Molecular characterization of genes with shared and diverse attributes of Oct4 regulation in pluripotent cells

• Applicant: Professor Dr. Frank Buchholz
• Subject Area: Developmental Biology ; Cell Biology
• Term: from 2012 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 214450084

Naïve mouse embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs) represent successive snapshots of pluripotency during embryogenesis 1. In the past few years, a number of RNA interference (RNAi) screens have been performed in mouse ESCs. Results of these screens have greatly extended our knowledge of ESC biology and have led to the identification of novel components required to maintain ESC identity. These screens have also provided new insights into understanding the interplay of multiple pathways of transcription factors, epigenetic regulators and cellular signals implicated in ESC pluripotency. Our group has recently extended the systematic investigation to EpiSCs that resemble pluripotent cells of the post implantation embryo.To reveal factors that control Oct4 expression in EpiSCs, we have performed a genome-scale RNAi screen using Oct4 reporter EpiSCs and compared the role of these factors to their function in ESCs. Surprisingly, beside the identification of shared factors required to maintain Oct4 expression in both cell types, our analyses also revealed numerous knockdowns that lead to increased Oct4 expression solely in EpiSCs2. This result indicates that, in contrast to ESCs, Oct4 is under active repressive control in EpiSCs, thereby establishing a fundamental difference in Oct4 regulation in these two pluripotent cell types. The goal of this proposal is to molecularly characterize two protein complexes in detail. One that exhibits shared and another that exhibits diverse attributes of Oct4 regulation in ESCs versus EpiSCs. We seek to understand better how differential Oct4 regulation is orchestrated by conducting comprehensive transcriptomics, proteomics, and chromatin binding studies. This work should help explain shared properties and fundamental changes in Oct4 regulation in pluripotent cells, providing a framework to dissect the molecular events when pluripotent cells transit from the naïve, pre-implantation to the primed, post-implantation state.

• DFG Programme: Research Grants