Abstract 4521: Substrate profiling for SETD8 reveals novel protein targets involved in DNA replication and DNA damage response

Protein lysine methyltransferases utilize S-adenosyl-L-methionine (SAM) to catalyze the addition of methyl groups to specific lysine residues in both histones and non-histone proteins. Recent evidence indicates that their dysregulation is closely linked to the development and progression of cancer, making them potential therapeutic targets. SETD8 is the sole methyltransferase responsible for mono-methylation of histone H4 lysine 20 (H4K20me1). Its activity is essential for cell viability and has been associated with many essential cellular processes including transcriptional regulation, heterochromatin formation, genomic stability and cell-cycle progression. SETD8 is overexpressed in several types of cancer and may play important roles on malignancy through its trans-methylation activity or by direct interaction with its binding partners. It has been reported that the association between SETD8 and TWIST, a regulator of epithelial-mesenchymal transition, enhances the invasiveness of breast cancer cells. In addition, it was shown that SETD8-mediated methylation of p53 suppresses p53 dependent transcription activation in cancer cells. Despite the identification of a few non-histone proteins such as p53, PCNA and Numb as SETD89s substrates, a comprehensive investigation of new potential substrates is lacking to date. Here we used Bioorthogonal Profiling of Protein Methylation (BPPM) with engineered SETD8 and synthetic SAM analogues to profile novel substrates for SETD8. We identified SETD8 mutants amenable to accommodate non-native SAM analogues containing a terminal alkyne moiety for click chemistry. The engineered SETD8 can transfer this distinct chemical moiety into target proteins for subsequent pulldown and identification of the modified substrates. The BBPM technology applied here revealed previously known substrates of SETD8 as well as novel SETD8 targets. Among the main targets of our interest are those involved in DNA replication and DNA damage response. Our finding is expected to bring new perspectives on the biological importance of SETD8 as well as on its role in carcinogenesis. Citation Format: Fabio Pittella Silva, Gil Blum, Chamara Senevirathne, Minkui Luo. Substrate profiling for SETD8 reveals novel protein targets involved in DNA replication and DNA damage response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4521.