CART gene promoter transcription is regulated by a cyclic adenosine monophosphate response element.
OBJECTIVE
To define the regulatory DNA sequence controlling the expression of the cocaine-amphetamine-regulated transcript (CART) gene expression.


METHODS
A rat genomic library was screened for genomic clones containing the CART gene and upstream DNA sequence. A clone containing exons 1 and 2 of the rat CART gene plus 1.2 kb of upstream sequence was isolated. The 1.2-kb upstream sequence and truncated segments of this sequence were cloned into a luciferase reporter vector for analysis of transcriptional activity in the CART expressing pituitary GH3 cell line. Luciferase reporter assays, gel shift assays, and site-directed mutagenesis were used to analyze potential regulatory regions in the 1.2-kb 5' DNA sequence.


RESULTS
Sequence analysis of the 1.2-kb upstream sequence reveals several potential regulatory elements. Luciferase reporter assays demonstrate that within the first 162 bp of the start codon is a functional cyclic adenosine monophosphate (cAMP) response element that can induce cAMP-regulated gene expression of the CART promoter in GH3 cells. Mutation of this cAMP response element abolishes cAMP-stimulated transcription [corrected]. Furthermore, the promoter is active in a neuronal cell line where it also demonstrates cAMP responsiveness.


CONCLUSIONS
cAMP-mediated transcription of the CART promoter may be an important aspect of the regulation of the CART gene. However, the cellular context of expression is also important because the CART promoter is not responsive to cAMP stimulation in all cell types. Other transcription factor binding sequences are likely to play a key role in CART promoter regulation.
Barrett
Perry
P
Rowett Research Institute, Aberdeen Centre for Energy Regulation and Obesity, Bucksburn, UK. p.barrett@rri.sari.ac.uk
Davidson
Jane
J
Morgan
Peter
P
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D005091:Exons; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D015723:Gene Library; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009474:Neurons; D009479:Neuropeptides / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D010902:Pituitary Gland; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins; D020218:Response Elements; D017385:Sequence Homology; D012984:Software; D014158:Transcription, Genetic; D014162:Transfection; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein
C495401:ADCYAP1 protein, human; C495402:Adcyap1 protein, mouse; C495403:Adcyap1 protein, rat; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein; D005576:Colforsin; D004247:DNA; D000242:Cyclic AMP; D008156:Luciferases