title: Identification and Functional Characterization of Cis-Regulatory Elements Controlling Expression of the Porcine ADRB2 Gene.
abstract: The beta-2 adrenergic receptor (beta-2 AR) modulates metabolic processes in skeletal muscle, liver, and adipose tissue in response to catecholamine stimulation. We showed previously that expression of the porcine beta-2 AR gene (ADRB2) is affected by cis-regulatory polymorphisms. These are most likely responsible for the association of ADRB2 with economically relevant muscle-related traits in pigs. The present study focused on characterization of promoter elements involved in basal transcriptional regulation of the porcine ADRB2 in different cell types to aid identification of its cis-regulatory polymorphisms. Based on in silico analysis, luciferase reporter gene assays and gel shift assays were performed using COS-7, HepG2, C2C12, and 3T3-L1 cells. Deletion mapping of the 5´ flanking region (-1324 to +33) of ADRB2 revealed the region between -307 and -269 to be the minimal promoter, including regulatory elements essential for the basal transcriptional activity in all four tested cell types. Directly upstream (-400 to -323) we identified an important enhancer element required for maximal promoter activity. In silico analysis and gel shift assays revealed that this GC-rich element harbors two evolutionarily conserved binding sites of Sp1, a constitutive transcriptional activator. Significant transcriptional activation of the porcine ADRB2 promoter was demonstrated by overexpression of Sp1. Our results demonstrate, for the first time, an important role of Sp1 and of the responsive enhancer element in the regulation of ADRB2 expression. Polymorphisms located in this domain of the porcine ADRB2 promoter represent candidate causal cis-regulatory variants.
authors.affiliation: Institute for Genome Biology, Leibniz-Institute for Farm Animal Biology, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Jaeger
authors.affiliation: Institute for Genome Biology, Leibniz-Institute for Farm Animal Biology, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Fritschka
authors.affiliation: Institute for Genome Biology, Leibniz-Institute for Farm Animal Biology, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany.
authors.forename: Siriluck
authors.initials: S
authors.lastname: Ponsuksili
authors.affiliation: Institute for Genome Biology, Leibniz-Institute for Farm Animal Biology, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Wimmers
authors.affiliation: Institute for Genome Biology, Leibniz-Institute for Farm Animal Biology, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany.
authors.forename: Eduard
authors.initials: E
authors.lastname: Muráni
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000235:genetics; D013552:Swine; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D018343:Receptors, Adrenergic, beta-2; D016329:Sp1 Transcription Factor
keywords: ADRB2; Sp1; beta-2 adrenergic receptor; porcine; promoter