Chromatin and transcriptional control of LGR5+ crypt base stem cells

描述(申请人提供)：炎症性肠病和结直肠癌等疾病反映了肠道干细胞(ISC)功能障碍。一个较小的Bmi1hi ISC池不经常分裂并占据较高的隐窝层，而较大的Lgr5+细胞池位于隐窝 BASE复制迅速，发挥着上皮细胞持续更新的“主力”功能。这两个ISC群体以惊人的容易相互转换，但我们对基本方面的理解是不完整的，例如ISC身份和功能的表观遗传和转录控制，或细胞位置和可转换性的决定因素。更好地理解这些 Aspects将改善肠道疾病的治疗，同时保留正常的消化功能。我们提出了对纯化的隐窝种群，特别是Lgr5+ISC进行染色质和其他分子分析的创新方法，并提出了先进的双光子共聚焦显微镜，以前所未有的细节原位成像ISC种群。数以千计的基因调节增强子元件并不是在ISC的肠上皮细胞(Ent)和分泌型(SEC)谱系中单独启动的；许多Ent或SEC活性的增强子已经在Lgr5+ISC中启动。增强子启动是序列特异的“先锋”转录因子的作用范围，它识别相关的顺式调控元件，并用组蛋白修饰标记核小体两侧，如组蛋白3赖氨酸4甲基化(H3K4me)。在目标1中，我们提出启动Lgr5+ISC转录程序的“先驱”因子是ASCL2，这是Lgr5+ISC生存和功能所必需的肠限制性因子。我们将在培养的细胞和转基因小鼠中测试ASCL2是否满足“先驱”属性，即它是否在Lgr5+ISC和其他隐窝细胞中最多结合启动的增强剂，以及这些增强剂上的活性组蛋白标记是否在体内没有组蛋白标记时消失。细胞分化需要基因激活和沉默，但ISC抑制不适当基因的机制尚不清楚。胚胎细胞中的一个主要抑制途径是使用多梳抑制复合体(PRC)2来放置组蛋白标记H3K27me3。肠上皮中缺乏PRC2会损害Lgr5+ISC和其他隐窝细胞的复制，产生发育迟缓、有缺陷的绒毛，这些绒毛很快就会被PRC2功能单位所取代。在目标2中，我们提出了对纯化的Lgr5+ISC及其后代隐窝和绒毛细胞的H3K27me3标记、PrC2依赖的基因表达和突变表型的综合分析。我们的具体目标是了解染色质动态变化的意义，并确定ISC中基因激活和沉默的机制。最后，在理解不同ISC池及其对应的利基之间的功能关系方面的进展在一定程度上受到Bmi1hi+4 ISC可视化的困难；我们已经改进了双光子共聚焦显微镜方法来检测这些细胞及其周围细胞的天然状态。在目标3中，我们将进一步完善这项技术，同时特别询问Bmi1hi+4 ISC如何应对隐窝扰动，例如Lgr5+ISC的复制缺陷或Paneth细胞的消融。

DESCRIPTION (provided by applicant): Disorders such as Inflammatory Bowel Disease and colorectal cancer reflect intestinal stem cell (ISC) dysfunction. One small pool of Bmi1hi ISC divides infrequently and occupies high crypt tiers, while a larger pool of Lgr5+ cells at the crypt base replicates briskly and serves the "workhorse" function of continuous epithelial renewal. The two ISC populations interconvert with surprising ease, but our understanding of fundamental aspects, such as epigenetic and transcriptional control of ISC identity and function, or determinants of cell location and convertibility, is incomplete. A better understanding of these aspects will improve treatment of intestinal disorders, while sparing normal digestive functions. We propose innovative approaches for chromatin and other molecular analyses of purified crypt populations, especially Lgr5+ ISC, and advanced 2-photon confocal microscopy to image ISC populations in situ in unprecedented detail. Thousands of gene-regulating enhancer elements are not separately primed in the Enterocyte (Ent) and Secretory (Sec) lineages that descend from ISC; many Ent- or Sec-active enhancers are already primed in Lgr5+ ISC. Enhancer priming is the purview of sequence-specific "pioneer" transcription factors (TFs), which identify relevant cis-regulatory elements and mark flanking nucleosomes with histone modifications such as Histone 3 Lysine 4 methylation (H3K4me). In Aim 1 we propose that the "pioneer" TF that primes the transcriptional program in Lgr5+ ISC is ASCL2, an intestine-restricted TF that is required for Lgr5+ ISC survival and function. We will test in cultured cells and transgenic mice whether ASCL2 fulfills "pioneer" attributes, i.e., if it binds at most primed enhancers in Lgr5+ ISC and other crypt cells, and if active histone marks at these enhancers are extinguished in its absence in vivo. Cell differentiation requires both gene activation and silencing, but mechanisms by which ISC repress inappropriate genes are unknown. One major repressive pathway in embryonic cells uses Polycomb Repressive Complex (PRC)2 to place the histone mark H3K27me3. Absence of PRC2 in the intestinal epithelium impairs replication of Lgr5+ ISC and other crypt cells, producing stunted, defective villi that are soon replaced by PRC2-competent units. In Aim 2 we propose an integrative analysis of H3K27me3 marks, PRC2-dependent gene expression, and mutant phenotypes in purified Lgr5+ ISC and their descendant crypt and villus cells. Our specific goals are to understand the significance of dynamic chromatin alterations and to define mechanisms of gene activation and silencing in ISC. Lastly, progress in understanding functional relationships among different ISC pools and their corresponding niches is hampered in part by difficult visualization of Bmi1hi +4 ISC; we have improved 2-photon confocal microscopy methods to detect these and surrounding cells in their native state. In Aim 3 we will further refine this technology, while specifically interrogating how Bmi1hi +4 ISC respond to crypt perturbations such as replicative deficiency in Lgr5+ ISC or ablation of Paneth cells.