MyoD and FoxO3 mediated hotspot interaction orchestrates super-enhancer activity during myogenic differentiation. Mus musculus

Super-enhancers (SEs) are cis-regulatory elements enriching lineage specific key transcription factors (TFs) to form hotspots. A paucity of identification and functional dissection promoted us to investigate SEs during myoblast differentiation. ChIP-seq analysis of histone marks leads to the uncovering of SEs which remodel progressively during the course of differentiation. Further analyses of TF ChIP-seq enable the definition of SE hotspots co-bound by the master TF, MyoD and other TFs, among which we perform in-depth dissection for MyoD/FoxO3 interaction in driving the hotspots formation and SE activation. Overall design: The CRISPR/Cas9 system was used to delete hotspots (H1_KO, H23_KO, H34_KO, H3_KO) and knockout MyoD expression (Myod1_KO) in C2C12 cell line following published protocols (Ran, F. A. et al., 2013; Huang, J. et al., 2016). Double deletion of H2 and H3 was generated by deleting H3 in H2 KO cell line. Similarly, double deletion of H3 and H4 was generated by deleting H3 in H4 KO cell line. Both WT and KO C2C12 cells were induced to differentiate by culturing in differentiation medium (DMEM, 2% horse serum and 1% pen/strep) and processed for H3K27ac ChIP-seq.