Figure 3: The −175T>C HPFH mutation increases promoter activity in K562 cells.

(a) Schematic of the normal (top) and engineered (bottom) β-globin locus in K562 cells. (b) Bar chart showing mean±s.d. γ-globin (tdTomato) promoter activity for clonal populations (n=5) of K562 WT/-175T>C:^Gγ-^Aγ cells as determined by measuring median tdTomato fluorescence intensity. Significance was determined by unpaired two-tailed t-test (*P<0.05). (c) Histogram showing γ-globin promoter activity in representative clonal populations of K562 WT/−175T>C:^Gγ-^Aγ cells, as determined by tdTomato fluorescence. Depicted is the median out of five clonal populations (for K562 WT ^Gγ-^Aγ or −175T>C:^Gγ-^Aγ, respectively). (d) Shown is the percentage mRNA expression of β-like globins in unmodified K562 cells (left) and also expression of tdTomato and β-like globins in clonal populations of K562 WT/−175T>C:^Gγ-^Aγ cells as determined by qPCR. The graph on the right depicts mean mRNA levels for clonal K562 WT/−175T>C:^Gγ-^Aγ cell populations (n=4). (e) Anti-TAL1 ChIP in K562 WT/-175T>C:^Gγ-^Aγ cells lines (n=4). Shown is mean±s.d. Enrichment of TAL1 at γ-globin promoter (HBG) is significantly higher in K562 −175T>C:^Gγ-^Aγ cells (P<0.005). Significance was determined by unpaired two-tailed t-test. (f) Representative sequencing pyrograms (left) of PCR products derived from input and ChIP samples in K562 cells heterozygous for the −175T>C mutation. Shown on the right is the mean frequency of cytosine (=mutated allele) at position −175 of the γ-globin promoter in input, control antibody ChIP (IgG) and TAL1 ChIP. The mutated allele is enriched only after TAL1 ChIP. Pyrosequencing was performed in triplicate and shown is the mean±s.d.