SCL establishes a transcriptional and epigenetic repressive environment in blood-fated cells to suppress alternative mesodermal lineages

Understanding the mechanisms underlying lineage specification is critical to shed light onto essential biological processes supporting tissue formation, and for development of cell-based therapies. The molecular basis of cell fate decision is, however, unclear. Our aim is to characterise the emergence of the blood programme from mesoderm precursors through the activity of SCL/TAL1, a transcriptional regulator at the apex of blood specification. SCL-null mouse embryos die at embryonic day E9.5 from absence of blood and exhibit mis-specification of mesodermal cells towards the cardiac lineage, thus offering an excellent entry point for mechanistic studies of cell fate determination. To identify the gene regulatory network governing blood specification, we have integrated SCL ChIP-, RNA- and ATAC-sequencing analyses from wild-type and Scl-/- mouse ES cell-derived FLK1+ mesoderm, at the time of blood specification (day 3.5 embryoid bodies, EBs). This revealed the SCL-driven activating and repressive genetic programmes. Interestingly, whilst directly repressing expression of regulators of alternative mesodermal lineages, SCL strongly activates expression of not only blood-specific regulators but also potent repressors, such as known partner ETO2 and Polycomb Repressive Complex 1 (PRC1) component RYBP, through robust binding at cis-regulatory elements. Consistent with a feedforward loop model, SCL, ETO2 and RYBP bind to cardiac loci normally repressed by SCL. Furthermore, Rybp knock-out and siRNA-mediated Rybp knock-down EB cells phenocopy the SCL-null cardiac phenotype. We are currently further assessing RYBP recruitment mechanisms and the link between SCL and the epigenetic landscape in FLK1+ cells. Finally, the concomitant activating and repressive functions of SCL suggested multi-lineage priming in mesodermal cells. Indeed, single molecule mRNA FISH revealed co-expression in single EB cells of Scl, Tbx6 and Mesp1, respectively master regulators of the blood, paraxial and cardiac programmes, prior to specification of the haematopoietic lineage. In conclusion, we have established strong genetic and functional links between SCL, repressors and chromatin remodelling factors in the orchestration of the mechanisms leading to blood specification. These are likely to stem from the promiscuous expression of distinct lineage-affiliated regulators in mesodermal cells.