Identification of a novel cyclic AMP-response element (CRE-II) and the role of CREB-1 in the cAMP-induced expression of the survival motor neuron (SMN) gene.
Spinal muscular atrophy, an autosomal recessive disorder, is caused by loss of the SMN1 (survival motor neuron) gene while retaining the SMN2 gene. SMN1 produces a majority of full-length SMN transcript, whereas SMN2 generates mostly an isoform lacking exon 7. Here, we demonstrate a novel cAMP-response element, CRE-II, in the SMN promoter that interacts with the cAMP-response element-binding (CREB) family of proteins. In vitro DNase I protection analysis and in vivo genomic footprinting of the SMN promoter using the brain and liver nuclei from SMN2 transgenic mice revealed footprinting at the CRE-II site. Site-directed mutation of the CRE-II element caused a marked reduction in the SMN promoter activity revealed by transient transfection assay. Activation of the cAMP pathway by dibutyryl cAMP (0.5 mm) alone or in combination with forskolin (20 microm) caused a 2-5-fold increase in the SMN promoter activity but had no effect on the CRE-II mutated promoter. Electrophoretic mobility shift assay and a UV-induced DNA-protein cross-linking experiment confirmed that CREB1 binds specifically to the CRE-II site. Transient overexpression of CREB1 protein resulted in a 4-fold increase of the SMN promoter activity. Intraperitoneal injection of epinephrine in mice expressing two copies of the human SMN2 gene resulted in a 2-fold increase in full-length SMN transcript in the liver. Combined treatment with dibutyryl cAMP and forskolin significantly increased the level of both the full-length and exon 7-deleted SMN (exonDelta7SMN) transcript in primary hepatocytes from mice expressing two copies of human SMN2 gene. Similar treatments of type I spinal muscular atrophy mouse and human fibroblasts as well as HeLa cells resulted in an augmented level of SMN transcript. These findings suggest that the CRE-II site in SMN promoter positively regulates the expression of the SMN gene, and treatment with cAMP-elevating agents increases expression of both the full-length and exonDelta7SMN transcript.
Majumder
Sarmila
S
Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio 43210, USA. jacob.42@osu.edu
Varadharaj
Saradhadevi
S
Ghoshal
Kalpana
K
Monani
Umrao
U
Burghes
Arthur H M
AH
Jacob
Samson T
ST
D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D022781:Hepatocytes / Q000166:cytology; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055532:SMN Complex Proteins; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; D014157:Transcription Factors / Q000737:chemistry* / Q000502:physiology*; D014162:Transfection; D014466:Ultraviolet Rays; D015854:Up-Regulation
C115163:CREB1 protein, human; C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; C526472:Smn1 protein, mouse; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; D014157:Transcription Factors; D000242:Cyclic AMP; D003850:Deoxyribonuclease I