Chinese Medical Sciences Journal ›› 2010, Vol. 25 ›› Issue (4): 193-198.doi: 10.1016/S1001-9294(11)60001-1

• Original Article •     Next Articles

NF-E2: a novel regulator of alpha-hemoglobin stabilizing protein gene expression

Guo-wei Zhao1, Rui-feng Yang1, Xiang Lü1, Mitchell J. Weiss2, De-peiLiu1*, and Chih-chuan Liang1#   

  1. 1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China
    2Children’s Hospital of Philadelphia, Philadelphia 19104, PA, USA
  • Received:2010-09-30 Online:2010-12-20 Published:2010-12-20
  • About author:*Correspondingauthor Tel: 86-10-65296415, Fax: 86-10-65105093, E-mail: liudp@pumc.edu.cn
  • Supported by:

    National Natural ScienceFoundation of China (30130026, U0632005, 30721063), National Basic ResearchProgram of China (973 Program) (2011CB964803), National Laboratory of MedicalMolecular Biology grant (2060204), and Beijingmunicipal government grant (YB20081002301).

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. Conclusion 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.

Key words: α-hemoglobin stabilizing protein, NF-E2, erythropoiesis, GATA-1, H3K4trimethylation

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