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

由蛋白质赖氨酸甲基转移酶（pkmt）催化的非组蛋白赖氨酸甲基化已成为影响多种细胞过程的重要修饰。Jeltsch和Levy实验室的初步研究强烈表明，PKMT SETD6甲基化转录因子E2F1的K117。此外，使用癌症基因组学的生物信息学分析显示，这些蛋白在许多癌症类型中发生突变，并且两者与前列腺癌高度相关。这些数据为我们的特定假设提供了基础，即SETD6功能和SETD6介导的E2F1和其他关键细胞蛋白的赖氨酸甲基化调节前列腺癌的进展。为了解决这一假设，我们提出了三个具体目标：目的1是阐明SETD6调节E2F1的分子机制。在这里，我们将使用生化和细胞方法来揭示SETD6对E2F1的细胞甲基化，测试其对E2F1稳定性和表达的影响，并研究潜在的甲基/乙酰基开关。此外，我们将开发重要的研究工具，包括调节SETD6和E2F1表达和甲基化特异性抗体的前列腺癌细胞系模型。目的2：探讨setd6介导的E2F1甲基化在前列腺癌中的细胞作用。在这个目标中，我们将利用生化、分子、细胞和基因组方法来定义E2F1甲基化在生理和病理环境中的下游后果。然后，我们将进行一系列细胞研究，以明确了解SETD6在K117位点的E2F1甲基化是否会影响癌症相关的细胞表型，以及它如何改变前列腺癌细胞的转录程序。最后，我们将应用改良的酵母- 3杂交筛选来鉴定特异性与甲基化E2F1相互作用并传递甲基化特异性信号的蛋白。目的3是在前列腺癌细胞中定义SETD6甲基组，并鉴定新的SETD6相互作用蛋白和甲基化底物。为此，我们将利用蛋白质组学和生化方法来定义SETD6依赖的甲基化模式，并在前列腺癌模型中识别新的SETD6伴侣。此外，我们将拓宽SETD6的特异性分析，研究SETD6甲基化极可变序列肽的分子机制。目前的提案汇集了两个研究领域——甲基化信号和癌症——它的成功完成将对基础研究和转化研究产生直接影响和广泛影响。特别是，它将鉴定出数十种新的SETD6底物、新的效应蛋白和新的相互作用蛋白，并定义具有潜在重要诊断、预后和治疗意义的前列腺癌SETD6甲基化图谱。

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.