Ectopic expression of histone H2AX mutants reveals a role for its post-translational modifications

Cancer Biol Ther. 2009 Mar;8(5):422-34. doi: 10.4161/cbt.8.5.7592. Epub 2009 Mar 9.


Recent evidence from a wide variety of biological systems has indicated important regulatory roles for post-translation histone modifications in cellular processes such as regulation of gene expression, DNA damage response and recombination. Phosphorylation of histone H2AX at serine 139 is a critical event in the response to DNA damage, but the functional implications of this modification are not yet clear. To investigate the role of H2AX phosphorylation we ectopically expressed epitope-tagged H2AX or mutants at the phosphorylation site. GFP-tagged wild type H2AX, H2AX Ser139Ala or H2AX Ser139Glu proteins were efficiently expressed, localizing exclusively to the interphase nucleus and to condensed chromosomes during mitosis. Biochemical fractionation indicated that epitope-tagged H2AX proteins are incorporated into nucleosomes. Expression of H2AX Ser139Ala, which disrupts the phosphorylation site partially suppressed early G(2)/M arrest following ionizing radiation, and cells expressing this mutant were more sensitive to DNA damage. Conversely, expression of H2AX Ser139Glu, designed as phosphorylation mimic, induced a decrease in the number of cells in mitosis in the absence of DNA damage. Interestingly, this decrease induced by H2AX Ser139Glu was independent of the formation of 53BP1-containing foci and was partially suppressed in CHK2-deficient cells, suggesting a role for CHK2 in this process. Further analyses revealed that expression of either mutant lead to apoptosis and induced higher caspase-3/7 activity compared to expression of wild type H2AX. In addition, we also identified Lys119 as a site for ubiquitination that controls H2AX half-life. Phosphorylation of Ser139 and ubiquitination of K119 are not interdependent. Taken together these results demonstrate a role for H2AX Serine 139 phosphorylation in cell cycle regulation and apoptosis, and for Lysine 119 in the control of H2AX turnover.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Apoptosis / genetics
  • Apoptosis / physiology
  • Blotting, Western
  • Cell Cycle / genetics
  • Cell Cycle / physiology
  • Cell Line
  • Chromosome Aberrations
  • Fluorescent Antibody Technique
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HCT116 Cells
  • HeLa Cells
  • Histones / genetics
  • Histones / metabolism*
  • Humans
  • Immunoprecipitation
  • Lysine / genetics
  • Lysine / metabolism
  • Mutation*
  • Phosphorylation
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Processing, Post-Translational*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Serine / genetics
  • Serine / metabolism
  • Transfection
  • Ubiquitination


  • H2AX protein, human
  • Histones
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Serine
  • Proteasome Endopeptidase Complex
  • Lysine