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

成熟和功能性肠上皮的形成需要未成熟上皮细胞整合一系列细胞外信息以产生分化和功能性肠上皮细胞（IEC）。这些信息通过修饰DNA调控序列和募集特定蛋白质（即组蛋白和转录因子）以允许基因表达，导致IEC分化所需的基因表达。我们发现三磷酸腺苷通过激活P2 X7（一种由分化的IEC表达的细胞表面受体）来传递这些信息。事实上，小鼠中P2 X7表达的缺失（P2 X7-/-）导致异常的上皮结构。在这项研究计划中，我们将确定P2 X7表达如何在IEC中调节，以及这种受体如何有助于建立功能性肠道。为了阐明调节P2 X7表达和功能的调节机制，我们将使用Caco-2，一种重现IEC分化的人上皮细胞模型，从P2 X7-/-小鼠和正常人肠活检中分离的IEC。控制P2 X7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。为了表征P2 X7在肠上皮结构中的作用，我们将在P2 X7-/-小鼠中使用经典方法，例如细胞间连接的表征、细胞增殖速率和细胞测定分析，并且在第二次中，我们将使用一种无偏的方法，通过分析整个基因组来确定P2 X7表达对IEC功能的整体影响（转录组）使用高通量RNA测序。最后，我们将在Caco-2人类肠上皮细胞模型中验证这些发现。这项研究计划的预期结果将首次确定P2 X7表达调控的分子和细胞机制及其在IEC中的假定作用。鉴于人类之间发现的多态性导致受体功能的丧失或获得，我们的研究结果将对该受体在人类生理学中的作用产生新的认识。

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.