Chipping away at gamma-H2AX foci.
The mammalian histone H2AX protein functions as a dosage-dependent genomic caretaker and tumor suppressor. Phosphorylation of H2AX to form gamma-H2AX in chromatin around DNA double strand breaks (DSBs) is an early event following induction of these hazardous lesions. For a decade, mechanisms that regulate H2AX phosphorylation have been investigated mainly through two-dimensional immunofluorescence (IF). We recently used chromatin immunoprecipitation (ChIP) to measure gamma-H2AX densities along chromosomal DNA strands broken in G(1) phase mouse lymphocytes. Our experiments revealed that (1) gamma-H2AX densities in nucleosomes form at high levels near DSBs and at diminishing levels farther and farther away from DNA ends, and (2) ATM regulates H2AX phosphorylation through both MDC1-dependent and MDC1-independent means. Neither of these mechanisms were discovered by previous if studies due to the inherent limitations of light microscopy. Here, we compare data obtained from parallel gamma-H2AX ChIP and three-dimensional IF analyses and discuss the impact of our findings upon molecular mechanisms that regulate H2AX phosphorylation in chromatin around DNA breakage sites.
Savic
Velibor
V
Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Sanborn
Keri B
KB
Orange
Jordan S
JS
Bassing
Craig H
CH
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D016193:G1 Phase; D006657:Histones / Q000032:analysis / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D025521:Tumor Suppressor Proteins; C508937:MDC1 protein, mouse; C576325:Atm protein, mouse; C475961:gamma-H2AX protein, mouse