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阐明组蛋白和非组蛋白甲基精氨酸和瓜氨酸的动态调节。肽基精氨酸脱氨酶 4（以下简称 PAD4）是一种组蛋白修饰酶，在大多数情况下其行为类似于转录辅阻遏物。 PAD4 已被证明可以对许多靶标进行脱亚胺化（“瓜氨酸化”），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，从而抵消共激活剂 CARM1 和 PRMT1 对这些位点的“激活”甲基化。在目标 1 中，我们开发并描述了我们的 MCF7 乳腺癌 PAD4 野生型和敲低细胞相匹配的系统。 MCF7 细胞中 PAD4 的敲低会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 基因靶标。使用染色质免疫沉淀 (ChIP) 并结合新一代高通量测序，我们将绘制整个基因组中的 PAD4 结合位点图谱，以及包含甲基化 H3 精氨酸 17 (H3R17me) 和 H4R3me（PAD4 靶标）以及组蛋白 H3 瓜氨酸 17 (H3Cit17) 和 H4Cit3（PAD4 产品）的图谱位点。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，以生成“真实”PAD4 基因靶标的完整基因组图谱。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。在目标 2 中，我们提出了“组蛋白密码假说”的概念进展，这是原始组蛋白密码与广泛存在的非组蛋白翻译后修饰 (PTM) 之间的混合体。非组蛋白含有短段的组蛋白序列，我们将其称为组蛋白盒模拟物，并且这些“组蛋白盒模拟物”可以通过靶向组蛋白序列的相同酶进行翻译后修饰。在这个目标中，描述基于候选的和无偏见的方法的例子，我们将识别关键下游靶标中的非组蛋白甲基精氨酸和瓜氨酸位点，以便识别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传导通路中的作用。最后，在目标 3 中，我们将通过识别 PAD4 翻译后修饰和将 PAD4 招募到染色质的蛋白质相互作用因子，剖析 PAD4 和瓜氨酸在蛋白质水平上的调节。我们还将鉴定瓜氨酸“阅读器”或效应分子，以促进组蛋白（和非组蛋白）瓜氨酸化下游信号事件的解析。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节，将是阐明正常发育和人类癌症发病机制所涉及途径的关键。

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.