Ectopic expression of Histone H2AX mutants reveal a role for its post-translational modifications

Abstract

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 G2/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 cell 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.