Role of E2F1 methylation by SETD6 and identification of novel SETD6 methylation substrates

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Lysine methylation of non-histone proteins catalyzed by Protein lysine methyl-transferases (PKMTs) has emerged as an important modification that impacts diverse cellular processes. Preliminary studies from the Jeltsch and the Levy labs, strongly suggest that the PKMT SETD6 methylates K117 of the transcription factor E2F1. Moreover, bioinformatics analysis using the cBioPortal for cancer genomics revealed that these proteins are mutated in many cancer types and both are highly relevant in prostate cancer. These data provide the ground for our specific hypothesis that SETD6 function and SETD6-mediated lysine methylation of E2F1 and additional key cellular proteins regulates prostate cancer progression. To address this hypothesis, we propose three specific objectives:Aim 1 is to elucidate the molecular mechanism by which SETD6 regulates E2F1. Here, we will use biochemical and cellular approaches to unravel the cellular methylation of E2F1 by SETD6, test its influence on E2F1 stability and expression and investigate a potential methyl/acetyl switch. Moreover, we will develop important research tools, including prostate cancer cell line models with modulated SETD6 and E2F1 expression and methylation specific antibodies.Aim 2 is to investigate the cellular effect of SETD6-mediated E2F1 methylation in prostate cancer. In this aim we will utilize biochemical, molecular, cellular and genomic approaches to define the downstream consequences of E2F1 methylation in both physiological and pathological settings. We will then perform a series of cellular studies to specifically understand if E2F1 methylation by SETD6 at K117 affects cancer related cellular phenotypes and how it alters transcriptional programs in prostate cancer cells. Finally, we will apply a modified yeast-three hybrid screen to identify proteins that specifically interact with methylated E2F1 and transmit the methylation specific signals.Aim 3 is to define the SETD6 methylome and to identify new SETD6-interacting proteins and methylation substrate in prostate cancer cells. In this aim, we will exploit proteomic, together with biochemical approaches to define SETD6-dependent methylation patterns and identify new SETD6 partners in prostate cancer models. In addition, we will broaden the specificity analysis of SETD6 and study the molecular mechanisms allowing SETD6 to methylate peptide with very variable sequences.The current proposal brings together two fields of research – methylation signaling and cancer – and its successful completion will have an immediate impact and broad implications for both basic and translational research. In particular, it will identify dozens of new SETD6 substrates, novel effector proteins, and new interacting proteins as well as define the SETD6 methylation landscape of prostate cancer with potentially important diagnostic, prognostic, and therapeutic implications.