Cloning and Characterization of the 5 0 -Flanking Region of the Pig Cocaine- and Amphetamine-Regulated Transcript Gene Fei Ling, 1, * Liqiong Wei, 1, * Tao Wang, 1 Yaosheng Chen, 2 Xiaoping Zhu, 3 Jiaqi Li, 3 Tingting Liu, 1 Hongli Du, 1 Haibo Wang, 1 and Jufang Wang 1 The cocaine- and amphetamine-regulated transcript (CART) gene encodes an anorexigenic peptide. It has a key role in the hypothalamic regulation of energy balance through reducing food intake and enhancing lipid substrate utilization. To detect the CART expression pattern in pigs, reverse transcription (RT)–polymerase chain reaction (PCR) and real-time PCR were performed in various tissues. Our RT-PCR results revealed that the pig CART gene was ubiquitously expressed in all examined tissues including hypothalamus, m. longissimus, backfat, heart, liver, spleen, lung, kidney, stomach, bladder, belly fat, brain, large intestine, lymph, and skin. Real-time quantitative PCR experiments revealed that the cDNA level of CART in both the hypothalamus and backfat of adult Landrace pig (lean-type) was significantly higher than that of Chinese indigenous Lantang pig (fat-type), and it was in the hypothalamus where the highest expression of CART was observed for both adult Lantang and Landrace pigs, compared with backfat and m. longissimus muscle. To understand the regulation of the pig CART gene, the 5 0 -flanking region was isolated from a pig bacterial artificial chromosome library and used in a luciferase reporter assay. A positive cis-acting element for efficient CART expression was identified at nucleo- tides 73 to 53, using 5 0 -serial deletion of the promoter. Electrophoretic mobility shift assays with competing oligonucleotides revealed that the critical region contained a cis-acting element for the zinc-binding protein factor, a zinc-finger transcription factor of the Kruppel family. This element has not been reported in human or mouse CART genes. Our results indicated that zinc-binding protein factor might be an essential regulatory factor for transcription of pig CART, providing important insight into mechanisms involved in energy homeostasis regulation in the porcine and human brain. Introduction T he cocaine- and amphetamine-regulated transcript (CART) encodes an anorexigenic peptide that has an important role in the hypothalamic regulation of energy balance. CART effects include reducing food intake and in- creasing lipid substrate utilization, and it is involved in im- munity (Bik et al., 2008), lipid metabolism and atherogenesis (Vasseur et al., 2007), direct and indirect prolactin secretion (Baranowska et al., 2007), and hypothalamic–pituitary– adrenal axis activation (Murphy, 2005; Iliff et al., 2008). CART peptide appears to have an important function in en- ergy homeostasis and interacts with several central appetite circuits. CART is expressed in various areas of the hypothalamus and is regulated by several peripheral peptide hormones involved in appetite regulation, including leptin (Murphy, 2005), cholecystokinin (CCK), and ghrelin (de Lartigue et al., 2007). CART and CCK have synergistic effects on appetite regulation (Maletinska et al., 2008). In addition, a few tran- scription factors including cyclic adenosine-monophosphate (cAMP)-responsive element–binding protein (CREB) tran- scription factor, glucocorticoid (Germano et al., 2007), neuron- restrictive silencer factor (Li et al., 2008), and estradiol E(2) are reported to be positive effectors of CART expression. For example, a CRE in the CART proximal promoter is involved in cAMP/protein kinase/CREB regulation in GH3 cells (Lakatos et al., 2002). CCK reciprocally regulates the ex- pression of CART and melanin-concentrating hormone within the same vagal afferent neuron (de Lartigue et al., 2007). CART gene expression in neuroendocrine cells is strictly controlled by neuron-restrictive silencer factor in a 1 School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, P.R. China. 2 State Key Laboratory of Bio-control, School of Life Science, Sun Yat-Sen University, Guangzhou, P.R. China. 3 College of Animal Science, South China Agricultural University, Guangzhou, P.R. China. *These authors contributed equally to this work. DNA AND CELL BIOLOGY Volume 30, Number 2, 2011 ª Mary Ann Liebert, Inc. Pp. 91–97 DOI: 10.1089/dna.2010.1101 91