Chromatin and transcriptional control of LGR5+ crypt base stem cells

描述（由申请人提供）：炎症性肠病和结直肠癌等疾病反映了肠干细胞（ISC）功能障碍。一个小的Bmi 1hi ISC池很少分裂并占据高的隐窝层，而在隐窝处的较大的Lgr 5+细胞池 基底细胞快速复制，发挥上皮细胞持续更新的“主力”作用。这两个ISC人口相互转换与令人惊讶的容易，但我们的理解的基本方面，如表观遗传和转录控制ISC的身份和功能，或决定因素的细胞位置和可转换性，是不完整的。更好地了解这些 这些方面将改善肠道疾病的治疗，同时保留正常的消化功能。我们提出了创新的方法，染色质和其他分子分析纯化的隐窝人口，特别是Lgr 5 + ISC，和先进的双光子共聚焦显微镜成像ISC人口在原位前所未有的细节。数以千计的基因调节增强子元件在ISC下降的肠上皮细胞（Ent）和分泌（Sec）谱系中没有单独启动;许多Ent或Sec活性增强子已经在Lgr 5 + ISC中启动。增强子引发是序列特异性“先锋”转录因子（TF）的范围，其鉴定相关的顺式调节元件并标记具有组蛋白修饰（例如组蛋白3赖氨酸4甲基化（H3 K4 me））的侧翼核小体。在目标1中，我们提出启动Lgr 5 + ISC中转录程序的“先锋”TF是ASCL 2，这是Lgr 5 + ISC存活和功能所需的一种精氨酸限制性TF。我们将在培养的细胞和转基因小鼠中测试ASCL 2是否满足“先锋”属性，即，如果它最多结合Lgr 5 + ISC和其他隐窝细胞中的引发增强子，并且如果这些增强子上的活性组蛋白标记在体内不存在时被消灭。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使用Polycomb Repressive Complex（PRC）2来放置组蛋白标记H3 K27 me 3。肠上皮中PRC 2的缺失损害Lgr 5 + ISC和其他隐窝细胞的复制，产生发育不良的有缺陷的绒毛，其很快被PRC 2感受态单位取代。在目标2中，我们提出了一个综合分析H3 K27 me 3标记，PRC 2依赖的基因表达，和突变体表型在纯化的Lgr 5 + ISC和他们的后代隐窝和绒毛细胞。我们的具体目标是了解动态染色质改变的意义，并确定在ISC基因激活和沉默的机制。最后，在理解不同ISC池及其相应的壁龛之间的功能关系的进展受到阻碍，部分困难的可视化Bmi 1hi +4 ISC;我们已经改进了双光子共聚焦显微镜方法，以检测这些和周围的细胞在其天然状态。在目标3中，我们将进一步完善这项技术，同时特别询问Bmi 1hi +4 ISC如何响应隐窝扰动，如Lgr 5 + ISC的复制缺陷或潘氏细胞的消融。

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.