Description

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

*Corresponding author

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.