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. Research Methods and Procedures: 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 positive control plasmid construct response element abolishes cAMP-stimulated transcription. Furthermore, the promoter is active in a neuronal cell line where it also demonstrates cAMP responsiveness. Discussion: 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.