Characterizing the function of transcription factor 15 (Tcf15) in pluripotent cells

Abstract

Pluripotent embryonic stem (ES) cells are heterogeneous mixtures of naïve and lineage-primed states defined by distinct transcription factor expression profiles. However, the events that prime pluripotent cells for differentiation are not well understood. Id proteins, which are inhibitors of basic helix-loop-helix (bHLH) transcription factors, contribute to pluripotency by blocking differentiation. Using Yeast-Two-Hybrid screening, our lab identified Tcf15 as an Id-regulated transcription factor. In this study, I first examined the expression of Tcf15 during differentiation in vitro and during early development in vivo in the mouse. Tcf15 expression is higher in primed pluripotent embryonic stem (ES) cells than in naïve ES cells or epiblast stem cells (EpiSCs). In addition, Tcf15 is expressed heterogeneously in ES cells and is also detected in the inner cell mass (ICM) of E4.5 mouse embryos. Expression of Tcf15 was upregulated during early stages of differentiation and downregulated before cells committed to any specific lineage. Using Tcf15-Venus reporter cells, I found that expression of Tcf15 is specifically associated with a novel subpopulation of ES cells primed for somatic lineages. Gain of function and loss of function studies were then performed to perturb Tcf15 expression in ES cells in order to assess the function of Tcf15 in self-renewal and during differentiation. An inducible Id-resistant form of Tcf15 accelerates somatic lineage commitment by maturating naïve pluripotent ES cells transit toward primed epiblast and later on epiblastderived somatic lineages whilst suppressing differentiation towards extraembryonic endoderm. Preliminary loss of function studies also suggest that down-regulation of Tcf15 may promote a naïve state within pluripotent cells. I investigated the mechanism by which Tcf15 expression becomes associated with the epiblast-primed state by identifying the upstream regulators and downstream targets of Tcf15. Tcf15 expression is dependent on FGF signalling. Microarray analysis identified that Tcf15 downregulates the naïve pluripotency determinant Nanog and upregulates the epiblast determinant Otx2. Taken together, our results suggest that Tcf15 acts in opposition to the pluripotency network to prime pluripotent cells towards differentiation.