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)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Olivier JL,Fan Q,Salvat C,Ziari M,Kong L,Mangeney M,Bereziat Gdoi
10.1021/bi00189a017subject
Has Abstractpub_date
1994-06-14 00:00:00pages
7134-45issue
23eissn
0006-2960issn
1520-4995journal_volume
33pub_type
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