Chromatin and transcriptional control of LGR5+ crypt base stem cells

LGR5 隐窝基底干细胞的染色质和转录控制

基本信息

批准号：
9135746
负责人：
Ramesh A Shivdasani
金额：
$ 5.49万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9135746
关键词：
Ablation Address Adult Affect BHLH Protein Binding Cell Differentiation process Cell Survival Cell physiology Cells Chromatin Colorectal Cancer Columnar Cell Complex Confocal Microscopy Cultured Cells DNA Dependency Detection Disease Distant EZH2 gene Elements Engineering Enhancers Enterocytes Enzymes Epigenetic Process Epithelial Frequencies Functional disorder Gastrointestinal tract structure Gene Activation Gene Expression Gene Expression Regulation Gene Silencing Gene Targeting Generations Genes Genetic Enhancer Element Goals Health Histones Human Image Imagery In Situ Inflammatory Bowel Diseases Intestinal Diseases Intestines Knockout Mice Lead Location Lysine Malignant Neoplasms Manuscripts Mediating Methods Methylation Microscopy Molecular Mus Mutate Natural regeneration Nucleosomes Organ failure Organoids Paneth Cells Pathway interactions Pattern Phenotype Polycomb Population Positioning Attribute Process Regulatory Element Research Personnel Role Small Intestines Specific qualifier value Stem cells Stress Technology Testing Tissues Toxic effect Transcriptional Regulation Transgenic Mice Uncertainty Villus base blastomere structure cancer therapy crypt cell drug development embryonic stem cell epigenome histone modification human ASCL1 protein improved in vivo inhibitor/antagonist innovation insight intestinal crypt intestinal epithelium molecular marker mutant nerve stem cell overexpression progenitor programs protein complex response self-renewal stem cell population transcription factor two-photon

项目摘要

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.

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道干细胞（ISC）功能障碍。一小群 Bmi1hi ISC 不经常分裂并占据较高的隐窝层，而隐窝处有一大群 Lgr5+ 细胞基底快速复制并发挥持续上皮更新的“主力”功能。这两个 ISC 群体的相互转化非常容易，但我们对基本方面的理解并不完整，例如 ISC 身份和功能的表观遗传和转录控制，或细胞位置和可转化性的决定因素。对这些有更好的了解方面将改善肠道疾病的治疗，同时保留正常的消化功能。我们提出了对纯化隐窝群体（尤其是 Lgr5+ ISC）进行染色质和其他分子分析的创新方法，以及先进的 2 光子共聚焦显微镜，以前所未有的细节对 ISC 群体进行原位成像。数以千计的基因调节增强子元件在 ISC 的肠细胞 (Ent) 和分泌 (Sec) 谱系中不是单独引发的；许多 Ent 或 Sec 活性增强子已在 Lgr5+ ISC 中引发。增强子引发是序列特异性“先锋”转录因子 (TF) 的范围，它可识别相关的顺式调控元件，并用组蛋白修饰（例如组蛋白 3 赖氨酸 4 甲基化 (H3K4me)）标记侧翼核小体。在目标 1 中，我们提出启动 Lgr5+ ISC 中转录程序的“先锋”TF 是 ASCL2，这是 Lgr5+ ISC 生存和功能所需的肠道限制性 TF。我们将在培养细胞和转基因小鼠中测试 ASCL2 是否满足“先锋”属性，即，它是否最多结合 Lgr5+ ISC 和其他隐窝细胞中的引发增强子，以及这些增强子上的活性组蛋白标记是否在其不存在的情况下在体内消失。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。胚胎细胞中的一种主要抑制途径使用多梳抑制复合物 (PRC)2 放置组蛋白标记 H3K27me3。肠上皮中 PRC2 的缺失会损害 Lgr5+ ISC 和其他隐窝细胞的复制，产生发育不良、有缺陷的绒毛，这些绒毛很快就会被 PRC2 活性单位所取代。在目标 2 中，我们建议对纯化的 Lgr5+ ISC 及其后代隐窝和绒毛细胞中的 H3K27me3 标记、PRC2 依赖性基因表达和突变表型进行综合分析。我们的具体目标是了解动态染色质改变的重要性并定义 ISC 中基因激活和沉默的机制。最后，Bmi1hi +4 ISC 的可视化困难部分阻碍了理解不同 ISC 池及其相应生态位之间功能关系的进展；我们改进了 2 光子共聚焦显微镜方法来检测这些细胞和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，同时专门探讨 Bmi1hi +4 ISC 如何响应隐窝扰动，例如 Lgr5+ ISC 的复制缺陷或潘氏细胞的消融。