Abstract 3946: Stem cell protein piwil2 enhances nucleotide excision repair through regulating chromatin remodeling following DNA damage

Abstract

Abstract The piwi family gene, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. Three piwi homologs have been identified in the mouse genome. Amongst these, mili (piwil2) protein expression appears to be associated with tumorigenesis as evidenced by the facts that piwil2 stably expresses in pre-cancerous stem cells and also appears at variable levels in different human and animal tumor cell lines. Based on our preliminary data showing that piwil2 is induced in normal cultured human fibroblasts in vitro by physical and chemical genotoxic agents, we hypothesized that piwil2 might function in DNA damage response. Here, we demonstrated that mammalian cells lacking piwil2 are hypersensitive to cisplatin and UV irradiation. In addition, nucleotide excision repair (NER) efficiency is significantly decreased in the absence of piwil2 as assessed by the removal of cisplatin-induced intra-strand cross-links (Pt-GG) and UV-induced cyclobutane pyrimidine dimers (CPDs) from genomic DNA. Although quantitative real-time PCR analysis indicated higher transcript levels of various NER proteins in piwil2 knockout mouse embryo fibroblasts (MEFs), the Western blotting did not show an obvious difference in the NER protein levels between mili+/+ and mili−/− MEFs. Since piwil2 is believed to orchestrate chromatin remodeling, we reasoned that low NER efficiency in mili−/− MEFs may result from disrupted chromatin remodeling in response to UV or cisplatin induced DNA damage. Thus, chromatin relaxation and histone acetylation was analyzed in these MEFs following treatment with UV or cisplatin. MEFs lacking piwil2 showed less sensitivity to micrococcal nuclease (MNase) digestion compared with piwil2-proficient MEFs, indicating a compact chromatin structure in these cells. Moreover, in contrast to mili+/+ MEFs, mili−/− MEFs exhibited decreased acetylated Histone H3 upon both UV and cisplatin treatment. This decrease could be blocked by histone deacetylase (HDAC) inhibition indicating that piwil2 may function in damage-induced chromatin remodeling via inhibiting the HDAC activity. In summary, our data suggests that piwli2 can participate in the NER through regulating chromatin relaxation which could be a pivotal factor in genotoxin-induced tumorigenesis as well as efficacy of cancer therapeutics. (Supported by NIH grants CA93413, ES2388 and ES12991 to AAW). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3946.