KLF1 binds to and activates the −198T>C γ-globin promoter in vitro. (A) The organization of the human β-globin locus (chromosome 11). The β-like globin genes are indicated by black boxes with their conventional gene symbols indicated above them. The locus-control region (LCR) is represented as a rectangle, with the DNase I hypersensitive sites (HS) within the LCR being represented by black boxes (HS1-4). The British HPFH (−198T>C) mutation in the promoter of the γ-globin gene is also depicted. (B) Aligned protein sequences of the 3 zinc finger (ZF) DNA-binding domains of all members of the SP/KLF family of transcription factors. Highlighted are residues −1, +3, and +6 of each zinc finger, which are responsible for making contact to DNA. (C) In vivo binding motifs of transcription factors SP1,¹⁸  KLF1,¹⁵  KLF3,¹⁶  and KLF4¹⁷  as determined by ChIP-Seq. (D) EMSA (electrophoretic mobility shift assay) showing that KLF1 can bind in vitro to a −198T>C probe but fails to bind to a WT probe containing the −198 region of the γ-globin promoter. Lanes 1 to 3 contain the WT probe for the −198 site (−209 to −187 bp) and lanes 4 to 6 contain the HPFH −198T>C mutant probe. Lanes 1 and 4 contain nuclear extracts from COS cells transfected with a pcDNA3 empty vector. Lanes 2 to 3 and 5 to 6 contain nuclear extracts from COS cells overexpressing KLF1. Binding of KLF1 to the −198T>C HPFH mutant probe can be observed in lane 5, with a supershift of KLF1 with an anti-KLF1 antibody in lane 6. Ab, antibody; WT, wild type.