Characterization of the mechanism regulating P2X7 expression and functions in intestinal epithelial cells.

成熟的功能性肠上皮的形成需要未成熟的肠上皮细胞整合一系列细胞外信息，形成分化的功能性肠上皮细胞(IECS)。这些信息通过修改DNA调控序列和通过招募特定的蛋白质，即组蛋白和转录因子来允许基因表达，从而导致IECS分化所需的基因的表达。我们发现，三磷酸腺苷通过激活由分化的IECs表达的细胞表面受体P2X7来传递这些信息。事实上，在小鼠中缺乏P2X7的表达(P2X7-/-)会导致上皮结构的异常。在这个研究计划中，我们将确定P2X7在IECS中的表达是如何调节的，以及该受体如何有助于建立一个功能正常的肠道。为了揭示调控P2X7表达和功能的机制，我们将使用Caco-2，一个概括IEC分化的人类上皮细胞模型，从P2X7-/-小鼠分离的IECs和正常的人类肠道活检组织。对控制P2X7表达的DNA调控序列的修饰将通过分析DNA序列和鉴定与该区域相关的组蛋白和转录因子来表征。为了确定P2X7在肠上皮结构中的作用，我们将使用经典的方法，如细胞到细胞连接的特征、细胞增殖率和细胞决定分析，并在第二次，我们将使用一种公正的方法，通过使用高通量RNA测序来描述整个基因组(转录组)，来确定P2X7表达在IECS功能中的全球影响。最后，我们将在Caco-2人类肠道上皮细胞模型中验证这些发现。这一研究计划的预期结果将首次确定调控P2X7表达的分子和细胞机制及其在IECS中的假定作用。考虑到人类之间发现的导致受体功能丧失或获得的多态，我们的结果将带来对该受体在人类生理学中作用的新理解。

The formation of a mature and functional intestinal epithelium requires the integration of an array of extracellular messages by immature epithelial cells to give rise to differentiated and functional intestinal epithelial cells (IECs). These messages lead to expression of genes required for IECs differentiation by modifying DNA regulatory sequences and by the recruitment of specific proteins, namely histones and transcription factors, to allow gene expression. We showed that adenosine triphosphate transmits such messages by activating P2X7, a cell surface receptor expressed by differentiated IECs. Indeed, the absence of P2X7 expression in mice (P2X7-/-) results in aberrant epithelium structure. In this research program, we will determine how P2X7 expression is regulated in IECs and how this receptor contributes to the establishment of a functional intestine. To unravel the regulatory mechanisms regulating P2X7 expression and functions, we will use Caco-2, a human epithelial cell model recapitulating IEC differentiation, IECs isolated from P2X7-/- mice and normal human intestinal biopsies. Modification to the DNA regulatory sequences controlling P2X7 expression will be characterized by analyzing the DNA sequence and by identifying histones and transcription factors associated to this region. To characterize P2X7 roles in intestinal epithelium structure, we will use classical approaches, such as the characterization of cell-to-cell junction, cell proliferation rate and cell determination analysis, in P2X7-/- mice and, in a second time, we will use an unbiased approach to define the global impact of P2X7 expression in IECs function by profiling the entire genome (transcriptome) using high-throughput RNA sequencing. Finally, we will validate these findings in the Caco-2 human model of intestinal epithelial cell. The expected results from this research program will define, for the first time, the molecular and cellular mechanisms governing the regulation of P2X7 expression and its putative role in IECs. Given the polymorphisms found between humans resulting in loss- or gain-of-receptor functions, our results will bring a new understanding on the role of this receptor in human physiology.