Functional characterization of the two alternative promoters of human p45 NF-E2 gene.
OBJECTIVE
The transcription factor NF-E2, a heterodimeric protein complex composed of p45 and small Maf family proteins, is considered crucial for the proper differentiation of erythrocytes and megakaryocytes in vivo. We report the results of studies aimed at understanding the regulatory mechanisms controlling p45 gene expression in erythroid cells.


METHODS
Human p45 mRNAs have two alternative isoforms, aNF-E2 and fNF-E2, and these isoforms are transcribed from the alternative promoters. We investigated lineage-specific expression of both isomers in human erythroid and megakaryocytic cells by reverse transcriptase polymerase chain reaction or Northern blot analysis. For functional characterization of both promoters, plasmids in which reporter genes were placed under the control of a series of truncated or mutated promoter fragments were transfected to human hematopoietic cell lines.


RESULTS
When CD34(+) cells isolated from human cord blood were induced to unilineage erythroid or megakaryocytic differentiation in liquid suspension culture, both transcripts, although barely detected at day 0, were induced in both erythroid and megakaryocytic cultures. fNF-E2 mRNA was found to be more abundant in erythroid cells than megakaryocytic cells at day 7 of culture. Although both isomers were expressed in human erythroid-megakaryocytic cell lines, megakaryocytic maturation with loss of erythroid phenotype induced by phorbol 12-myristate 13-acetate (PMA) resulted in exclusive downregulation of fNF-E2, suggesting that fNF-E2 promoter is more erythroid specific. Functional analysis of fNF-E2 promoter showed that the promoter is active only in erythroid-megakaryocytic cells and that the double GATA site in the proximal region is necessary for its efficient activity.


CONCLUSIONS
These results suggest that GATA proteins, which govern the differentiation of erythroid lineage cells, are required for full promoter activity of the p45 gene.
Toki
T
T
Department of Pediatrics, Hirosaki University School of Medicine, Hirosaki, Japan.
Arai
K
K
Terui
K
K
Komatsu
N
N
Yokoyama
M
M
Katsuoka
F
F
Yamamoto
M
M
Ito
E
E
D001665:Binding Sites; D015152:Blotting, Northern; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004912:Erythrocytes / Q000737:chemistry; D050981:Erythroid-Specific DNA-Binding Factors; D005312:Fetal Blood / Q000166:cytology; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D008533:Megakaryocytes / Q000737:chemistry; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D012333:RNA, Messenger; D014157:Transcription Factors; D013755:Tetradecanoylphorbol Acetate