title: Characterizing a distal muscle enhancer in the mouse Igf2 locus.
abstract: Insulin-like growth factor-2 (IGF2) is highly expressed in skeletal muscle and was identified as a quantitative trait locus for muscle mass. Yet little is known about mechanisms of its regulation in muscle. Recently, a DNA segment found ∼100 kb from the Igf2 gene was identified as a possible muscle transcriptional control element. Here we have developed an in vivo reporter system to assess this putative enhancer by substituting nuclear (n) EGFP for Igf2 coding exons in a bacterial artificial chromosome containing the mouse Igf2 - H19 chromosomal locus. After stable transfection into a mesenchymal stem cell line, individual clones were converted to myoblasts and underwent progressive muscle-specific gene expression and myotube formation in differentiation medium. Transgenic mRNA and nuclear-targeted enhanced green fluorescent protein were produced coincident with endogenous Igf2 mRNA, but only in lines containing an intact distal conserved DNA element. Our results show that a 294 bp DNA fragment containing two E-boxes is a necessary and sufficient long-range enhancer for induction of Igf2 gene transcription during skeletal muscle differentiation and provides a robust experimental platform for its further functional dissection.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon; and.
authors.forename: Damir
authors.initials: D
authors.lastname: Alzhanov
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon; and Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas peter.rotwein@ttuhsc.edu.
authors.forename: Peter
authors.initials: P
authors.lastname: Rotwein
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002875:Chromosomes / Q000378:metabolism; D003470:Culture Media; D004742:Enhancer Elements, Genetic; D005091:Exons; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*; D062085:RNA, Long Noncoding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D003470:Culture Media; C094335:H19 long non-coding RNA; C513121:IGF2 protein, mouse; D062085:RNA, Long Noncoding; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007335:Insulin-Like Growth Factor II
keywords: IGF2; distal enhancer; gene transcription; muscle differentiation; recombineering