Analysis of intestinal genes regulated by the transcription factor CDX2

描述（由申请人提供）：对负责精氨酸特异性基因调控和细胞分化的分子机制的认识有限。一种重要的调节因子，转录因子CDX 2，局限于肠上皮，在其中它在整个隐窝-绒毛单位中表达，并且在体内是肠特异性柱状上皮分化所需的。CDX 2的转录靶点和机制的知识是不完整的，因为它是如何作为一个主转录调节功能的理解。我们已经使用全基因组染色质免疫沉淀（ChIP），以高置信度，在结肠上皮细胞中的CDX 2占用区域。CDX 2结合区是高度保守的，并显示了一些其他序列特异性DNA结合蛋白先前涉及肠道基因调控的基序的显着聚类。因此，我们的研究正确地确定了许多CDX 2靶点，并揭示了3个特定的候选伴侣转录因子在精氨酸特异性基因调控。我们将扩展初步数据和见解，以测试CDX 2功能和分子机制的特定假设。目的1旨在确定在约1，100个CDX 2结合位点中哪些是肠细胞中真正的顺式元件。我们将鉴定在表达该因子的细胞中对CDX 2耗竭做出反应的转录物，以及在不表达该因子的细胞中对强迫CDX 2表达做出反应的转录物。我们还将测试推定的顺式元件的功能报告分析，并严格检查是否CDX 2靶基因反映了预期的主调节器的活动。出乎意料的是，我们发现CDX 2通常占据非常接近Tcf蛋白结合位点的DNA，Tcf蛋白是经典Wnt途径的转录效应子。Wnt信号在许多组织中传递，但在肠道内稳态中至关重要。我们将测试新的假设，CDX 2赋予肠道特异性内的全球Wnt反应。我们还发现了CDX 2激活基因附近的核受体HNF 4和CDX 2似乎抑制的基因附近的加塔蛋白的显著共占据。目的2采用几种方法来检验HNF 4和加塔因子联合收割机与CDX 2结合分别激活和沉默基因的假设。最后，对分离的小鼠肠隐窝和绒毛组分的全基因组ChIP表明Cdx 2控制这两个功能区室中的不同基因。在目标3中，我们将测试这一假设并解决潜在的机制。我们将描绘Cdx 2的伴侣蛋白，并询问是否需要Cdx 2的结合，以产生隐窝和绒毛特异性染色质结构域，或者相反，如果Cdx 2响应其他因素创建这样的结构域。为此，我们已经建立了全基因组分析的可行性信息染色质标记和产生的小鼠肠道Cdx 2水平可以调制。这些研究代表了一个详细和全面的方法，以阐明机制的甜菜碱特异性基因调控。 公共卫生相关性：常见的胃肠道疾病，包括炎症、吸收不良、溃疡和癌症，会造成相当大的痛苦和死亡。虽然这些条件是从根本上与正常基因表达和细胞行为的机制，有其分子基础有限的赞赏。CDX 2蛋白几乎只存在于肠道中，是正常和疾病状态下的关键调节因子。对其调节功能的研究将提高对许多常见胃肠道疾病的理解，并最终提高其治疗效果。

DESCRIPTION (provided by applicant): Appreciation of the molecular mechanisms responsible for intestine-specific gene regulation and cell differentiation is limited. One important regulator, the transcription factor CDX2, is restricted to intestinal epithelium, where it is expressed throughout the crypt-villus unit and required in vivo for differentiation of gut- specific columnar epithelium. Knowledge of CDX2's transcriptional targets and mechanisms is incomplete, as is understanding of how it functions as a master transcriptional regulator. We have used whole-genome chromatin immunoprecipitation (ChIP) to identify, with high confidence, regions of CDX2 occupancy in colonic epithelial cells. CDX2-binding regions are highly conserved and show significant clustering of motifs for a handful of other sequence-specific DNA-binding proteins previously implicated in intestinal gene regulation. Our studies hence correctly identify numerous CDX2 targets and reveal 3 specific candidate partner transcription factors in intestine-specific gene regulation. We will extend the preliminary data and insights to test specific hypotheses on CDX2 function and molecular mechanisms. Aim 1 seeks to identify which among ~1,100 CDX2-binding sites are bona fide cis-elements in intestine cells. We will identify transcripts that respond to CDX2 depletion in cells that express the factor and to forced CDX2 expression in cells that don't. We will also test putative cis-elements in functional reporter assays and critically examine whether CDX2 target genes reflect the activities expected of a master regulator. Unexpectedly, we find that CDX2 commonly occupies DNA very close to binding sites for Tcf proteins, transcriptional effectors of the canonical Wnt pathway. Wnt signals are transmitted in many tissues but are critical in intestinal homeostasis. We will test the novel hypothesis that CDX2 imparts intestinal specificity within a global Wnt response. We also find significant co-occupancy of the nuclear receptor HNF4) near CDX2-activated genes and of GATA proteins near genes that CDX2 appears to repress. Aim 2 takes several approaches to test the hypothesis that HNF4) and GATA factors combine with CDX2 to activate and silence genes, respectively. Lastly, genome-wide ChIP on isolated mouse intestinal crypt and villus fractions implies that Cdx2 controls distinct genes within these two functional compartments. In Aim 3 we will test this hypothesis and address the underlying mechanisms. We will delineate Cdx2 partner proteins and ask if Cdx2 binding is needed to generate crypt- and villus-specific chromatin domains or, conversely, if Cdx2 responds to the creation of such domains by other factors. To this end, we have established the feasibility of whole-genome analysis of informative chromatin marks and generated mice in which intestinal Cdx2 levels can be modulated. These studies represent a detailed and comprehensive approach to elucidate mechanisms of intestine-specific gene regulation. PUBLIC HEALTH RELEVANCE: Common disorders of the gastrointestinal (GI) tract, including inflammation, malabsorption, ulcers and cancer, cause considerable suffering and death. Although these conditions are fundamentally related to mechanisms of normal gene expression and cell behavior, there is limited appreciation of their molecular underpinnings. The CDX2 protein, which is found almost exclusively in the intestine, is a pivotal regulator in normal and disease states. Investigation of its regulatory functions will improve understanding and, ultimately, treatment of many common GI diseases.