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. Author manuscript; available in PMC: 2009 Apr 10.
Published in final edited form as: Nat Med. 2008 Aug 31;14(10):1112–1117. doi: 10.1038/nm.1866

Figure 4.

Figure 4

CART and KISS1 genes are direct targets of TORC1 and CREB in the hypothalamus. A. and B. Transient transfection assay of HEK293T cells using CART-luciferase (A) and KISS1-luciferase (B) reporters. Exposure to FSK (1μM) and A23187 (1μM), alone or in combination, indicated. Relative effect of wild-type TORC1 and phosphorylation-defective S151A TORC1 expression on reporter activity shown. Co-transfection of dominant negative A-CREB expression plasmid indicated. Data are representative of at least three independent experiments. P<0.05 for multiple comparisons between the groups is considered statistically significant. Pertinent comparisons are discussed in the text. C. Top, chromatin immunoprecipitation (ChIP) assay of GT1-7 cells exposed to FSK (F; 1μM), A23187 (A; 1μM), or vehicle (V) for 1 hour. Recovery of CART and KISS1 promoter fragments from immunoprecipitates of CREB and TORC1 relative to control IgG shown. Input amounts of CART and KISS1 promoter DNA indicated. Bottom, schematic of CART and KISS1 promoters showing location of conserved CREB binding sites (CRE) relative to the transcription start site. D. ChIP assay of hypothalamic tissue from ob/ob mice injected IP with saline (Veh) or leptin (3μg/g) for 1 hour. Relative occupancy of hypothalamic CREB and TORC1 over CART and KISS1 promoters in control and leptin-injected mice indicated. CREB and TORC1 occupancy is statistically significant compared (P<0.05) to the relevant IgG controls. * indicates P<0.05 compared to hypothalami from vehicle-treated ob/ob mice. E. and F. Transient transfection assay of HEK293T cells using CART (E) or KISS1 (F) luciferase reporters. Effect of leptin treatment (100nM) on reporter activity in control and leptin receptor (LRb) expressing cells shown; co-treatment with FSK (1μM) indicated. Expression of wild-type TORC1 or phosphorylation-defective S151A TORC1 indicated. Effect of dominant negative A-CREB on reporter activity shown. P<0.05 for multiple comparisons between the groups is considered statistically significant. Pertinent comparisons are discussed in the text.