Epigenetic inactivation of ERF reactivates γ-globin expression in β-thalassemia

Am J Hum Genet. 2021 Apr 1;108(4):709-721. doi: 10.1016/j.ajhg.2021.03.005. Epub 2021 Mar 17.


The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of β-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA+ erythroid cells from β-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of β-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in β-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in β-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for β-hemoglobinopathies.

Keywords: CD34+ HSPCs; ERF; GYPA+ cells; engraftment mice; epigenetics; fetal hemoglobin; genome editing; methylation; whole-genome bisulfite sequencing; β-thalassemia.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, CD34 / metabolism
  • Base Sequence
  • CRISPR-Cas Systems / genetics
  • Cell Differentiation
  • Cell Line
  • Child
  • DNA (Cytosine-5-)-Methyltransferases / genetics
  • DNA Methylation
  • DNA Methyltransferase 3A
  • Epigenesis, Genetic*
  • Erythroid Precursor Cells / cytology
  • Erythroid Precursor Cells / metabolism
  • Female
  • Fetal Hemoglobin / genetics
  • Gene Editing
  • Gene Expression Profiling*
  • Humans
  • Male
  • Mice
  • Promoter Regions, Genetic / genetics
  • Repressor Proteins / deficiency*
  • Repressor Proteins / genetics*
  • Reproducibility of Results
  • Sulfites
  • Whole Genome Sequencing
  • beta-Thalassemia / genetics*
  • beta-Thalassemia / pathology
  • gamma-Globins / genetics*


  • Antigens, CD34
  • DNMT3A protein, human
  • Dnmt3a protein, mouse
  • ERF protein, human
  • Repressor Proteins
  • Sulfites
  • gamma-Globins
  • Fetal Hemoglobin
  • DNA (Cytosine-5-)-Methyltransferases
  • DNA Methyltransferase 3A
  • hydrogen sulfite