C/EBP factor suppression of inhibition of type II secreted phospholipase A2 promoter in HepG2 cells: possible role of single-strand binding proteins.
We previously reported that the type II secreted phospholipase A2 (sPLA2) promoter from positions (-326 to +20) ([-326;+20] promoter) is negatively regulated by two adjacent regulatory elements, C (-210 to -176) and D (-247 to -210). This study examines in greater detail the way in which this negative regulation operates. Successive 5' deletions of the [-326;+20] type II sPLA2 promoter indicated that the region upstream of position -195 inhibits the transcription activity sixfold in HepG2 cells but not in HeLa cells. Although the whole [-326;-176] region decreased the activity of a heterologous thymidine kinase promoter, this effect was orientation and position sensitive. C/EBP beta, C/EBP alpha, and C/EBP delta, which bind to element C, prevented the inhibition of promoter activity. Electrophoretic mobility shift experiments identified the binding of NF1-like proteins to the [-225;-218] site, which overlaps an insulin response-like sequence, 5'-TGTTTTG-3'. This sequence bound a factor which also recognized the promoters of the apolipoproteins C-III and A-II. Substitutions preventing the binding of this factor or the NF1-like proteins did not increase the transcription activity, but substitution in the [-217;-204] sequence blocked the transcription inhibition. This sequence did not bind any double-strand binding factor, but its antisense strand is critical for the binding of single-strand binding proteins to the [-232;-191] region. We therefore suggest that these single-strand binding proteins are involved in the inhibitory mechanism.
Fan
Q
Q
URA CNRS 1283, U.F.R. Saint Antoine, Université Pierre et Marie Curie,Paris, France.
Paradon
M
M
Salvat
C
C
Bereziat
G
G
Olivier
J L
JL
D000818:Animals; D001053:Apolipoproteins / Q000235:genetics; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D006528:Carcinoma, Hepatocellular; D015342:DNA Probes; D004274:DNA, Recombinant / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D025542:Neurofibromin 1; D009687:Nuclear Proteins / Q000378:metabolism*; D010741:Phospholipases A / Q000235:genetics*; D054467:Phospholipases A2; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017384:Sequence Deletion / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured
D001053:Apolipoproteins; D022762:CCAAT-Enhancer-Binding Proteins; D015342:DNA Probes; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D025542:Neurofibromin 1; D009687:Nuclear Proteins; D011506:Proteins; D014157:Transcription Factors; D010741:Phospholipases A; D054467:Phospholipases A2