It has been documented that apoptotic cells themselves can induce phosphorylation of serine 139 on H2AX (γH2AX) due to DNA fragmentation during apoptosis (doi: 10.1074/jbc.275.13.9390). As γH2AX is a well-established marker for DNA damage, should we necessarily consider apoptosis-induced γH2AX signal when conducting DNA damage-related research?
In my study, I depleted a gene in a tissue of mouse, both apoptosis (cleaved caspase-3) and γH2AX signals were observed at the same time point. I am wondering whether this gene regulates DNA damage response or if the observed γH2AX signals were merely a consequence of apoptosis.
Could anyone in this field give some suggestions? I would be extremely grateful.
Dear Li Jessury
I briefly wanted to reach out concerning your inquiry. In order to bona fide relate your phenotype to apoptosis, please check (if possible) a) ROS production and b) p53 expression as your phenotype is very similar to one we observed with Mysm1 deficiency (in mice, which is phenotypically copied in humans with rare loss of function mutations).
For experimental protocols and experiments please consider Figure 7 of
Article The critical role of histone H2A-deubiquitinase Mysm1 in hem...
For a detailed analysis of apoptotic cells by FACS, please use my protocol/ experimental design of Figure 5 (which you can easily combine with our flow assay for γH2AX) of
Article Regulation of B Lymphocyte Development by Histone H2A Deubiq...
And for our work on MYC-MYSM1 interaction and its effect on ribosomal protein synthesis (rather than DNA damage response, we initially suspected) please see:
Article Loss of MYSM1 inhibits the oncogenic activity of cMYC in B c...
Article Repression of p53-target gene Bbc3/PUMA by MYSM1 is essentia...
All the best & good luck with your experiments,
Michael
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