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。有缺陷的染色质 和基因失调是结直肠癌等肿瘤不可或缺的方面，而表达 EZH2-多梳抑制复合体(PRC)2的催化亚基，它将抑制物 组蛋白标记H3K27me3-在结直肠癌和其他癌症中也普遍升高。然而， 表观遗传调控在肠干细胞分化和分化中的作用及机制 动态平衡仍然知之甚少，特别是H3K27me3与其他表观遗传学的关系 DNA甲基化(DNAME)等修饰。对癌细胞系或ES细胞的研究暗示 通过这些替代过程来协调基因抑制。然而，决定因素和互惠 其中许多相互作用还没有在体内进行过严格的研究，而抑制染色质的研究 MARKS远远落后于影响各种表观遗传学的药物的临床前和临床开发 过程，包括小分子EZH2抑制剂。这项建议建立在我们的一个总原则之上 最近发现H3K27me3在成人组织中的功能，以及另外两个意想不到的 观察：在成熟的PrC2缺失的肠道细胞中大量下调的基因不受 当PRC2缺失时，ISCS和H3K27me3标记的基因获得广泛的DNA新甲基化。 我建议研究DNAME和PRC2介导的H327me3在ISC和 成熟细胞基因表达、分化和肿瘤发生。我会先用转基因小鼠 模型和免疫器官培养以消除这些修饰中的一个或两个的活性(Aim1)。 我将使用CHIP-SEQ研究H3K27me3和其他染色质状态的后果 修饰、用于深层转录组分析的rna-seq和全基因组亚硫酸盐测序 (WGBS)评估异常的DNAME。这些实验将阐明大脑的功能和时间 H3K27me3与DNAME在成年哺乳动物细胞中的首次相互作用其次，我会 将问题扩展到我针对EZH2活动增加或缺失而生成的小鼠模型 在肠道肿瘤中(目标2)。出乎意料的是，我发现EZH2的过度表达减少了，而PRC2 虚实增多，肿瘤负荷明显。我将描述在每种情况下出现的肿瘤的特征， 建立具有每种调制的肿瘤的基因表达特征，并测试它们的依赖性 H3K27me3介导的基因沉默及其改变的DNAME在基因调控中的作用 肿瘤来源的ISCs。总之，这些研究将为人类基因沉默的机制提供新的线索。 组织动态平衡和肿瘤中特定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.