Dissecting the molecular mechanisms regulating somatic cell reprogramming

Somatic nuclei can be reprogrammed to a pluripotent or embryonic stage. Somatic cell reprogramming has been shown to be possible via animal cloning, cell fusion, and ectopic expression of a few embryonic stem cell (ESC) factors. The reprogramming time of a cell nucleus is normally relatively long and it depends upon cell type, methods of reprogramming, and culture conditions used. These observations led us to envisage that the process according to which somatic cells change their fate involves many factors and sequential events. Thus, transcription factors, chromatin-modifying complexes and signalling molecules are likely to operate in a specific order to induce and maintain the reprogramming of a somatic nucleus. We have recently shown that the periodic activation of Wnt/beta-catenin signalling controls fusion-mediated somatic-cell reprogramming. Only this periodic and limited nuclear accumulation of beta-catenin allows ESCs to reprogramme somatic cells after fusion. Furthermore, in ESCs, either overexpression of Axin2, which is part of the beta-catenin destruction complex, or deletion of GSK3, which is devoted to the phosphorylation of beta-catenin, inhibits ESC-fusion-mediated somatic-cell reprogramming. In contrast, deletion of a specific beta-catenin modulator, in ESCs strongly enhances their reprogramming ability. Our hypothesis is that signalling dependent genes encode for key reprogramming factors, which we aim to identify. We will perform transcriptome, microRNAome and epigenome analyses of ESCs pre-treated with positive and negative modulators of the Wnt pathway. In addition, we will perform the same type of analysis using wild-type and mutant ESCs which carry the activated or inhibited Wnt signalling pathway. All of the collected data will be analysed by ARACNe, MINDy and MRA reverse engineering algorithms, to reconstruct the reprogramming regulatory network and to identify pathways and master reprogrammer genes. Finally, the reprogrammer candidates will be cloned and validated for their ability to induce fusion-mediated reprogramming or direct reprogramming.