CCACC-binding or simian-virus-40-protein-1-binding proteins cooperate with human GATA-1 to direct erythroid-specific transcription and to mediate 5' hypersensitive site 2 sensitivity of a TATA-less promoter.
Previous studies have shown that a -112 to +78 DNA fragment from the erythroid promoter of the human porphobilinogen deaminase (PBGD) gene has erythroid-specific activity. This PBGD-(-112 to +78) promoter contains a CCACC binding site (position -100), a GATA binding site (position -70) and an initiator element around the cap site. Using a cotransfection assay, we find that the human factor GATA-1 trans-activates the PBGD-(-112 to +78) promoter in non-erythroid cells. We show that, if trans-activation is abolished by mutations that destroy either the -100 CCACC binding or the -70 GATA binding sites, replacement of the -100 CCACC binding site by a simian-virus-40-protein-1 (Sp1) binding site maintains both the erythroid-specific activity of this promoter and the human GATA-1 trans-activation. Thus, human GATA-1 acts on the PBGD promoter in association with Sp1 or CCACC binding proteins. This PBGD-(-112 to +78) promoter is activated 20-fold by a cis-linked 5' hypersensitive site 2 (5'HS-2) of the human beta-globin locus control region. This activation depends on the -70 GATA and -100 CCACC or Sp1 binding sites. When a longer -714 to +78 fragment of the PBGD promoter is used, the -70 GATA mutant still displays erythroid-specific activity and is cis-activated by the 5'HS-2 enhancer, while the -100 CCACC mutant is completely inactive in the absence or in the presence of the 5'HS-2 enhancer. Thus, the -100 CCACC binding site is indispensable for the correct activity and sensitivity of the human PBGD promoter to the 5'HS-2 enhancer, whereas the -70 GATA binding site can functionally be replaced by upstream cis-acting elements.
Institut National de la Santé et de la Recherche Médicale, Unité 91, Hôpital Henri Mondor, Créteil, France.
J F
JF
Eleouet
P H
PH
Roméo
D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics* / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000378:metabolism*; D016385:TATA Box; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C080480:SV40 protein-1-binding protein, human; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D011163:Hydroxymethylbilane Synthase