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JUN-CCAAT/enhancer-binding protein complexes inhibit surfactant-associated protein B promoter activity.

ABSTRACT: The murine surfactant-associated protein B (Sftpb) gene promoter, spanning nucleotides -653 to +42, is composed of functionally distinct proximal and distal regions. Although both regions contain consensus/putative activator protein 1 (AP-1) sites, the distal, but not the proximal, region mediates the inhibition by jun proto-oncogene (JUN) of Sftpb promoter activity. In transient cotransfection assays, JUN inhibited the luciferase reporter activity of plasmid constructs containing Sftpb promoter fragments that lacked the distal putative AP-1 site, indicating that another regulatory motif mediates JUN-dependent inhibition. Electrophoretic mobility shift assays and in silico analyses identified a DNA target sequence (Sftpb nucleotides -339 to -316) and transcription factors that regulate Sftpb promoter activity. The identified sequence contains a CCAAT/enhancer-binding protein (C/EBP) consensus recognition element. Mutation of the site reduced Sftpb promoter activity and sensitivity to inhibition by JUN. Purified recombinant JUN, which did not recognize the -339 to -316 target sequence when added alone, supershifted the mobility of in vitro translated C/EBP-? and C/EBP-? proteins complexed with the identified cis-regulatory element. These findings support the idea that heterodimerization between JUN and C/EBP-? and/or C/EBP-? targets JUN to the Sftpb promoter, thereby mediating its inhibitory regulatory role.

SUBMITTER: Bein K

PROVIDER: S-EPMC3175569 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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JUN-CCAAT/enhancer-binding protein complexes inhibit surfactant-associated protein B promoter activity.

Bein Kiflai K Leight Hayley H Leikauf George D GD

American journal of respiratory cell and molecular biology 20101210 2

The murine surfactant-associated protein B (Sftpb) gene promoter, spanning nucleotides -653 to +42, is composed of functionally distinct proximal and distal regions. Although both regions contain consensus/putative activator protein 1 (AP-1) sites, the distal, but not the proximal, region mediates the inhibition by jun proto-oncogene (JUN) of Sftpb promoter activity. In transient cotransfection assays, JUN inhibited the luciferase reporter activity of plasmid constructs containing Sftpb promoter ...[more]

PMID: 21148742

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Project description:The liver-specific expression of the GLUT2 glucose transporter gene is suppressed in cultured hepatoma cell lines as well as in hepatocytes in primary culture. To understand the underlying mechanism involved in this process, we analysed the rat GLUT2 promoter region. A DNase I footprinting assay with rat liver nuclear extract revealed eight protected regions within a -500 bp region of the GLUT2 promoter (sites A to H). Three of these sites (B, F and H) were occupied by transcription factors that are considerably enriched in liver cells compared with spleen or kidney. The proteins binding to these sites were investigated by a combination of DNase I footprinting assay and electrophoretic mobility-shift assay with the addition of specific oligonucleotide competitors and specific antibody against known transcription factors. As a result it was revealed that hepatocyte nuclear factor 3 binds to site B (-120 to -70), and CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta bind to site F (-375 to -356) and site H (-500 to -471). The binding of C/EBP to sites F and H was markedly decreased within 4 h when liver cells were subjected to primary culture, suggesting that C/EBP might be responsible for the decreased expression of GLUT2 in this process. In contrast, Western blot analysis revealed that C/EBPalpha began to decrease after 1 h of hepatocyte culture, and C/EBPbeta was not changed significantly throughout the culture period, suggesting that C/EBP could be regulated at the transcriptional level as well as the post-translational level when hepatocytes were put in culture. To confirm the role of C/EBP in the regulation of GLUT2 promoter activity, sites F and H were ligated to a chloramphenicol acetyltransferase (CAT) reporter gene and co-transfected with a C/EBP expression vector into HepG2 cells. The co-expression of C/EBPalpha and C/EBPbeta resulted in 9.1-fold and 3. 8-fold increases of CAT activities in the site F-CAT and site H-CAT constructs respectively. These results indicate that C/EBPalpha and C/EBPbeta regulate the promoter activity of the GLUT2 gene and might be responsible for the down-regulation of the GLUT2 gene when hepatocytes are subjected to primary culture.

Dataset Information

JUN-CCAAT/enhancer-binding protein complexes inhibit surfactant-associated protein B promoter activity.

Publications

JUN-CCAAT/enhancer-binding protein complexes inhibit surfactant-associated protein B promoter activity.

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