Signaling in digestive epithelial development and homeostasis

描述（由申请人提供）：肠道疾病如炎症性肠病（IBD）和癌症经常反映隐窝干细胞和祖细胞活性失调。这些细胞之间的关系目前正在成为焦点，但仍不清楚关键细胞群如何调节谱系特异性基因，以选择替代肠上皮细胞和分泌细胞的命运。肠隐窝稳态最显著地需要Notch和Wnt信号传导以及其他途径。这两个典型的信号合作，以促进细胞复制，但Wnt信号促进肠分泌细胞的分化，Notch信号有利于肠上皮细胞分化的分泌细胞为代价，这些差异的基础是未知的。Notch抑制转录因子Atoh 1（Math 1）的表达，Atoh 1的活性或缺失似乎足以通过进化上保守的侧向抑制过程分配细胞谱系。操纵GI信号通路用于临床应用将需要更深入地了解基本原理和机制，以确定最不破坏正常消化生理的治疗靶点。该提案采用创新的方法分离小鼠肠隐窝祖细胞，并研究细胞命运决定的表观遗传和转录基础的新细节。在具体目标1中，我们将鉴定纯化的分泌祖细胞中Atoh 1的谱系限制性增强子元件和全基因组结合位点，以确定其指定完整分泌谱系的有效能力的机制。我们将分析Atoh 1占用顺式元件相对于核小体组蛋白动力学的过渡，从干细胞到承诺的祖细胞。我们将定义Atoh 1靶基因，并研究这些基因在显示异常分泌分化的人类病理标本中的作用，包括IBD样本和粘液性/印戒结直肠癌。为了解释Wnt和Notch信号在肠道上皮祖细胞中的多方面关系，我们将检验Atoh 1引导Wnt效应转录因子Tcf 4远离增殖基因的顺式元件并朝向Wnt依赖性分泌细胞程序的顺式元件的假设。在具体目标2中，我们提出分离最早的干细胞后代，能够分化为分泌细胞或肠上皮细胞的双能祖细胞。我们将研究表观遗传基础和Atoh 1的依赖性，为他们随后的承诺，一个单一的命运。我们将检验Atoh 1控制组蛋白标记和核小体动力学不仅激活分泌性顺式元件而且抑制肠上皮细胞基因的假设。使用遗传小鼠模型和最新的方法在表观基因组和转录因子分析，我们提出的实验，将给出明确的答案和测试具体的预测和假设。这些研究共同解决了基本问题，探索了体内分子细节的发育信号传导和调控机制，测试了特定和令人信服的假设，并将有助于改善对常见胃肠道疾病的理解和治疗。

DESCRIPTION (provided by applicant): Intestinal disorders such as inflammatory bowel disease (IBD) and cancer frequently reflect dysregulated activity of crypt stem and progenitor cells. Relationships among these cells are presently coming into focus but it remains unclear how the key cell populations regulate lineage-specific genes to choose among alternative enterocyte and secretory cell fates. Intestinal crypt homeostasis most notably requires Notch and Wnt signaling, among other pathways. The two canonical signals cooperate to promote cell replication, but whereas Wnt signaling promotes intestinal secretory cell differentiation, Notch signaling favors enterocyte differentiation at the expense of secretory cells; the basis for these differences is unknown. Notch inhibits expression of transcription factor Atoh1 (Math1), whose activity or absence seems sufficient to allocate cell lineages by the evolutionarily conserved process of lateral inhibition. Manipulation of GI signaling pathways for clinical application will require a deeper appreciation of underlying principles and mechanisms to identify therapeutic targets that will least disrupt normal digestive physiology. This proposal applies innovative methods to isolate mouse intestinal crypt progenitors and to study in fresh detail the epigenetic and transcriptional basis of cell fate decisions. In Specific Aim 1 we will identify lineage- restricted enhancer elements and genome-wide binding sites for Atoh1 in purified secretory progenitors, with the view to determine mechanisms for its potent ability to specify the full secretory lineage. We will analyze Atoh1 occupancy at cis-elements with respect to nucleosome-histone dynamics in the transition from stem cells to committed progenitors. We will define Atoh1 target genes and investigate the roles of such genes in human pathology specimens showing aberrant secretory differentiation, including IBD samples and mucinous/signet-ring colorectal cancer. To explain the multi-faceted relationship of Wnt and Notch signaling in gut epithelial progenitors, we will test the hypothesis that Atoh1 steers the Wnt-effector transcription factor Tcf4 away from cis-elements of proliferative genes and toward those of a Wnt- dependent secretory cell program. In Specific Aim 2 we propose to isolate the earliest stem-cell progeny, bipotential progenitors able to differentiate into secretory cells or enterocytes. We will study the epigenetic basis and Atoh1 dependencies for their subsequent commitment to a single fate. We will test the hypothesis that Atoh1 controls histone marks and nucleosome dynamics not only to activate secretory cis- elements but also to repress enterocyte genes. Using genetic mouse models and up-to-date approaches in epigenomic and transcription factor analysis, we propose experiments that will give clear answers and test specific predictions and hypotheses. These studies collectively address fundamental questions, explore developmental signaling and regulatory mechanisms in molecular detail in vivo, test specific and cogent hypotheses, and will contribute toward improved understanding and treatment of common GI disorders.