Mesoscale chromatin confinement facilitates target search of pioneer transcription factors in live cells [ATAC-seq]

To dissect the molecular mechanisms of the transcription factor FOXA2 accessing nucleosomal targets and controlling cell fates, we combined live-cell single-molecule imaging, genome editing, chromatin binding, chromatin accessibility, and RNA sequence analysis. Using the hESC differentiation cellular system to anterior primitive streak (APS) and definitive endoderm (DE), we demonstrated by RNA-seq that FOXA2 is a lineage-determining transcription factor for DE formation and that its intrinsically disordered C-terminal domain (CTD) is essential for its normal function. Through ChIP-seq and ATAC-seq at multiple time points of exogenous expression, we also showed that the CTD is required for FOXA2 effectively accessing targets in closed chromatin and altering chromatin accessibility. Together with the single-molecule kinetics captured by fluorescence microscopy, this study uncovers new mechanistic insights on the activity of lineage-determining transcription factors in engaging chromatin and accessing nucleosomal targets.