Regulation of intestinal genes by CDX2 and other tissue-restricted transcription factors

项目说明 常见的消化道疾病，如炎症性肠病、结直肠癌和 Barrett‘s食道反映了严重的上皮功能障碍和基因失调。改进的治疗方法 需要更好地了解肠道特异性基因控制的基础。某些转录因子 (TFS)-CDX2、HNF4和HNF1-作用于定义肠道上皮特性的基因的远程增强子 和功能，但增强子组装、特异性和活性的机制尚不清楚。非 是否已知肠道内胚层中染色质和转铁蛋白的动态变化如何使独特的消化系统出现？ 上皮细胞。这项建议解决了体内小鼠肠道细胞中的一些基本问题。 在数千个控制肠道基因的顺式元件中，我们发现了一类新的组织特异性 需要CDX2从大基因组中排除抑制性组蛋白标记H3K27me3的增强子 结构域；H3K27me3以前在成人组织中没有涉及到远程增强子。这些发现 将CDX2的一种新的、意想不到的功能归因于控制最多的1A型抗抑制增强子 Cdx2-/-绒毛细胞中数量脆弱的基因。具体目标1解决了要求和 这些新型促进剂的作用机理。我们建议进行遗传和生化实验来测试两个 中心假设：(A)大约500个关键的肠道基因座特别容易受到抑制，因为 H3K27me3的广泛局部沉积，以及(B)CDX2招募特定的H3K27去甲基酶 KDM6A/B来对抗这种全基因效应。我们将使用缺乏甲基化H3K27的Eed-/-肠道来 确定这一特征是否具有因果抑制作用，并尝试挽救最受干扰的基因 CDX2-/-肠道。因为1A型增强剂在H3K27me3水平上显示出很大的差异 肠隐窝中的肠细胞前体细胞和沿着绒毛的终末成熟的肠细胞，我们建议 对这种抑制标记限制肠道细胞基因高表达的假设进行了研究 复制隐窝细胞。特定目标2考虑肠道的发育起源和决定因素 增强剂，并询问为什么某些基因组结构域获得组织特异性H3K27me3。这一目标建立在 我们的发现是所有消化上皮的新生促进剂最初在整个早期都可以获得 内胚层和组织特异性部位随后在每个器官中增强，而不适当增强剂 关门了。我们将检验这一假设：成人肠道中依赖于1A型增强子的区域 是在开发过程中使用的增强剂的残留物。我们还将检验以下假设：(A) 除非局部TF加强开放的内胚层染色质，否则增强剂会在吻尾波中关闭， 以及(B)染色质在任何时候的状态决定了发育中的肠道对缺乏 CDX2。因此，该提案应用了创新的最先进的方法来追求令人兴奋的发现和 解决有关体内肠道基因调控的基本问题。

Project Description Common disorders of the digestive tract, such as Inflammatory Bowel Disease, colorectal cancer and Barrett's esophagus reflect profound epithelial dysfunction and gene dysregulation. Improved treatments require better understanding of the basis for intestine-specific gene control. Certain transcription factors (TFs) – CDX2, HNF4 and HNF1 – act at the distant enhancers of genes that define gut epithelial identity and function, but mechanisms of enhancer assembly, specificity, and activity are not well understood. Nor is it known how chromatin and TF dynamics in gut endoderm allow the emergence of distinctive digestive epithelia. This proposal addresses some of these fundamental questions in mouse intestinal cells in vivo. Among thousands of cis-elements that control intestinal genes, we identify a new class of tissue-specific enhancers that require CDX2 to exclude the repressive histone mark H3K27me3 from large genomic domains; H3K27me3 was not previously implicated at distant enhancers in adult tissues. These findings ascribe a novel, unexpected function for CDX2 at `anti-repressive' Type 1A enhancers that control the most quantitatively vulnerable genes in Cdx2-/- villus cells. Specific Aim 1 addresses the requirements and mechanisms of these novel enhancers. We propose genetic and biochemical experiments to test two central hypotheses: (a) that ~500 key intestinal loci are particularly susceptible to repression by virtue of extensive local deposition of H3K27me3, and (b) that CDX2 recruits the specific H3K27 demethylases KDM6A/B to oppose that locus-wide effect. We will use Eed-/- intestines, which lack methylated H3K27, to determine whether this feature is causally repressive and to attempt rescue of the genes most perturbed in Cdx2-/- intestines. Because Type 1A enhancers show a large difference in H3K27me3 levels between enterocyte progenitors in intestinal crypts and terminally mature enterocytes along the villi, we propose studies to test the hypothesis that this repressive mark limits high expression of enterocyte genes in replicating crypt cells. Specific Aim 2 considers the developmental origins and determinants of intestinal enhancers and asks why certain genomic domains acquire tissue-specific H3K27me3. This Aim builds on our discovery that nascent enhancers for all digestive epithelia are initially accessible throughout the early endoderm and that tissue-specific sites later strengthen in each organ while inappropriate enhancers are shut down. We will test the hypothesis that regions dependent on Type 1A enhancers in the adult intestine are the vestiges of enhancers that were used in development. We will also test the hypotheses that: (a) unless regional TFs reinforce open endodermal chromatin, enhancers shut down in a rostral-caudal wave, and (b) the state of chromatin at any time determines how the developing intestine responds to absence of CDX2. This proposal thus applies innovative state-of-the-art approaches to pursue exciting discoveries and address fundamental questions about intestinal gene regulation in vivo.