Promoter elements and factors involved in hepatic transcription of the human ApoA-I gene positive and negative regulators bind to overlapping sites.
DNase I footprinting analysis of the proximal apoA-I promoter sequences with rat liver nuclear extracts identified four protected regions: A, -22 to +17; B, -128 to -77; C, -175 to -148; and D, -220 to -190. Region D (-220 to -190) binds at least two distinct activities, designated AID1 and AID2, respectively, which can be separated by ion exchange chromatography. Region C (-175 to -148) forms five DNA protein complexes. Three of the complexes (2, 4, and 5) originate from the binding of more than one heat-stable nuclear factor, and two (1 and 3), from the binding of two heat-labile factors. The heat-stable factors bind in the -175 to -148 region and can be distinguished from C/EBP, which recognizes the same region, with DNA binding gel electrophoretic assays. Both factors 1 and 3 bind in the -168 to -148 apoA-I region. Despite the lack of a CCAAT motif in this region, the binding of factor 1 is competed out by oligonucleotides containing the binding sites of NFY and NFY*. Mutagenesis of the promoter region showed that mutations in the -171 to -166 and -158 to -153 regions diminished the binding of the heat-stable factors and reduced hepatic transcription to 14 and 8% of control, respectively. In contrast, a mutation in the -164 to -159 region abolished the binding of factor 1 and was associated with a 4.6-fold increase in hepatic transcription. These findings suggest that the heat-stable factors act as positive regulators, whereas factor 1 acts as a negative regulator in apoA-I gene transcription.
Papazafiri
P
P
University of Crete Medical School, Greece.
Ogami
K
K
Ramji
D P
DP
Nicosia
A
A
Monaci
P
P
Cladaras
C
C
Zannis
V I
VI
D000818:Animals; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000235:genetics*; D001667:Binding, Competitive; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D016172:DNA Fingerprinting; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D015500:Chloramphenicol O-Acetyltransferase