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.

Keywords: IGF2; distal enhancer; gene transcription; muscle differentiation; recombineering.

Fig. 1.

Development of transgenic cell lines encoding recombinant bacterial artificial chromosomes (BACs) containing a modified Igf2 gene. A: schematic of Igf2 - H19 locus and adjacent genes on mouse chromosome 7. Genes encoding proteins are indicated by a black box; genes expressed as noncoding RNAs (H19 and Nctc1) are labeled by a white box. The red oval signifies a putative muscle enhancer. B, top: diagram of wild-type (WT) BAC, with its genomic coordinates, illustrating modifications made to replace Igf2 coding exons with nuclear-targeted enhanced green fluorescent protein (nEGFP) and to insert a pGK-Neo selection cassette. The gray color of Lsp1 indicates that only part of the gene is present. Middle: diagram of enhancer knockout (Enh-KO) BAC, demonstrating replacement of putative muscle enhancer with cassette containing bar-coded DNA. The broken outline of Nctc1 indicates disruption of its transcription unit. Bottom middle: diagram of Enhancer Ebox knockout (Enh-Ebox-ko) BAC, demonstrating deletion of paired E-boxes within the muscle enhancer. Bottom: schematic of enhancer knock-in BAC (Enh-KI). The broken outline of Nctc1 indicates that its transcription unit is perturbed. C, left: organization of mouse Igf2 gene [6 exons, 5 introns, 3 promoters (29)]. Arrows show locations of primers for Fig. 1D. Below are schematics of major muscle Igf2 mRNA (containing exons 3–6), and IGF2 protein precursor (SP, signal peptide; IGF2, 67 amino acid mature protein; E peptide, COOH-terminal 89 amino acid extension). Right: organization of Igf2-nEGFP gene. Arrows show locations of primers for Fig. 1C. Below are Igf2-nEGFP mRNA, and nuclear-localized EGFP protein (EGFP-nls). D: copy number determination for lines encoding wild-type (WT) BAC, BAC lacking putative muscle enhancer (KO), and BAC in which putative enhancer has been relocated (KI). Left: PCR-amplified endogenous Igf2 DNA; right: amplified transgene DNA. Calculated copy numbers: WT, 2; KO, 4; KI, 2; E-ko, 4 per haploid genome (see materials and methods for details).

Fig. 2.

Production of nEGFP by the recombinant Igf2 locus during muscle differentiation. A: experimental scheme. Ad-MyoD, recombinant adenovirus encoding mouse MyoD; DM, differentiation medium. B: schematic of native and modified mouse Igf2 genes, showing locations of primers for RT-PCR. C: time course of expression for Igf2, Igf2-nEGFP, myogenin, and S17 transcripts, measured by RT-PCR. Con, negative and positive controls. D: time course of myotube formation by immunocytochemistry for troponin-T (red) and nuclear staining by Hoechst (blue). Expression of EGFP in nuclei is indicated by green tint. E: higher magnification view of 48 h time point shown in D. For D and E, scale bar = 50 μM.

Fig. 3.

Restoration of Igf2 expression during muscle differentiation by knock-in of the distal enhancer. A: expression of Igf2, Igf2-nEGFP, myogenin, and S17 RNAs, assessed by RT-PCR in differentiating cells encoding either WT or enhancer KI BACs. B: time course of myotube formation by immunocytochemistry for troponin-T (red) and nuclear staining by Hoechst (blue). Expression of EGFP in nuclei is indicated by green tint. Scale bar = 50 μM. C: relative gene expression for Igf2-nEGFP by quantitative RT-PCR at 0 and 24 h after incubation in DM for cells encoding either WT, enhancer KO, or enhancer KI BACs. Note log scale on ordinate.