Micro-Capture-C to determine the long-range interaction of HBG1/2 promoter with the locus control region (LCR), a powerful upstream enhancer in -175 A>G mutantant HUDEP-2 cells.

Naturally occurring mutations in the γ-globin promoters can result in hereditary persistence of fetal hemoglobin (HPFH), a benign condition that ameliorates the severity of β-hemoglobinopathies through increased post-natal fetal hemoglobin (HbF) expression. Base editing to recreate the HPFH mutations is a promising approach to induce HbF. Compared with Cas9-generated indels, base-editing approaches were more potent, with the −175 A>G conversion resulting in the strongest induction of HbF by creating a new TAL1 binding motif that stimulates long-range interaction with the locus control region (LCR), a powerful upstream enhancer. Micro-Capture-C analysis showed that introducing the −175 A>G variant stimulated long-range interaction between the γ-globin promoters and the LCR in erythroid progenitor cell line. Together, these findings show that the −175 A>G variant creates a bipartite GATA–TAL1 motif, resulting in the assembly of a GATA1/TAL1/LMO2/LDB1 complex, which activates γ-globin gene transcription by enhancing its physical association with the LCR. Micro-Capture-C experiments were performed in wild type hemizygous HUDEP-2 cells (HUDEP2∆εγδβ cells), HUDEP2∆εγδβ cells harboring -175 A>G mutation at -globin promoter the γ-globin promotesr (HBG2/HBG1). Wild type HUDEP-1 is used as a control.