Figure 1 : The -175T>C mutation in the γ-globinpromoter creates an E-Box motif.

From: Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin

Figure 1

(a) Schematic of the human β-globin locus. Enlarged is the γ-globin proximal promoter. The position of the −175T>C mutation is indicated by a downward arrow. The newly created TAL1 consensus site is shown by a red box. (b) TAL1 consensus binding motif as determined by MEME analysis of published ChIP-seq data40. (c) The −175T>C mutation creates a complete E-Box consensus on the antisense strand of the γ-globin promoter. Nucleotides at position −175 are shown in boxes. (d) EMSA. The DNA-binding domains (bHLH) of TAL1 and E47 were coexpressed in bacteria and purified by ion exchange chromatography. Binding of E47/E47 homodimer (E/E) and TAL1/E47 heterodimer (T/E) to the WT and −175T>C γ-globin promoters is shown in lanes 2 and 5, respectively. Lanes 1 and 4 show probe alone; specific binding of TAL1/E47 to the mutant probe is confirmed by supershift (T*/E) using an anti-His antibody (lane 6). The probe spans region −166 to −215. (e) EMSA showing interaction of TAL1/E47 with LMO2-LDB1 and the mutant −175T>C promoter. LMO2 and LDB1 were bacterially expressed as a tethered protein14 and then purified by ion exchange chromatography. Binding of E47/E47 homodimer (E/E) and TAL1/E47 heterodimer (T/E) to the WT and −175T>C γ-globin promoters is shown in lanes 2 and 5, respectively. Lanes 1 and 4 show probe alone. The retarded band in lane 5 supershifts upon addition of LMO2-LDB1 (lane 6) indicating an interaction of TAL1/E47 with LMO2-LDB1 (T/E/L-L). The probe spans region −163 to −195.