Inhibitory effect of AP-1 complex on 5-aminolevulinate synthase gene expression through sequestration of cAMP-response element protein (CRE)-binding protein (CBP) coactivator.
Activation protein-1 (AP-1) transcription factors are early response genes involved in a diverse set of transcriptional regulatory processes. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is often used to induce AP-1 activity. The purpose of this work was to explore the molecular mechanisms involved in the TPA regulation of ubiquitous 5-aminolevulinate synthase (ALAS) gene expression, the first and rate-controlling step of the heme biosynthesis. Previous analysis of the 5'-flanking sequence of ALAS revealed the existence of two cAMP-response elements (CRE) required for basal and cAMP-stimulated expression. The fragment -833 to +42 in the 5'-flanking region of rat ALAS gene was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector pALAS/CAT produced a significant CAT activity in transiently transfected HepG2 human hepatoma cells, which was repressed by TPA. Sequence and deletion analysis detected a TPA response element (TRE), located between -261 and -255 (TRE-ALAS), that was critical for TPA regulation. We demonstrated that c-Fos, c-Jun, and JunD are involved in TPA inhibitory effect due to their ability to bind TRE-ALAS, evidenced by supershift analysis and their capacity to repress promoter activity in transfection assays. Repression of ALAS promoter activity by TPA treatment or Fos/Jun overexpression was largely relieved when CRE protein-binding protein or p300 was ectopically expressed. When the TRE site was placed in a different context with respect to CRE sites, it appeared to act as a transcriptional enhancer. We propose that the decrease in ALAS basal activity observed in the presence of TPA may reflect a lower ability of this promoter to assemble the productive pre-initiation complex due to CRE protein-binding protein sequestration. We also suggest that the transcriptional properties of this AP-1 site would depend on a spatial-disposition-dependent manner with respect to the CRE sites and to the transcription initiation site.
Guberman
Alejandra S
AS
Laboratorio de Biologia Molecular, Departamento de Quimica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II Piso 4, Ciudad Universitaria, Argentina.
Scassa
Maria E
ME
Giono
Luciana E
LE
Varone
Cecilia L
CL
Cánepa
Eduardo T
ET
D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis* / Q000235:genetics*; D015153:Blotting, Western; D050882:CREB-Binding Protein; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D005799:Genes, Dominant; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009281:Naphthalenes / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D009281:Naphthalenes; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D000242:Cyclic AMP; D000624:5-Aminolevulinate Synthetase; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C058819:calphostin C