title: Monoubiquitination of H2AX protein regulates DNA damage response signaling.
abstract: Double strand breaks (DSBs) are the most deleterious of the DNA lesions that initiate genomic instability and promote tumorigenesis. Cells have evolved a complex protein network to detect, signal, and repair DSBs. In mammalian cells, a key component in this network is H2AX, which becomes rapidly phosphorylated at Ser(139) (γ-H2AX) at DSBs. Here we show that monoubiquitination of H2AX mediated by the RNF2-BMI1 complex is critical for the efficient formation of γ-H2AX and functions as a proximal regulator in DDR (DNA damage response). RNF2-BMI1 interacts with H2AX in a DNA damage-dependent manner and is required for monoubiquitination of H2AX at Lys(119)/Lys(120). As a functional consequence, we show that the H2AX K120R mutant abolishes H2AX monoubiquitination, impairs the recruitment of p-ATM (Ser(1981)) to DSBs, and thereby reduces the formation of γ-H2AX and the recruitment of MDC1 to DNA damage sites. These data suggest that monoubiquitination of H2AX plays a critical role in initiating DNA damage signaling. Consistent with these observations, impairment of RNF2-BMI1 function by siRNA knockdown or overexpression of the ligase-dead RNF2 mutant all leads to significant defects both in accumulation of γ-H2AX, p-ATM, and MDC1 at DSBs and in activation of NBS1 and CHK2. Additionally, the regulatory effect of RNF2-BMI1 on γ-H2AX formation is dependent on ATM. Lacking their ability to properly activate the DNA damage signaling pathway, RNF2-BMI1 complex-depleted cells exhibit impaired DNA repair and increased sensitivity to ionizing radiation. Together, our findings demonstrate a distinct monoubiquitination-dependent mechanism that is required for H2AX phosphorylation and the initiation of DDR.
authors.affiliation: Department of Systems Biology, Unit 950, The University of Texas MD Anderson Cancer Center, Houston, Texas 77054, USA.
authors.forename: Mei-Ren
authors.initials: MR
authors.lastname: Pan
authors.forename: Guang
authors.initials: G
authors.lastname: Peng
authors.forename: Wen-Chun
authors.initials: WC
authors.lastname: Hung
authors.forename: Shiaw-Yih
authors.initials: SY
authors.lastname: Lin
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D000818:Animals; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D063150:Polycomb Repressive Complex 1; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D004268:DNA-Binding Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D063150:Polycomb Repressive Complex 1; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D044767:Ubiquitin-Protein Ligases; C069077:BMI1 protein, human; C489421:Bmi1 protein, mouse; C468783:H2AX protein, human; C413163:MDC1 protein, human; C508937:MDC1 protein, mouse; C489841:RNF2 protein, human; C489842:Rnf2 protein, mouse; C578634:CHEK2 protein, human; C578633:Chek2 protein, mouse; C475961:gamma-H2AX protein, mouse