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

描述(申请人提供)：在这项提案中，我们试图解开组蛋白和非组蛋白甲基精氨酸和瓜氨酸在基因组和机制水平上的动态调节。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，它的行为类似于转录辅助抑制物。PAD4已被证明能去亚胺(‘瓜氨酸’)一些靶标，包括组蛋白H3(H3R2、H3R17和H3R26)和H4(H4R3)N-末端的精氨酸和甲基精氨酸，这抵消了辅助激活剂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匹配的MCF7乳腺癌PAD4野生型和敲除细胞系统。在MCF7细胞中敲除PAD4会导致细胞的运动和侵袭，或者说是“转移”表型。我们将利用这个系统来识别PAD4基因靶点。使用染色质免疫沉淀(CHIP)，结合下一代高通量测序，我们将绘制整个基因组的PAD4结合位点，以及包含甲基化H3精氨酸17(H3R17me)和H4R3me(PAD4靶标)的位点，以及组蛋白H3瓜氨酸17(H3Cit17)和H4Cit3(PAD4产物)。我们将把这一全球芯片数据与我们的PAD4基因表达阵列数据结合起来，以产生一个完整的PAD4基因靶标的基因组图谱。这个转录网络的生物学功能将根据PAD4基因敲除细胞的转移表型进行检测。在目标2中，我们提出了“组蛋白密码假说”的概念上的进展，这是原始组蛋白密码和广泛存在的非组蛋白翻译后修饰(PTM)之间的杂交。非组蛋白蛋白包含一小段组蛋白序列，我们称之为组蛋白盒模拟物，这些‘组蛋白盒模拟物’可以被靶向组蛋白序列的相同酶翻译后修饰。在这一目标中，描述了基于候选和无偏见的方法的例子，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控的重要生物底物，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来剖析PAD4和瓜氨酸在蛋白质水平上的调节。我们还将识别瓜氨酸“阅读器”或效应器分子，促进组蛋白(和非组蛋白)瓜氨酸化下游信号事件的剖析。对精氨酸甲基化和瓜氨酸化在组蛋白和非组蛋白上的功能和调节的详细了解将是解开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.