Erythroid-specific activity of the glycophorin B promoter requires GATA-1 mediated displacement of a repressor.
We have performed a detailed analysis of the cis-acting sequences involved in the erythroid-specific expression of the human glycophorin B (GPB) promoter and found that this promoter could be divided into two regions. The proximal region, -1 to -60, contains a GATA binding sequence around -37 and an SP1 binding sequence around -50. This region is active in erythroid and non-erythroid cells. The distal region, -60 to -95, contains two overlapping protein binding sites around -75, one for hGATA-1 and one for ubiquitous proteins. This distal region completely represses the activity of the proximal promoter in non-erythroid cells and defines the -95 GPB construct as a GPB promoter that displays erythroid specificity. Using site directed mutagenesis, we show that the -37 GATA and the -50 SP1 binding sites are necessary for efficient activity of the -95 GPB construct. Mutations that impair the -75 GATA-1 binding result in extinction of the -95 GPB construct activity if the -75 ubiquitous binding site is not altered, or in loss of erythroid specificity if the -75 ubiquitous binding site is also mutated. Using a cotransfection assay, we found that hGATA-1 can efficiently activate transcription of the -95 GPB construct in non-erythroid cells. This transactivation is abolished by mutations that impair either the -37 GATA-1 or the -50 SP1 binding. Mutations that impair the -75 GATA-1 binding and still allow the -75 ubiquitous binding also abolish the transactivation of the -95 GPB construct, indicating that hGATA-1 can remove repression of the GPB promoter by displacement of the ubiquitous proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Rahuel
C
C
INSERM U.76, Institut National de Transfusion Sanguine, Paris, France.
Vinit
M A
MA
Lemarchandel
V
V
Cartron
J P
JP
Roméo
P H
PH
D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005822:Genetic Vectors; D006021:Glycophorins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers / Q000235:genetics* / Q000502:physiology*
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D006021:Glycophorins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I