Sequence specific transcription factors (TFs) bind to short specific DNA motifs, yet only a tiny fraction of the possible binding sites within the genome are ever bound. Factors that influence binding are affinity, co-operative interactions with additional TFs, and physical impediments provided by chromatin. Pioneer TFs are a special class of TF that can interact with ‘silent’ chromatin, increasing local accessibility that can facilitate binding of other TFs that do not possess pioneering activity (settler TFs). We engineered a Klf1-/- cell line with inducible KLF1 (known as K1ER cells) to study the immediate effects on chromatin of its introduction pinto the nucleus 1. Using ATAC-seq, ChIP-seq and RNA-seq we show binding of KLF1 to cis-regulatory modules induces an increase in local chromatin accessibility that facilitates assembly of a complex of TFs (including GATA1 and SCL, but not NF-E2) that enable enhancer activity. We confirmed these findings in primary erythroid cells from Klf1-/- fetal liver. This pioneering function occurs at ~300 key erythroid enhancers and super-enhancers such the one at -26kb in the alpha-globin LCR and one within the body of the E2f2 gene, but rarely at promoters. We show neomorphic mutations in the DNA-binding domain lead to ectopic pioneering and aberrant gene activation. We generated a series of N-terminal deletions in KLF1 and employed ATAC-seq to map the domain/s within KLF1 responsible for the pioneering activity, and show it is distinct from DNA-binding activity. We show this domain is responsible for SMARCA4/Brg1 recruitment, the likely effector of chromatin remodelling. Pioneering activity has been show for other SMARC subunits in other contexts 2. This work has broad implications for how the KLF/SP family of TFs work to reprogram cells and direct differentiation.
References:
1. Coghill et al. Blood 97, 1861-1868 (2001)
2. Wang et al. Nature Genetics 49, 289-295 (2017)