Characterization of the survival motor neuron (SMN) promoter provides evidence for complex combinatorial regulation in undifferentiated and differentiated P19 cells.
There exist two SMN (survival motor neuron) genes in humans, the result of a 500 kb duplication in chromosome 5q13. Deletions/mutations in the SMN1 gene are responsible for childhood spinal muscular atrophy, an autosomal recessive neurodegenerative disorder. While the SMN1 and SMN2 genes are not functionally equivalent, up-regulation of the SMN2 gene represents an important therapeutic target. Consequently, we exploited in silico, in vitro and in vivo approaches to characterize the core human and mouse promoters in undifferentiated and differentiated P19 cells. Phylogenetic comparison revealed four highly conserved regions that contained a number of cis-elements, only some of which were shown to activate/repress SMN promoter activity. Interestingly, the effect of two Sp1 cis-elements varied depending on the state of P19 cells and was only observed in combination with a neighbouring Ets cis-element. Electrophoretic mobility-shift assay and in vivo DNA footprinting provided evidence for DNA-protein interactions involving Sp, NF-IL6 and Ets cis-elements, whereas transient transfection experiments revealed complex interactions involving these recognition sites. SMN promoter activity was strongly regulated by an NF-IL6 response element and this regulation was potentiated by a downstream Ets element. In vivo results suggested that the NF-IL6 response must function either via a protein-tethered transactivation mechanism or a transcription factor binding an upstream element. Our results provide strong evidence for complex combinatorial regulation and suggest that the composition or state of the basal transcription complex binding to the SMN promoter is different between undifferentiated and differentiated P19 cells.
Rouget
Raphaël
R
Centre de Recherche, Hôpital Sainte-Justine and Université de Montréal, Montréal, QC, Canada H3T 1C5.
Vigneault
François
F
Codio
Circé
C
Rochette
Camille
C
Paradis
Isabelle
I
Drouin
Régen
R
Simard
Louise R
LR
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004622:Embryo, Mammalian / Q000166:cytology / Q000294:innervation; D004742:Enhancer Elements, Genetic / Q000235:genetics; D023281:Genomics / Q000379:methods; D006801:Humans; D006822:Hybrid Cells / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009046:Motor Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010802:Phylogeny; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics*; D055532:SMN Complex Proteins; D013234:Stem Cells / Q000737:chemistry / Q000378:metabolism; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000379:methods
D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; 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