor Suppressor p53-Binding Protein 1; D012694:Serine; D000409:Alanine