Coordinate roles of histone H3K27me3 and DNA methylation in intestinal homeostasis and tumorigenesis

项目摘要 不同转录因子的表达和染色质状态的变化共同决定了细胞 干细胞和祖细胞分化过程中的特异性基因表达。缺陷染色质 和基因失调是肿瘤如结肠直肠癌的组成部分， EZH 2-Polycomb Repressive Complex（PRC）2的催化亚基，其将抑制性的 组蛋白标记H3 K27 me 3-在结直肠癌和其他癌症中也通常升高。然而，在这方面， 表观遗传调节在肠干细胞分化中的作用和机制， 体内平衡仍然知之甚少，特别是H3 K27 me 3与其他表观遗传的关系， DNA甲基化（DNA methylation，DNAme）对癌细胞系或胚胎干细胞的研究表明， 通过这些替代过程协调基因抑制。然而，决定因素和互惠 的相互作用还没有在体内进行严格的研究，抑制染色质的研究 marks远远落后于影响各种表观遗传的药物的临床前和临床开发， 包括小分子EZH 2抑制剂。这项建议建立在一项一般原则之上， 最近发现的关于H3 K27 me 3在成人组织中的功能和另外两个意想不到的 观察结果：在成熟的PRC 2缺失的肠细胞中大量去阻遏的基因不受影响， ISC和H3 K27 me 3标记的基因在PRC 2不存在时获得广泛的DNA新甲基化。 我建议研究DNAme和PRC 2介导的H327 me 3在ISC中的相互依赖性， 成熟细胞基因表达、分化和肿瘤发生。我将首先使用工程鼠 模型和隐窝类器官培养物以消除这些修饰之一或两者的活性（Aim 1）。 我将使用ChIP-seq研究H3 K27 me 3和其他染色质状态的后果 用于深度转录组分析的RNA-seq和全基因组亚硫酸氢盐测序 （WGBS）来评估异常DNA。这些实验将阐明功能和时间 H3 K27 me 3与DNAme的相互作用首次在成年哺乳动物细胞中发现。第二，我会 我将问题扩展到我所建立的EZH 2活性增加或缺乏的小鼠模型 肠肿瘤（目标2）。出乎意料的是，我发现EZH 2过表达减少，而PRC 2 缺素增加，肿瘤负荷显著。我将描述每种情况下出现的肿瘤， 建立具有每种调节的肿瘤的基因表达特征，并测试它们的依赖性 H3 K27 me 3介导的基因沉默以及改变的DNAme在基因调控中的作用， 肿瘤来源的ISCs。总之，这些研究将揭示基因沉默的机制， 组织稳态和肿瘤中特异性抑制性染色质修饰的功能。

Project Summary Expression of distinct transcription factors and changes in chromatin states together determine cell- specific gene expression during differentiation of stem and progenitor cells. Defective chromatin and gene dysregulation are integral aspects of tumors such as colorectal cancer, and expression of EZH2 – a catalytic subunit of Polycomb Repressive Complex (PRC) 2, which places the repressive histone mark H3K27me3 – is also commonly elevated in colorectal and other cancers. However, the roles and mechanisms of epigenetic modulation in intestinal stem cell (ISC) differentiation and homeostasis remain poorly understood, particularly the relation of H3K27me3 to other epigenetic modifications such as DNA methylation (DNAme). Studies in cancer cell lines or ES cells hint at coordinated gene repression by these alternative processes. However, determinants and reciprocity of the interactions have not been studied critically in vivo, and investigation of repressive chromatin marks lags far behind pre-clinical and clinical development of drugs that affect various epigenetic processes, including small-molecule EZH2 inhibitors. This proposal builds on a general principle we recently uncovered regarding H3K27me3 function in adult tissues and on two additional unexpected observations: Genes massively derepressed in mature PRC2-null intestinal cells are unaffected in ISCs, and H3K27me3-marked genes acquire extensive DNA neomethylation when PRC2 is absent. I propose to study the interdependence of DNAme and PRC2-mediated H327me3 in ISC and mature cell gene expression, differentiation, and tumorigenesis. I will first use engineered mouse models and crypt organoid cultures to eliminate activity of either or both these modifications (Aim1). I will study the consequences using ChIP-seq for the H3K27me3 and other chromatin state modifications, RNA-seq for deep transcriptome analysis, and whole-genome bisulfite sequencing (WGBS) to assess aberrant DNAme. These experiments will elucidate the functional and temporal interactions of H3K27me3 with DNAme for the first time in adult mammalian cells. Secondly, I will extend the questions to mouse models that I have generated for increased or absent EZH2 activity in intestinal tumors (Aim 2). Unexpectedly, I find that EZH2 overexpression reduces, while PRC2 deficiency increases, tumor load significantly. I will characterize tumors that arise in each condition, establish gene expression signatures for tumors with each modulation, and test their dependence on H3K27me3-mediated gene silencing as well as the role of altered DNAme in gene regulation in tumor-derived ISCs. Together, these studies will shed new light on mechanisms of gene silencing in tissue homeostasis and on the functions of specific repressive chromatin modifications in tumors.