Deciphering the Erf-NCoR1/2 complex cooperation in TS cells

The placenta is the site of exchange between the maternal and foetal bloodstreams. The specialized trophoblast cell types of the placenta originate from common precursors called trophoblast stem cells (TSCs). In the presence of specific signalling molecules, mouse TSCs self-renew indefinitely while sustaining an undifferentiated, multipotent state. Upon removal of these signalling factors, TSCs differentiate into various trophoblast cell types. The molecular mechanism of how abrogation of signalling drives changes in gene expression that result in TSC differentiation remains largely unknown. Our preliminary data confirmed that the Ets2 repressor factor (Erf) is an important mediator of signalling in TSCs. While abrogation of Erf results in a trophoblast differentiation defect, its exact mode of action is poorly understood. To address the underlying mechanism, we identified components of the multi-subunit complexes Nuclear Receptor Co-Repressor 1 and 2 (NCoR1/2) as strong interacting partners of Erf. Depletion of the NCoR1/2 component transducin ß-like 1 X-linked (Tbl1x) resulted in TSC differentiation failure as assessed by an abnormal gene expression profile. Based on the preliminary data and previous studies, we hypothesise that Erf translocates into the nucleus where it recruits the NCoR1/2 complex and causes transcriptional silencing of key TSC genes. These molecular changes result then in TSC differentiation. To test this hypothesis, we aim to: i) address the requirement of the Erf-NCoR1/2 co-operation in TSC self-renewal and differentiation, by depletion of its components and ii) identify the genes directly co- regulated by Erf and NCoR1/2. The anticipated results will uncover the role of the NCoR1/2 complex in TSCs and reveal its close, functional cooperation with Erf at the onset of TSC differentiation. Finally, the discovery of this direct functional link may have a broader medical impact including on studies of Erf and NCoR1/2-dependent oncogenesis.

The placenta is a vital organ to sustain pregnancy and ensure embryonic development. It enables exchange of nutrients, gases, and metabolites exploiting via a range of highly specialized cell types differentiating during development from a shared progenitor. The progenitor population is represented in vitro by trophoblast stem cells (TSCs). In the presence of specific signalling molecules, murine TSCs self-renew indefinitely while sustaining an undifferentiated, multipotent state. Upon removal of these signalling factors, TSCs differentiate into various trophoblast cell types, providing an in vitro model of placental development. How signalling abrogation drives changes in gene expression that result in TSC differentiation remained largely unknown. Here we used murine TSCs to understand how the Erf protein transforms the signaling cues into expression of specific genes and thus ensures proper placental development. We showed that upon inhibition of the Fgf/Erk signalling pathway the Erf protein translocates into the cell nucleus where it interacts with the Nuclear Receptor Co-Repressor Complex 1 and 2 (NCoR1/2) and recruits it to key trophoblast genes. Genetic ablation of Erf or Tbl1x (a component of the NCoR1/2 complex) abrogates the Erf/NCoR1/2 interaction. This leads to mis-expression of Erf/NCoR1/2 target genes, resulting in a TSC differentiation defect. Mechanistically, Erf regulates expression of these genes by recruiting the NCoR1/2 complex to the gene regulatory regions called enhancers. The Erf/NCoR1/2 complex removes a repressive chromatin mark from the enhancers, resulting in their deactivation and in turn in silencing of the associated genes (genes they are associated with). Our findings uncovered the molecular function of the Fgf/Erf/NCoR1/2 axis and the molecular mechanism underlying cell fate transitions during murine placental development. The discovery of this direct functional link may have a broader medical relevance including for Erf and NCoR1/2-dependent oncogenesis.