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

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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.