Analysis of intestinal genes regulated by the transcription factor CDX2

描述(由申请人提供)：对肠道特异基因调控和细胞分化的分子机制的评价有限。转录因子CDX2是一种重要的调节因子，它仅限于肠道上皮，在隐窝-绒毛单位中表达，在体内是分化肠道特异性柱状上皮所必需的。对CDX2的S转录靶点和转录机制的了解是不完整的，对它如何作为主要转录调控因子的理解也是不完整的。我们已经使用全基因组染色质免疫沉淀(CHIP)来高度可信地识别结肠上皮细胞中CDX2的占位区域。CDX2结合区是高度保守的，并显示出显着的基序聚集的少数其他序列特异性DNA结合蛋白以前涉及肠道基因调控。因此，我们的研究正确地识别了大量的CDX2靶标，并揭示了肠道特异性基因调控中的3个特定候选伙伴转录因子。我们将扩展初步的数据和见解，以检验关于CDX2功能和分子机制的具体假设。目的1鉴定在~1100个CDX2结合位点中，哪些是肠道细胞中真正的顺式元件。我们将在表达该因子的细胞中识别对CDX2耗竭做出反应的转录本，在不表达CDX2的细胞中识别对强制表达CDX2做出反应的转录本。我们还将在功能报告分析中测试假定的顺式元件，并严格检查CDX2靶基因是否反映了主要调控因子预期的活性。出乎意料的是，我们发现CDX2通常占据非常接近Tcf蛋白结合部位的DNA，Tcf蛋白是典型的Wnt途径的转录效应因子。WNT信号在许多组织中传递，但在肠道内稳态中起关键作用。我们将测试这一新的假设，即CDX2在全球WNT反应中赋予肠道特异性。我们还发现CDX2激活的基因附近的核受体HNF4)和CDX2似乎抑制的基因附近的GATA蛋白显著共存。目的2采用多种方法验证假设，即HNF4和GATA因子分别与CDX2结合来激活和沉默基因。最后，对分离的小鼠肠道隐窝和绒毛部分进行的全基因组芯片表明，CDX2控制着这两个功能区块中的不同基因。在目标3中，我们将测试这一假设，并解决潜在的机制。我们将描述CDX2伙伴蛋白，并询问是否需要CDX2结合来产生隐窝和绒毛特异的染色质结构域，或者相反，CDX2是否对其他因素创建这样的结构域做出反应。为此，我们已经建立了对信息性染色质标记进行全基因组分析的可行性，并产生了肠道CDX2水平可以调节的小鼠。这些研究为阐明肠道特异性基因调控机制提供了一种详细而全面的途径。 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。虽然这些情况从根本上与正常基因表达和细胞行为的机制有关，但人们对它们的分子基础认识有限。CDX2蛋白几乎只在肠道中发现，在正常和疾病状态下是一个关键的调节因子。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，并最终促进对其的治疗。

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.