Characterizing a distal muscle enhancer in the mouse Igf2 locus

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...

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Bibliographic Details
Published in:Physiological genomics 2016-02, Vol.48 (2), p.167-172
Main Authors: Alzhanov, Damir, Rotwein, Peter
Format: Article
Language:English
Subjects:
Animals
Cell Differentiation
Cell Nucleus - metabolism
Chromosomes - metabolism
Culture Media
Enhancer Elements, Genetic
Exons
Gene Expression Regulation
Green Fluorescent Proteins - metabolism
Immunohistochemistry
Insulin-Like Growth Factor II - genetics
Mice
Mice, Transgenic
Muscle Fibers, Skeletal - metabolism
Muscle, Skeletal - metabolism
Muscles - metabolism
Physiological Genomics of Cell States and Their Regulation and Single Cell Genomics
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
RNA, Long Noncoding
Transcription, Genetic
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Summary: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.
ISSN:1094-8341
1531-2267
DOI:10.1152/physiolgenomics.00095.2015