Epigenetic Inactivation Of ERF Reactivates γ-globin Expression In β-thalassemia

BACKGROUNDReactivation of fetal hemoglobin(HbF)has therapeutic potential for treatingβ-thalassemia and sickle cell disease.However,the molecular mechanism underlying the epigenetic dysregulation of the fetal-to-adult hemoglobin switch in disease states is unexplored.METHODSWe recruited three cases of thalassemia maj or with low HbF(0.3±0.1 g/dL)and three cases of thalassemia intermedia with high HbF(9.0±0.1 g/dL).We used whole-genome bisulfite sequencing and RNA sequencing to analyze bone marrow-derived GYPA+cells from all patients with β0-thalassemia stratified into lowand high-HbF(HbFL and HbFH)groups.We further extended our in-vivo validation to an independent cohort of 47 samples(HbFL:n=34;HbFH:n=13)withβ-thalassemia by bisulfite pyrosequencing.We analyzed primary human erythroid CD34+cells or HUDEP-2 cells through various functional assays and performed in vivo assays to study the target gene’s role as an HbF repressor and its epigenetic mechanisms regulating the reactivation of γ-globin expression.RESULTSWe identified ETS2 repressor factor(ERF)as an HbF repressor.ERF depletion or ERF inactivation via ERF promoter methylation markedly increased HbF production in human erythroid cells.ERF repressed y-globin expression through direct binding to β-globin cluster,independently of the major HbF repressors BCL11A and ZBTB7A.We further characterized a long noncoding RNA,RP11-196G18.23,which specifically bound the ERF promoter,and we identified the epigenetic mechanism underlying the high HbF production to be long noncoding RNA-mediated transcriptional derepression through enrichment of DNMT3A in the ERF promoter.CONCLUSIONSEpigenetic inactivation of ERF can reactivate y-globin gene expression inβ-thalassemia and is responsible for the increased HbF levels,thereby ameliorating the severity of β-thalassemia major.Our findings suggested ERF and RP11-196G18.23 as potential therapeutic targets for hemoglobinopathies.