Unravelling the dual function of the regulator SCL/TAL1 in lineage specification through single molecule mRNA FISH and proteomics analyses
Maiken Kristiansen1*, Hedia Chagraoui1, Johanna Richter1, Nicki Gray2, Emmanouela Repapi2, Dominic Waithe2 and Catherine Porcher1
1MRC Molecular Haematology Unit and 2Computational Biology Research Group, MRC Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, UK
Understanding the mechanisms underlying lineage specification is critical to shed light onto essential biological processes and to refine in vitro production of lineage-fated cells. To address these questions, we study the emergence of the blood program from mesodermal precursors through the activity of SCL, a transcription factor essential for blood specification. Scl-/- mouse embryos die at day E9.5 from complete absence of blood and exhibit mis-specification of mesodermal cells towards the cardiac lineage, thus offering an excellent entry point for mechanistic studies of fate determination. We first determined whether the cardiac phenotype observed in Scl-/- embryos reflected multi-lineage priming in mesodermal progenitors. Using single molecule mRNA FISH, we identified ES cell-derived single cells co-expressing low levels of master regulators of blood (Scl), cardiac (Mesp1) and paraxial (Tbx6) lineages at the onset of blood specification. Interestingly, high levels of Scl transcripts (indicating blood-fated cells) were exclusive of the other transcripts, suggesting that repressive mechanisms may actively prevent expression of alternative lineage-affiliated genes at branching points. RNA- and ChIP-sequencing confirmed that SCL acts both as an activator (of the blood programme) and a repressor (of alternative mesodermal lineages). To understand how SCL achieves this dual role, we performed proteomics analysis and identified a large array of SCL interaction partners (co-activators, co-repressors and chromatin remodelling factors). We are currently characterising SCL-containing complexes biochemically and functionally to get molecular insight into the mechanisms orchestrating blood cell fate determination.