Mechanisms of Intestinal Epithelial Differentiation

肠上皮细胞的适当分化是肠道健康的几乎所有方面的基础。比如说， 不适当的上皮屏障功能是炎症性肠病的关键因素， 分化导致最终消化和营养吸收不良，并增加对癌症的易感性。 促进肠上皮细胞分化的转录因子是已知的，但在肠上皮细胞中存在缺口。 了解这些因子如何与转录机制相互作用以驱动肠道分化。 新技术现在使测量染色质成环事件和RNA聚合酶动力学成为可能， vivo.这些过程在肠道中未被探索，可能具有精氨酸特异性机制。 我们工作的长期目标是解决和操纵肠分化的机制;我们的 中心假设是，未探索的转录机制是动态调节的细胞， 在肠道中分化。这些机制包括：1）增强子-启动子染色质环，2）RNA 聚合酶加载，和3）转录延伸。该提案还将审查亲- 分化BMP/SMAD 4信号通路与转录因子交叉以驱动分化。 一种新的HNF 4A和HNF 4G转录因子的复合敲除导致HNF 4A和HNF 4G的严重破坏。 肠分化ChIP-seq和RNA-seq识别了数百个在绒毛上激活的基因 分化并依赖HNF 4表达。这种新的转基因模型提供了一个严格的系统 应用新技术并定义驱动分化的肠道调节机制。 这个提议的基本原理是发现能够在转录水平上调节细胞的丝氨酸特异性转录调控机制， 为了人类健康而被利用。新的转基因小鼠将被用来定义调控 需要激活体内分化基因的机制。Aim 1将应用新技术来解决 未探索的驱动分化的转录机制。目标2将探索一种新的模式， 通过BMP信号传导和HNF 4因子的协调作用促进细胞分化。我们希望能发现新的 肠上皮细胞分化的机制。 目标1和2的重要性在于它们可以帮助我们了解遗传变异对增强子的影响， 确定新的协同调节因子，并导致改变肠道内稳态平衡的治疗方法， 这些分化与IBD、癌症和再生医学的治疗相关。HNF 4和SMAD 4 都与人类IBD和结肠癌密切相关。 拟议的研究是创新的使用新的转基因模型，国家的最先进的表观基因组学 方法和人类类器官培养。创新理念包括差异化管控新范式 通过HNF 4和BMP信号的协同努力，并提供了一个新的概念，通过该领域可以移动 期待找到控制上皮分化的治疗机会。

Proper differentiation of the intestinal epithelium underlies nearly all aspects of intestinal health. For example, improper epithelial barrier function is a critical contributor to inflammatory bowel diseases, and improper differentiation leads to poor terminal digestion and nutrient absorption, and increases susceptibility to cancers. Transcription factors that promote differentiation in the intestinal epithelium are known, but a gap exists in understanding how these factors interact with the transcriptional machinery to drive intestinal differentiation. New technologies now make it feasible to measure chromatin looping events and RNA Polymerase kinetics in vivo. These processes are unexplored in the intestine and likely to have intestine-specific mechanisms. The long term goal of our work is to resolve and manipulate the mechanisms of intestinal differentiation; our central hypothesis is that unexplored transcriptional mechanisms are dynamically regulated as cells differentiate in the intestine. These mechanisms include: 1) enhancer-promoter chromatin looping, 2) RNA polymerase loading, and 3) transcriptional elongation. The proposal will also examine how the pro- differentiation BMP/SMAD4 signaling pathway intersects with transcription factors to drive differentiation. A new, compound knockout of both HNF4A and HNF4G transcription factors leads to severe disruption of intestinal differentiation. ChIP-seq and RNA-seq identify hundreds of genes that are activated upon villus differentiation and depend upon HNF4 for expression. This novel transgenic model provides a rigorous system to apply new technologies and define intestinal regulatory mechanisms driving differentiation. The rationale of this proposal is to discover intestine-specific transcriptional regulatory mechanisms that can be exploited for the gain of human health. The novel transgenic mouse will be leveraged to define regulatory mechanisms required to activate differentiation genes in vivo. Aim 1 will apply new technologies to tackle unexplored transcriptional mechanisms driving differentiation. Aim 2 will explore a new paradigm for intestinal differentiation through the coordinated actions of BMP signaling and HNF4 factors. We expect to identify new mechanisms through which differentiation of enterocytes is achieved. Aims 1 & 2 are significant in that they can help us understand the impacts of genetic variants at enhancers, identify novel co-regulators, and lead to therapeutics that alter the homeostatic balance of intestinal differentiation that are relevant for treatments of IBD, cancer, and in regenerative medicine. HNF4 and SMAD4 are both strongly implicated in human IBD and Colon Cancer. The proposed research is innovative in the use of novel transgenic models, state-of-the-art epigenomics approaches, and human organoid cultures. Innovative ideas include the new paradigm of differentiation control by concerted efforts of HNF4 and BMP signaling, and provide a new concept through which the field can move forward to find therapeutic opportunities to control epithelial differentiation.