Molecular mechanisms of direct reprogramming of fibroblasts to trophoblast stem cells

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Lineage-identity of a differentiated somatic cell is being stabilized by specific chromatin configuration, DNA-methylation and networks of self-sustaining transcription factors. Experimental settings, such as somatic cell nuclear transfer or the overexpression of transcription factors are able to interfere with these stabilizing networks result in reprogramming other somatic lineages or to pluripotent cells. Recently, we demonstrated that four transcription factors (Tfap2c, Eomes, Gata3 and Ets2; abbreviated: TEGE) are sufficient for reprogramming of murine fibroblasts to trophoblast stem cells. Direct reprogramming is a promising approach for regenerative medicine and human disease modeling: die molecular mechanism of such direct reprogramming is only poorly understood. Here we propose, to take advantage of our recently published fibroblast-to-trophoblast reprogramming model and subject it to an highly sophisticated integrative genomic approach. At various time-points during reprogramming we propose to determine expression (by RNA-seq), DNA binding of the TEGE-factors (by ChIP-seq) and the nucleosome-coverage (by ATAC-seq). We will be able to define the so-called pioneer-factors, which are able to bind to nucleosome-covered (and silenced) DNA-elements, transactivate transcription and initiate the epigenetic reprogramming. Thus, we will be able to define direct targets of the TEGE-Factors in this system. These targets should be able to replace one or more of the TEGE-factors when overexpressed. Knowledge of such dynamics enables us to define the molecular steps in the reprogramming. The experiments will allow an insight about the close interconnection of transcription factor circuits, alterations in nucleosome coverage, histones and DNA-Methylation. We aim to establish a model, which precisely describes the dynamics of this reprogramming. With this in hand, we will be able to predict the efficiency of reprogramming of other cell types.