Supplementary Figure 11: The Erg+85 enhancer is active during haematopoietic development

(a) UCSC Genome Browser tracks of re-analysed ChIP-Seq data for HoxB4⁵ indicates that HoxB4 binds to the Erg+85 enhancer (shown in grey) during a 26-day differentiation time course that produces HSCs from ES cells, with HSCs able to contribute to long-term engraftment in recipient mice. Binding is absent at day 6, but becomes apparent by day 16. (b) Flow cytometric analysis of Flk1 and CD41 expression across a time-course of EB differentiation from Figure 4B indicates that different clones have similar kinetics of differentiation, with Flk1⁺ cells giving rise to Flk1⁺CD41⁺ and then Flk1⁻CD41⁺ cells. (c) Flow cytometric analysis of YFP expression in all live cells across a time-course of EB differentiation from Figure 4b illustrates reduction of YFP positive cells in ES cell clones where YFP expression is driven by mutant enhancer constructs. In (b) and (c), a representative experiment is shown from three biological repeats of 2-3 clones per construct.