Elucidating the role of H3K27me3 demethylation in the intestinal epithelium

项目摘要 哺乳动物肠道是研究细胞命运的分子事件的理想模型系统 在发展和成人稳态的规范。在围产期肠发育期间， 上皮，增殖的绒毛间结构域内陷到下面的间充质中以产生 利伯库恩与隐窝形成同时出现的是成熟的表达Lgr 5的肠干细胞 （ISCs），其位于这些隐窝的基部，并在成人体内平衡期间每天分裂以自我更新， 产生定向祖细胞。随着祖细胞沿着沿着隐窝壁向上迁移，它们进一步分裂 并经历渐进分化。在离开隐窝并进入绒毛突起时，祖细胞 完成终末分化并产生成熟的吸收和分泌细胞。尽管取得了进展， 鉴定促进ISC发育和分化的转录和分子机制， 指导这些过程的表观遗传机制仍然缺乏特征。拟定的研究将 因此，研究UTX和JMJD 3，两种组蛋白去甲基化酶，在肠上皮中的作用。UTX和 JMJD 3去甲基化三甲基化组蛋白3赖氨酸27（H3 K27 me 3），这是一种组蛋白修饰， 基因沉默通过去除这种抑制性组蛋白标记，UTX和JMJD 3去抑制谱系定义， 转录因子促进发育过程中的分化。此外，UTX和JMJD 3有助于 其他再生成人组织的分化，因此对于维持成人组织至关重要 体内平衡UTX和JMJD 3是否是肠上皮细胞分化的重要表观遗传调节因子 上皮仍是未知的。目的1将检验UTX和JMJD 3介导的H3 K27 me 3 去甲基化激活了胎儿祖细胞中的成熟干细胞转录程序， 是围产期肠道发育所必需的。组成型、精氨酸特异性Utx和Jmjd 3的小鼠模型 将采用消融来确定UTX和JMJD 3是否促进围产期隐窝形成和ISC 发展为了验证和扩展体内结果，体外类器官形成测定将确定是否 UTX和JMJD 3是胎儿肠球状体成熟为成熟的出芽类器官所必需的。 此外，单细胞RNA测序（scRNA-Seq）和ChIP-Seq将用于鉴定转录 以及UTX和JMJD 3消融后围产期肠中诱导的表观基因组变化。目标2将测试 假设UTX和JMJD 3介导的H3 K27 me 3去甲基化是激活分化所必需的 基因和维持成人肠上皮细胞的适当分化。UTX和Jmjd 3表达式将是 在成年小鼠的肠上皮内条件性消融。这些小鼠将通过以下方法进行分析： 免疫组织化学、scRNA-Seq和ChIP-Seq，以确定分化程序是否是 维持在细胞和基因组水平。总之，这些研究将提供新的见解， 在发育和稳态过程中指导肠分化的重要表观遗传机制。

Project Summary The mammalian intestine is an ideal model system to study the molecular events that control cell fate specification during development and adult homeostasis. During perinatal development of the intestinal epithelium, proliferative intervillus domains invaginate into the underlying mesenchyme to produce crypts of Lieberkühn. Concomitant with crypt formation is the emergence of mature Lgr5-expressing intestinal stem cells (ISCs), which situate at base of these crypts and divide daily during adult homeostasis to self-renew and produce committed progenitor cells. As progenitor cells migrate up along the crypt walls, they divide further and undergo progressive differentiation. Upon exiting the crypt and entering villus projections, progenitor cells complete terminal differentiation and produce mature absorptive and secretory cells. Despite advances in identifying the transcriptional and molecular mechanisms that facilitate ISC development and differentiation, the epigenetic mechanisms directing these processes remain poorly characterized. The proposed study will therefore examine the role of UTX and JMJD3, two histone demethylases, in the intestinal epithelium. UTX and JMJD3 demethylate trimethylated histone 3 lysine 27 (H3K27me3), a histone modification that is required for gene silencing. By removing this repressive histone mark, UTX and JMJD3 de-repress lineage defining transcription factors to promote differentiation during development. Furthermore, UTX and JMJD3 facilitate differentiation in other regenerative adult tissues and are therefore critical for maintaining adult tissue homeostasis. Whether UTX and JMJD3 are important epigenetic regulators of differentiation in the intestinal epithelium remains unknown. Aim 1 will test the hypothesis that UTX- and JMJD3-mediated H3K27me3 demethylation activates a mature stem cell transcriptional program in fetal progenitor cells and is therefore required for perinatal intestinal development. A mouse model of constitutive, intestine-specific Utx and Jmjd3 ablation will be employed to determine whether UTX and JMJD3 promote perinatal crypt formation and ISC development. To validate and extend in vivo results, an in vitro organoid formation assay will identify whether UTX and JMJD3 are required for the maturation of fetal intestinal spheroids into mature budding organoids. Furthermore, single-cell RNA-Sequencing (scRNA-Seq) and ChIP-Seq will be used to identify transcriptional and epigenomic changes induced in the perinatal intestine following UTX and JMJD3 ablation. Aim 2 will test the hypothesis that UTX- and JMJD3-mediated H3K27me3 demethylation is required to activate differentiation genes and maintain proper differentiation of adult intestinal epithelial cells. Utx and Jmjd3 expression will be conditionally ablated within the intestinal epithelium of adult mice. These mice will be analyzed by immunohistochemistry, scRNA-Seq, and ChIP-Seq to determine whether the differentiation program is maintained at both the cellular and genomic levels. Together, these studies will provide novel insights into an important epigenetic mechanism directing intestinal differentiation during development and homeostasis.