Analysis of intestinal genes regulated by the transcription factor CDX2

描述（申请人提供）：对肠道特异性基因调控和细胞分化的分子机制的认识是有限的。一个重要的调节因子，转录因子CDX2，仅限于肠上皮，它在整个隐窝绒毛单位中表达，并且是肠道特异性柱状上皮分化所必需的。对CDX2的转录靶点和机制的了解尚不完整，对它如何作为主要转录调控因子发挥作用的理解也不完整。我们使用全基因组染色质免疫沉淀（ChIP）技术以高置信度鉴定了CDX2在结肠上皮细胞中的占据区域。cdx2结合区是高度保守的，并且显示出少数其他序列特异性dna结合蛋白的显着聚类基序，这些基序先前涉及肠道基因调控。因此，我们的研究正确地确定了许多CDX2靶点，并揭示了3个特定的候选伴侣转录因子在肠道特异性基因调控中。我们将扩展初步数据和见解，以测试关于CDX2功能和分子机制的特定假设。Aim 1旨在确定肠细胞中约1100个cdx2结合位点中哪些是真正的顺式元件。我们将在表达CDX2因子的细胞中识别对CDX2缺失有反应的转录本，在不表达CDX2因子的细胞中识别对CDX2缺失有反应的转录本。我们还将在功能报告基因分析中测试假定的顺式元件，并严格检查CDX2靶基因是否反映了预期的主调控因子的活动。出乎意料的是，我们发现CDX2通常占据非常接近Tcf蛋白结合位点的DNA， Tcf蛋白是典型Wnt通路的转录效应物。Wnt信号在许多组织中传递，但在肠道内稳态中至关重要。我们将验证CDX2在全球Wnt反应中赋予肠道特异性的新假设。我们还发现核受体HNF4靠近CDX2激活的基因，GATA蛋白靠近CDX2抑制的基因。Aim 2采用了几种方法来验证HNF4和GATA因子分别与CDX2结合激活和沉默基因的假设。最后，对分离的小鼠肠隐窝和绒毛进行全基因组ChIP分析表明，Cdx2控制着这两个功能区室中的不同基因。在目标3中，我们将检验这一假设并解决潜在的机制。我们将描述Cdx2伴侣蛋白，并询问是否需要Cdx2结合来产生隐窝和绒毛特异性染色质结构域，或者相反，Cdx2是否通过其他因素响应这些结构域的产生。为此，我们建立了对信息染色质标记进行全基因组分析的可行性，并产生了可以调节肠道Cdx2水平的小鼠。这些研究为阐明肠道特异性基因调控机制提供了详细而全面的途径。

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.