Figure 4: Chromatin looping of the locus control region to the γ-globin promoter.

(a) 3C assay measuring locus-wide crosslinking frequencies in MEL WT:^Aγ cells (grey), MEL −175T>C:^Aγ cells (black) and unmodified transgenic MEL cells (green). A schematic of the human β-globin locus is shown on top of the graph. The x axis indicates distances in kb from the ɛ-gene. Vertical lines represent HindIII restriction sites. The dark brown bar denotes the anchor HindIII fragment containing hypersensitive site (HS) 2. Beige bars denote analysed HindIII fragments. Replicates are from two independently generated clonal cell populations for WT:^Aγ and −175T>C:^Aγ cells (n=2), respectively. Shown is mean±s.e.m. (b) 3C assay measuring relative crosslinking frequencies of ^Gγ-globin and LCR in K562 WT and −175T>C:^Gγ-^Aγ cells. Vertical lines represent HindIII restriction sites. The dark brown bar denotes the anchor HindIII fragment containing the ^Gγ-globin promoter. Replicates are from independently generated clonal cell populations of K562 WT (n=2) and −175T>C:^Gγ-^Aγ (n=3). Shown is mean±s.e.m. (c) Model of LCR looping to the γ-globin promoter upon introduction of the −175T>C mutation in the γ-globin promoter. In the fetal environment, nuclear factors mediate looping of the LCR to the γ-globin genes (left panel). In the WT adult environment, the LCR loops to the β-globin gene and γ-globin is silenced. The −175T>C mutation drives recruitment of the LCR to the γ-promoter via assembly of a looping complex consisting of TAL1 and associated cofactors⁴¹.