Removal of promoter DNA methylation by epigenome editing reverses HBG silencing [ATAC-seq]

Beta-hemoglobinopathies caused by mutations in adult-expressed HBB can be treated by re-activating the adjacent paralogous genes HBG1 and HBG2 (HBG), which are normally silenced perinatally. Although HBG expression is induced by global demethylating drugs, their mechanism is poorly understood and toxicity limits their use. We identified the DNMT1-associated maintenance methylation protein UHRF1 as a mediator of HBG repression in a genome-wide screen. Loss of UHRF1 in the adult-type erythroid cell line HUDEP2 caused global demethylation and HBG activation that was reversed upon localized promoter re-methylation. Conversely, targeted demethylation of the HBG promoters activated their genes in HUDEP2 or primary CD34+ cell-derived erythroblasts. Mutation of MBD2, a CpG-methylation reading component of the NuRD complex, to impair methylation sensitivity recapitulated the effects of promoter demethylation. Our studies demonstrate that localized CpG-methylation at the HBG promoters facilitates developmental silencing and identify a targeted therapeutic approach for b-hemoglobinopathies via epigenomic editing. Overall design: ATAC-seq experiments were performed inCD34+ derived erythroidblast cells treated with NT sgRNA or UHRF1 sgRNA1, or targeted by TETv4 dTETv4 with gRNAs targeting HBG promoter