Elucidating the role of H3K27me3 demethylation in the intestinal epithelium

项目摘要 哺乳动物肠道是研究控制细胞命运的分子事件的理想模型系统 在发育和成体动态平衡过程中的规格。在围产期肠道发育过程中 上皮，增殖的绒毛间域插入到下面的间质中以产生隐窝 利伯库恩。伴随着隐窝形成的是成熟的表达Lgr5的肠道干细胞的出现 (ISCs)，位于这些隐窝的底部，在成体动态平衡期间每天分裂，自我更新和 产生忠诚的祖细胞。随着祖细胞沿着隐窝壁向上迁移，它们会进一步分裂 并经历渐进式分化。在离开隐窝并进入绒毛突起时，祖细胞 完成顶端分化，产生成熟的吸收细胞和分泌细胞。尽管取得了进展，但 确定促进ISC发展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没有得到充分的描述。拟议的研究将 因此，研究两种组蛋白去甲基酶UTX和JMJD3在肠道上皮细胞中的作用。UTX和 JMJD3三甲基化组蛋白3赖氨酸27(H3K27me3)，一种组蛋白修饰，需要 基因沉默。通过去除这个抑制性组蛋白标记，UTX和JMJD3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JMJD3有助于 在其他再生的成体组织中分化，因此对维持成体组织至关重要 动态平衡。UTX和JMJD3是否是肠道分化的重要表观遗传调控因子 上皮组织尚不清楚。目标1将检验UTX和JMJD3介导的H3K27me3的假设 去甲基化激活胎儿祖细胞中的成熟干细胞转录程序，因此 是围产期肠道发育所必需的。结构性肠特异性UTX和Jmjd3小鼠模型的建立 消融将被用来确定UTX和JMJD3是否促进围产期隐窝形成和ISC 发展。为了验证和扩展体内的结果，体外有机物形成试验将识别 UTX和JMJD3是胎儿肠道球体成熟为成熟萌芽器官所必需的。 此外，单细胞RNA测序(scRNA-Seq)和CHIP-Seq将用于鉴定转录 以及UTX和JMJD3消融后围产期肠道的表观基因组变化。AIM 2将测试 UTX和JMJD3介导的H3K27me3去甲基化是激活分化所必需的假设 基因，并维持成人肠道上皮细胞的适当分化。UTX和Jmjd3表达式将为 在成年小鼠的肠道上皮内有条件地消融。这些小鼠将被分析为 免疫组织化学、scRNA-Seq和ChIP-Seq确定分化方案是否为 维持在细胞和基因组水平。总而言之，这些研究将为 重要的表观遗传机制，在发育和动态平衡过程中指导肠道分化。

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.