Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner.
The human globin locus control region-binding protein, NF-E2, was purified by DNA affinity chromatography. Its tissue-specific component, p45 NF-E2, was cloned by use of a low-stringency library screen with murine p45 NF-E2 cDNA (N. C. Andrews, H. Erdjument-Bromage, M. B. Davidson, P. Tempst, and S. H. Orkin, Nature [London] 362:722-728, 1993). The human p45 NF-E2 gene was localized to chromosome 12q13 by fluorescent in situ hybridization. Human p45 NF-E2 and murine p45 NF-E2 are highly homologous basic region-leucine zipper (bZIP) proteins with identical DNA-binding domains. Immunoprecipitation experiments demonstrated that p45 NF-E2 is associated in vivo with an 18-kDa protein (p18). Because bZIP proteins bind DNA as dimers, we infer that native NF-E2 must be a heterodimer of 45- and 18-kDa subunits. Although AP-1 and CREB copurified with NF-E2, no evidence was found for heterodimer formation between p45 NF-E2 and proteins other than p18. Thus, p18 appears to be the sole specific partner of p45 NF-E2 in erythroid cells. Cloning of human p45 NF-E2 should permit studies of the role of NF-E2 in globin gene regulation and erythroid differentiation.
Ney
P A
PA
Clinical Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Andrews
N C
NC
Jane
S M
SM
Safer
B
B
Purucker
M E
ME
Weremowicz
S
S
Morton
C C
CC
Goff
S C
SC
Orkin
S H
SH
Nienhuis
A W
AW
D000595:Amino Acid Sequence; D001483:Base Sequence; D002881:Chromosomes, Human, Pair 12; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D050981:Erythroid-Specific DNA-Binding Factors; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics; D006410:Hematopoiesis; D006801:Humans; D016350:Leucine Zippers; D050985:MafK Transcription Factor; D008969:Molecular Sequence Data; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D009687:Nuclear Proteins / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics*
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; C494695:MAFK protein, human; D050985:MafK Transcription Factor; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D005914:Globins