title: NF-E2: a novel regulator of alpha-hemoglobin stabilizing protein gene expression.
abstract: OBJECTIVE
  To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2.
  
  
  METHODS
  We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45.
  
  
  RESULTS
  We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45.
  
  
  CONCLUSIONS
  NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.
authors.affiliation: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Guo-wei
authors.forename: Yang
authors.initials: Y
authors.lastname: Rui-feng
authors.forename: Lv
authors.initials: L
authors.lastname: Xiang
authors.forename: Weiss J
authors.initials: WJ
authors.lastname: Mitchell
authors.forename: Liu
authors.initials: L
authors.lastname: De-pei
authors.forename: Liang
authors.initials: L
authors.lastname: Chih-chuan
mesh_terms: D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics*; D017931:DNA Primers; D050982:GATA1 Transcription Factor / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D018832:Molecular Chaperones / Q000235:genetics*; D050984:NF-E2 Transcription Factor, p45 Subunit / Q000502:physiology*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C468768:AHSP protein, human; D001798:Blood Proteins; D017931:DNA Primers; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D018832:Molecular Chaperones; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human