The −113A>G HPFH mutation does not disrupt BCL11A binding but rather creates a de novo binding site for the transcriptional activator GATA1 in vitro. (A) Five mutations and a 13-bp deletion (Δ13bp) at the −115 site of the γ-globin promoter have been reported to induce HPFH. (B) The −113A>G HPFH mutation alters a critical residue within the BCL11A in vivo binding motif TG(A/T)CCA.^5,6  (C) The −113A>G HPFH mutation creates a “GATA” motif at the −115 site of the γ-globin promoter. The γ-globin promoter sequences (−128 to −100 bp) of other introduced mutations, −113A>C and −113A>T/−110A>C, which creates an “artificial GATA” site (TATC as the reverse complement), are also shown. The BCL11A TGACCA motif is highlighted in blue and GATA motifs are highlighted in red. (D) EMSA showing that BCL11A zinc fingers (ZF) bind to the wild-type (WT) −115 site of the γ-globin promoter (−128 bp to −100 bp) and that the −113A>G HPFH mutation, −113A>C mutation, and “artificial GATA” site do not disrupt BCL11A binding in vitro. Nuclear extracts were prepared from COS-7 cells transfected with a pcDNA3 empty vector as a control or from COS-7 cells transiently overexpressing BCL11A ZF (amino acids 740-835). A supershift of the BCL11A ZF:probe complex was performed with an antibody (Ab) against endogenous BCL11A. (E) GATA1 does not bind to the WT −115 site of the γ-globin promoter (−128 bp to −100 bp) or the −113A>C mutation, however, the −113A>G HPFH mutation and an “artificial GATA” site create a de novo binding site for GATA1 in vitro. Nuclear extracts were prepared from COS-7 cells transfected with a pcDNA3 empty vector as a control or from COS-7 cells transiently overexpressing full-length GATA1. A supershift of the GATA1:probe complex was performed with an Ab against endogenous GATA1.