Abstract 4985: Identification of PARP-1 as a novel KLF8 interacting and regulating protein

Abstract

Abstract KLF8 transcription factor regulates important genes including cyclin D1 and E-cadherin and plays a critical role in cell cycle progression, oncogenic transformation and EMT. Transcription factors need to interact with both DNA and other proteins in order to regulate transcription. However, little is known about KLF8-protein interaction. Using mass spectrometry and co-immunoprecipitation (co-IP), we identified PARP-1, a chromatin-associated enzyme that catalyzes protein poly(ADP-ribosyl)ation (PARylation), as one of novel proteins that interact with KLF8. Co-IP and western blotting indicated that KLF8 is a PARylation substrate. Mutation of the cystein residues in the first and second zinc-finger motifs of KLF8 abolishes its interation with PARP1. Luciferase assays showed KLF8 activation on the cyclin D1 promoter was significantly reduced when PARP1 was physically deleted or functionally inhibited or when KLF8-PARP-1 interation was disrupted. Surprisingly, immunofluorescent staining demonstrated that KLF8 was localized in the nuclei of the wild-type mouse embryonic fibroblasts (MEF), but was mislocalized in the perinuclear cytoplasm of the PARP-1−/− MEFs, which could be rescued by re-expression of PARP1. Interestingly, while the PARP1-interation deficient KLF8 mutant was also mislocalized in the cytoplasm of the wild-type MEFs, inhibition of PARP1 activity in the MEFs did not affect the nuclear localization of wild-type KLF8. Cychloheximade (CHX)-chase assay revealed the stability of KLF8 dramatically reduced by loss of PARP-1 or when KLF8-PARP-1 interaction was disrupted. Ubiquitination assay showed heavier ubiquitin chains in PARP-1−/− MEFs or when KLF8-PARP-1 interaction was disrupted. Furthermore, DNA damage assay under Doxorubin treatment indicated KLF8 involved in PARP-1-dependent DNA damage repair pathway. Those results suggest that PARP-1 work as a co-activator in KLF8's transcriptional activity on Cyclin D1 promoter, keep KLF8's nuclear localization, inhibit KLF8's ubiquitination and contribute to KLF8's stability. This work has identified PARP1 as a novel binding partner and post-translational modifier of KLF8 and a critical regulator of KLF8 localization, function and stability in the cells. 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 4985.