Dynamic Regulation of Methyl-arginine and Citrulline in Breast Cancer Cells

描述（由申请人提供）：在本提案中，我们试图在基因组和机制水平上揭示组蛋白和非组蛋白甲基精氨酸和瓜氨酸的动态调控。肽基精氨酸脱氨酶4（以下简称PAD4）是一种组蛋白修饰酶，在大多数情况下表现为转录共抑制因子。研究表明，PAD4可以消除（“瓜氨酸化”）许多靶标，包括组蛋白H3 （H3R2、H3R17和H3R26）和H4 (H4R3) N端尾部的精氨酸和甲基精氨酸，从而抵消了这些位点被共激活因子CARM1和PRMT1“激活”的甲基化。在Aim 1中，我们开发并描述了匹配MCF7乳腺癌PAD4野生型和敲低细胞的系统。MCF7细胞中PAD4的敲低导致细胞运动和侵袭，或“转移”表型。我们将利用该系统来识别PAD4基因靶点。利用染色质免疫沉淀（ChIP），结合下一代高通量测序，我们将绘制PAD4在基因组中的结合位点，以及包含甲基化H3精氨酸17 （H3R17me）和H4R3me （PAD4靶点）和组蛋白H3瓜氨酸17 （H3Cit17）和H4Cit3 （PAD4产物）的位点。我们将把这些全球ChIP数据与我们的PAD4基因表达阵列数据结合起来，以产生一个完整的“真正的”PAD4基因靶点基因组图谱。该转录网络的生物学功能将根据PAD4敲低细胞的转移表型进行检查。在Aim 2中，我们提出了“组蛋白编码假说”的概念进展，这是原始组蛋白编码和广泛存在的非组蛋白翻译后修饰（ptm）之间的混合体。非组蛋白含有组蛋白序列的短片段，我们将其称为组蛋白盒模拟物，这些“组蛋白盒模拟物”可以被针对组蛋白序列的相同酶在翻译后修饰。在这个目标中，我们将描述基于候选和无偏倚方法的例子，我们将确定关键下游靶标中的非组蛋白甲基精氨酸和瓜氨酸位点，以确定受翻译后修饰调节的重要生物底物，并开始揭示它们在细胞信号通路中的作用。最后，在Aim 3中，我们将通过鉴定PAD4翻译后修饰和将PAD4招募到染色质的蛋白质相互作用物，在蛋白质水平上解剖PAD4和瓜氨酸的调控。我们还将识别瓜氨酸“读取器”或效应分子，促进组蛋白（和非组蛋白）瓜氨酸化的下游信号事件的解剖。详细了解组蛋白和非组蛋白上精氨酸甲基化和瓜氨酸化的功能和调控将是揭示参与正常发育和人类癌症发病机制的途径的关键。

DESCRIPTION (provided by applicant): In this proposal, we seek to unravel the dynamic regulation of histone and non-histone methyl-arginine and citrulline, both at genomic and mechanistic levels. Peptidyl arginine deaminase 4 (hereafter PAD4), is a histone-modifying enzyme that behaves like a transcriptional co-repressor in most instances. PAD4 has been shown to deiminate ('citrullinate') a number of targets, including arginines and methyl-arginines on the N- terminal tails of histones H3 (H3R2, H3R17, and H3R26) and H4 (H4R3), which counteracts the 'activating' methylation of these sites by the coactivators CARM1 and PRMT1. In Aim 1, we develop and describe our system of matched MCF7 breast cancer PAD4 wild type and knockdown cells. Knock down of PAD4 in MCF7 cells results in a cellular motility and invasion, or 'metastatic' phenotype. We will utilize this system in order to identify PAD4 gene targets. Using chromatin immunoprecipitation (ChIP), combined with next-generation high throughput sequencing, we will map PAD4 binding sites across the genome, as well as map sites containing methylated H3 arginine 17 (H3R17me), and H4R3me (PAD4 targets), and histone H3 citrulline 17 (H3Cit17) and H4Cit3 (PAD4 products). We will combine this global ChIP data with our PAD4 gene expression array data in order to produce a complete genomic map of 'true' PAD4 gene targets. Biological functions of this transcriptional network will be examined in light of the metastatic phenotype of PAD4 knockdown cells. In Aim 2, we propose a conceptual advance of the 'histone code hypothesis,' a hybrid between the original histone code and the extensive presence of non-histone post-translational modifications (PTMs). Non-histone proteins contain short stretches of histone sequences, which we have termed histone cassette mimics, and these 'histone cassette mimics' can be post-translationally modified by the same enzymes that target histone sequences. In this aim, describing examples of both candidate-based and unbiased approaches, we will identify non-histone methyl-arginine and citrulline sites in key downstream targets in order to identify important biological substrates that are regulated by post translational modifications and to begin to unravel their roles in cell signaling pathways. Finally, in Aim 3, we will dissect the regulation of PAD4 and citrulline on the protein level, by identifying PAD4 post-translational modifications and protein interactors that recruit PAD4 to chromatin. We will also identify citrulline 'readers', or effector molecules, facilitating the dissection of the downstream signaling events of histone (and nonhistone) citrullination. Detailed insights into the function and regulation of arginine methylation and citrullination on both histone and nonhistone proteins will be key to unraveling pathways involved in normal development and the pathogenesis of human cancers.