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Recent evidence from a wide variety of biological systems has indicated an important regulatory role 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 cellular response to DNA damage, yet little is known about the functional implications of this modification. To investigate the role of histone 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 are efficiently expressed localizing exclusively to the interphase nucleus and to condensed chromosomes during mitosis. Moreover, biochemical fractionation indicates that epitope-tagged H2AX proteins are incorporated into nucleosomes. Expression of H2AX Ser139Ala, which disrupts the phosphorylation site, partially abrogated G2/M arrest following ionizing radiation. Conversely, expression of H2AX Ser139Glu, designed as a phosphorylation mimic, induced G2/M arrest in the absence of DNA damage. G2/M arrest induced by H2AX Ser139Glu was independent of the formation of 53BP1-containing foci and ATM activation. In addition, stable cell lines expressing H2AX Ser139Glu or H2AX Ser139Ala had a lower mitotic index than wild-type H2AX. Further analyses revealed that expression of either mutant induced apoptosis and induced higher caspase-3/7 activity compared to expression of wild-type H2AX. Taken together these results demonstrate a role for H2AX Serine 139 phosphorylation in cell cycle regulation and apoptosis.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]