Molecular mechanism of Foxd3 transcription factor enhancer selection in the embryonic stem cell to epiblast cell transition

• Applicant: Dr. Deniz Gökbuget
• Subject Area: Cell Biology
• Term: from 2017 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 363120021

Transcription factors (TFs) are crucial drivers of cell identity in health and disease. Intriguingly, TFs only populate a small fraction of all potential binding sites in the genome in a given cell type, thereby critically driving cell fate acquisition. In the transition of embryonic stem cells (ESCs) to epiblast cells (EpiCs), pioneer-like TF Foxd3 binds to almost mutually exclusive enhancer regions, thereby priming proximal genes for future activation or repression. Loss or gain of Foxd3 function during this transition results in precocious differentiation and a failure to maintain self-renewal highlighting the importance of properly utilizing physiological amounts of Foxd3. This proposal aims to dissect the molecular mechanism underlying Foxd3 localization in ESCs and EpiCs. It is based on the hypothesis that localization of pioneer-like TFs depends on association with cofactors in the form of posttranslational modifications, non-coding RNA scaffolds and/or collaborating TFs. First, differential posttranslational modifications of Foxd3 will be addressed in ESCs and EpiCs using mass spectrometry after purification of endogenously tagged Foxd3. Posttranslational modifications of Foxd3 will be addressed in ESCs and EpiCs using mass spectrometry, followed by subsequent analyses of upstream signaling pathways and evaluation of their functional relevance for Foxd3 localization and ESC physiology. Foxd3 co-regulators, particularly collaborating TFs, will be identified using a combined proteomics and transcriptomics approach after prior immunoprecipitation of endogenously tagged Foxd3 or affinity purification of biotinylated proteins and RNAs proximal to BioID2 or APEX2-tagged endogenous Foxd3. Subsequently, functional relevance of selected candidates for regulation of Foxd3 localization will be addressed using CRISPRi-mediated repression followed by Foxd3 ChIP sequencing. Ultimately, these data will provide critical insights into the basic molecular mechanism of cell-type specific TF localization, which is highly relevant for a broad range of cell fate transitions in health and disease.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Robert Blelloch