5-aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB)
The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene...
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todo:paper_02646021_v353_n2_p307_Giono2023-10-03T15:13:00Z 5-aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB) Giono, L.E. Varone, C.L. Cánepa, E.T. Gene expression Haem biosynthesis Hepatic cell Protein kinase A Transcription factor 5 aminolevulinate synthase cyclic AMP cyclic AMP dependent protein kinase cyclic AMP responsive element binding protein DNA animal cell article cyclic AMP responsive element enzyme activation enzyme induction enzyme phosphorylation gene deletion gene expression regulation genetic transfection heme synthesis housekeeping gene human human cell nonhuman priority journal promoter region protein binding protein DNA binding rat repressor gene site directed mutagenesis transcription regulation 5-Aminolevulinate Synthetase Animals Binding Sites CREB-Binding Protein Cyclic AMP Response Element-Binding Protein Cyclic AMP-Dependent Protein Kinases Gene Expression Humans Mutation Nuclear Proteins Oligonucleotides, Antisense Plasmids Promoter Regions (Genetics) Rats Signal Transduction Trans-Activators Transcription, Genetic Tumor Cells, Cultured Animalia Fungi Mammalia The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions - 38 and - 142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_02646021_v353_n2_p307_Giono |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Gene expression Haem biosynthesis Hepatic cell Protein kinase A Transcription factor 5 aminolevulinate synthase cyclic AMP cyclic AMP dependent protein kinase cyclic AMP responsive element binding protein DNA animal cell article cyclic AMP responsive element enzyme activation enzyme induction enzyme phosphorylation gene deletion gene expression regulation genetic transfection heme synthesis housekeeping gene human human cell nonhuman priority journal promoter region protein binding protein DNA binding rat repressor gene site directed mutagenesis transcription regulation 5-Aminolevulinate Synthetase Animals Binding Sites CREB-Binding Protein Cyclic AMP Response Element-Binding Protein Cyclic AMP-Dependent Protein Kinases Gene Expression Humans Mutation Nuclear Proteins Oligonucleotides, Antisense Plasmids Promoter Regions (Genetics) Rats Signal Transduction Trans-Activators Transcription, Genetic Tumor Cells, Cultured Animalia Fungi Mammalia |
| spellingShingle |
Gene expression Haem biosynthesis Hepatic cell Protein kinase A Transcription factor 5 aminolevulinate synthase cyclic AMP cyclic AMP dependent protein kinase cyclic AMP responsive element binding protein DNA animal cell article cyclic AMP responsive element enzyme activation enzyme induction enzyme phosphorylation gene deletion gene expression regulation genetic transfection heme synthesis housekeeping gene human human cell nonhuman priority journal promoter region protein binding protein DNA binding rat repressor gene site directed mutagenesis transcription regulation 5-Aminolevulinate Synthetase Animals Binding Sites CREB-Binding Protein Cyclic AMP Response Element-Binding Protein Cyclic AMP-Dependent Protein Kinases Gene Expression Humans Mutation Nuclear Proteins Oligonucleotides, Antisense Plasmids Promoter Regions (Genetics) Rats Signal Transduction Trans-Activators Transcription, Genetic Tumor Cells, Cultured Animalia Fungi Mammalia Giono, L.E. Varone, C.L. Cánepa, E.T. 5-aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB) |
| topic_facet |
Gene expression Haem biosynthesis Hepatic cell Protein kinase A Transcription factor 5 aminolevulinate synthase cyclic AMP cyclic AMP dependent protein kinase cyclic AMP responsive element binding protein DNA animal cell article cyclic AMP responsive element enzyme activation enzyme induction enzyme phosphorylation gene deletion gene expression regulation genetic transfection heme synthesis housekeeping gene human human cell nonhuman priority journal promoter region protein binding protein DNA binding rat repressor gene site directed mutagenesis transcription regulation 5-Aminolevulinate Synthetase Animals Binding Sites CREB-Binding Protein Cyclic AMP Response Element-Binding Protein Cyclic AMP-Dependent Protein Kinases Gene Expression Humans Mutation Nuclear Proteins Oligonucleotides, Antisense Plasmids Promoter Regions (Genetics) Rats Signal Transduction Trans-Activators Transcription, Genetic Tumor Cells, Cultured Animalia Fungi Mammalia |
| description |
The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions - 38 and - 142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression. |
| format |
JOUR |
| author |
Giono, L.E. Varone, C.L. Cánepa, E.T. |
| author_facet |
Giono, L.E. Varone, C.L. Cánepa, E.T. |
| author_sort |
Giono, L.E. |
| title |
5-aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB) |
| title_short |
5-aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB) |
| title_full |
5-aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB) |
| title_fullStr |
5-aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB) |
| title_full_unstemmed |
5-aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB) |
| title_sort |
5-aminolaevulinate synthase gene promoter contains two camp-response element (cre)-like sites that confer positive and negative responsiveness to cre-binding protein (creb) |
| url |
http://hdl.handle.net/20.500.12110/paper_02646021_v353_n2_p307_Giono |
| work_keys_str_mv |
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