Characterizing a distal muscle enhancer in the mouse Igf2 locus

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Insulin-like Growth Factor-2 (IGF2) is crucial for skeletal muscle mass.
• Mechanisms regulating IGF2 expression in muscle remain largely unknown.
• A distal DNA element was previously proposed as a muscle-specific transcriptional control element for Igf2.

Purpose of the Study:

• To investigate the function of a putative muscle transcriptional control element located 100 kb upstream of the Igf2 gene.
• To functionally characterize this DNA segment's role in regulating Igf2 gene transcription during muscle development.

Main Methods:

• Developed an in vivo reporter system using a bacterial artificial chromosome (BAC) containing the mouse Igf2-H19 locus.
• Substituted nuclear-enhanced green fluorescent protein (nEGFP) for Igf2 coding exons in the BAC.
• Transfected the modified BAC into a mesenchymal stem cell line and induced differentiation into myoblasts and myotubes.

Main Results:

• Transgenic mRNA and nEGFP expression were detected coincident with endogenous Igf2 mRNA in differentiated myotubes.
• This expression was dependent on the presence of an intact distal conserved DNA element.
• A 294 bp DNA fragment containing two E-boxes within this element was identified as a key regulatory region.

Conclusions:

• A distal 294 bp DNA fragment functions as a necessary and sufficient long-range enhancer for Igf2 gene transcription during skeletal muscle differentiation.
• This enhancer is critical for the induction of Igf2 during myogenesis.
• The developed reporter system provides a platform for further functional studies of IGF2 regulation in muscle.