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All rights reserved. http://resource.belframework.org/belframework/20131211/knowledge/large_corpus.bel http://purl.org/dc/elements/1.1/title BEL Framework Large Corpus Document http://resource.belframework.org/belframework/20131211/knowledge/large_corpus.bel http://purl.org/pav/authoredBy http://www.tkuhn.ch/bel2nanopub/RAC455PsnDi6qOeI5Tok4DD08llvT16e_88Xqu3R6Bpdc#_5 http://resource.belframework.org/belframework/20131211/knowledge/large_corpus.bel http://purl.org/pav/version 20131211 http://www.tkuhn.ch/bel2nanopub/RAC455PsnDi6qOeI5Tok4DD08llvT16e_88Xqu3R6Bpdc#_4 http://www.w3.org/ns/prov#value In this report, the control of PGC-1-alpha expression by CaMKIV and CnA in muscle cells is investigated. We show that the PGC-1-alpha promoter is subject to positive regulation by these key calcium-signaling factors. In addition, PGC-1-alpha is demonstrated to regulate its own promoter through interactions with components of the calcium-signaling pathway, resulting in an autoregulatory loop that potentially can provide a certain stability to the expression of genes characteristic of type I muscle fibers. Methods Plasmids and Reagents. Various fragments of the 5 flanking sequence of the mouse PGC-1-alpha gene were amplified by PCR and subcloned into the pGL3basic reporter gene vector (Promega). Thus, the plasmids containing the regions between 78 and 2,533 or6,483 with respect to the transcriptional start site are referred to as 2- and 6-kb promoters, respectively. Expression plasmids for MEF2C, the dominant-negative MEF2C KR23,24ID (dnMEF2C), MEF2D, NFATc3, constitutively active CaMKIV, and constitutively active CnA were kind gifts from Eric N. Olson (University of Texas Southwestern Medical Center, Dallas). The dominant-negative cAMP response element (CRE)-binding protein (CREB), termed ACREB, was generously provided by Charles Vinson (National Cancer Institute, National Institutes of Health, Bethesda). All other reagents were obtained from Sigma. Site-Directed Mutagenesis. Site-directed mutagenesis was performed as described (21). Briefly, PCR amplifications were performed by using overlapping primers at the target sites, and the inserts were subcloned into new reporter gene vectors. The CRE and the MEF-binding site were termed CRE and MEF2, respectively. Cell Culture, Transfection, and Reporter Gene Assays. C2C12 cells were maintained in DMEM supplemented with 10% FCS and 1 M sodium-pyruvate in subconfluent cultures. For myotube differentiation, confluent C2C12 myoblasts were cultured in differentiation medium (DMEM with 2% horse serum and 1 M sodium-pyruvate) for 5 days with daily changes of the medium. Cells were transfected by using Lipofectamine 2000 transfection reagent (Invitrogen), and luciferase activity was determined 48 h after transfection. Expression of reporter genes was normalized to -galactosidase levels driven by a cotransfected pSV-- galactosidase expression vector (Promega). Finally, these relative expressions were normalized vs. empty pGL3basic reporter gene vector expression. Electrophoretic Mobility-Shift Assays. The sequences of the MEF2 oligonucleotide probe were derived from the mouse PGC-1-alpha promoter and consisted of the MEF2 site and the mutated MEF2 site flanked by 20 bp on each side. Radiolabeling, incubation with proteins, and gel electrophoresis were performed as described (22). PGC-1-alpha protein at concentrations of 0.8, 2, 4, and 6 g was added as a bacterially expressed GST-PGC-1-alpha fusion protein encoding for amino acids 1-180 or 31-797 of PGC-1-alpha , respectively. Antibodies against MEF2 and HNF3 (used as a nonspecific antibody) were from Santa Cruz Biotechnology. Analysis of PGC-1-alpha Gene Expression in Wild-Type and Transgenic PGC-1-alpha Mice. Wild-type and transgenic mice from strain 31 (see ref. 16) that express PGC-1-alpha in muscle were killed, and plantaris muscle was collected. Total RNA was isolated and subsequently reverse-transcribed. Primers for the ABI Prism 7700 sequence detector (Applied Biosystems) were designed for the respective target genes. Transcript levels were determined from at least three wild-type and transgenic mice and subsequently normalized to 18S rRNA levels. Adenoviral Generation and Infections. Adenoviral constructs containing GFP, PGC-1-alpha , MEF2C, and dnMEF2C were generated by using the Ad-Easy system as published (23). C2C12 cells were infected with adenovirus; 1 day postinfection, medium was changed to differentiation medium and cells were harvested after 5 days. 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