title: Positive and negative hepatic regulation of the human type II phospholipase A2 gene.
abstract: To identify the elements which regulate the liver transcription of the human type II phospholipase A2 gene and its stimulation by interleukin 6, the 5' flanking region from -1614 to +806 and several 3' and 5' deleted fragments have been analyzed in CAT assays. Negative regulatory elements have been located in the regions -1614 to -326 and +20 to +806. The fragment -326 to +20 contains the main elements required for the transcription as well as for the stimulation by interleukin 6. Footprinting assays have been performed on this region and showed four protected elements, A [-35;-6], B [-125;-86], C [-209;-176], and D [-247;-211]. Deletion of element D enhanced the transcription of the reporter gene 10.5-fold compared to the [-326;+20]-CAT construct. Further deletions up to position -87 which removed both the elements B and C or the substitution of element C by a nonspecific sequence lowered the promoter activity to 23% and 70% of the control, respectively. These results indicate that element C binds positive regulatory factors and element D binds a negative regulatory factor. Furthermore, stimulation by interleukin 6 is lost when element C is substituted or deleted. As shown by the footprinting and band shift assays, the transcription factors C/EBP alpha and C/EBP beta can bind to elements C and D but the dissociation constant (Kd) of C/EBP alpha is 10 times lower for element C (0.6 nM) than for element D (5.8 nM). Band shift experiments using rat liver nuclear extracts showed that element C formed four heat stable complexes, some of which could be supershifted by anti C/EBP alpha antibodies. The binding of C/EBP factors to element C was confirmed by competition with previously described oligonucleotide and nucleotide substitution of element C. Band shift experiments using rat liver nuclear extracts showed that element D formed one major DNA-protein complex. This complex could be competed out by oligonucleotides containing a cAMP responsive element (CRE) but not by oligonucleotides containing the binding site of C/EBP. However, anti-CREB antibodies did not supershift this complex. Methylation interference experiments showed the involvement of a G nucleotide upstream to the sequence homologous to CRE in the binding of the hepatic nuclear factors.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Laboratoire de Biochimie, CHU Saint-Antoine, Université Pierre et Marie Curie, URA CNRS 1283, Paris, France.
authors.forename: J L
authors.initials: JL
authors.lastname: Olivier
authors.forename: Q
authors.initials: Q
authors.lastname: Fan
authors.forename: C
authors.initials: C
authors.lastname: Salvat
authors.forename: M
authors.initials: M
authors.lastname: Ziari
authors.forename: L
authors.initials: L
authors.lastname: Kong
authors.forename: M
authors.initials: M
authors.lastname: Mangeney
authors.forename: G
authors.initials: G
authors.lastname: Bereziat
mesh_terms: D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002478:Cells, Cultured; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism; D054467:Phospholipases A2; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D012045:Regulatory Sequences, Nucleic Acid
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010741:Phospholipases A; D054467:Phospholipases A2