Abstract
The differentiation, maintenance, and repair of skeletal muscle is controlled by interactions between genetically determined transcriptional programs regulated by myogenic transcription factors and environmental cues activated by growth factors and hormones. Signaling through the insulin-like growth factor 1 (IGF1) receptor by locally produced IGF2 defines one such pathway that is critical for normal muscle growth and for regeneration after injury. IGF2 gene and protein expression are induced as early events in muscle differentiation, but the responsible molecular mechanisms are unknown. Here we characterize a distal DNA element within the imprinted mouse Igf2-H19 locus with properties of a muscle transcriptional enhancer. We find that this region undergoes a transition to open chromatin during differentiation, whereas adjacent chromatin remains closed, and that it interacts in differentiating muscle nuclei but not in mesenchymal precursor cells with the Igf2 gene found more than 100 kb away, suggesting that chromatin looping or sliding to bring the enhancer in proximity to Igf2 promoters is also an early event in muscle differentiation. Because this element directly stimulates the transcriptional activity of an Igf2 promoter-reporter gene in differentiating myoblasts, our results indicate that we have identified a bona fide distal transcriptional enhancer that supports Igf2 gene activation in skeletal muscle cells. Because this DNA element is conserved in the human IGF2-H19 locus, our results further suggest that its muscle enhancer function also is conserved among different mammalian species.
Highlights
IGF2 [5, 6],3 two closely related single-chain secreted proteins that bind with high affinity to the insulin-like growth factor 1 (IGF1) receptor [7], leading to activation of several intracellular signal transduction pathways that act downstream of this membrane-spanning protein-tyrosine kinase [7]
In mouse fetal liver all three promoters are active, as evidenced by positive RT-PCR products with primers derived from each leader exon coupled to a shared coding exon primer, whereas in adenoviruses for MyoD (Ad-MyoD)-converted 10T1/2 cells, only transcripts directed by P3 accumulate during muscle differentiation (Fig. 2)
A role for IGF2 has been established in skeletal muscle based on evidence that its production has been linked temporally and functionally with muscle regeneration after injury [32] and on the association of a DNA polymorphism in the porcine IGF2 gene with increased muscle IGF2 mRNA expression and enhanced muscle mass [12]
Summary
Chemicals and Reagents—DMEM, Superscript III first strand synthesis kit, TRIzol reagent, trypsin/EDTA solution, and horse serum were from Invitrogen, and FBS and newborn calf serum were from Hyclone (Logan, UT). C3H 10T1/2 mouse embryonic fibroblasts (catalogue number CCL226; ATCC, Manassas, VA) were incubated on gelatin-coated tissue culture dishes in growth medium (DMEM with 10% heat-inactivated FBS) at 37 °C in humidified air with 5% CO2. They were converted to myoblasts by infection at ϳ50% of confluent density with a recombinant adenovirus for mouse MyoD (AdMyoD), as described [25], followed by incubation in DM after reaching confluent density as above. All of the subfragments were generated by restriction enzyme digestion or PCR and were purified after preparative agarose gel electrophoresis by ion exchange chromatography (Qiaex II gel extraction kit; Qiagen) and subcloned into Igf promoter-luciferase plasmids. C2 cell extracts were harvested 1 day later (undifferentiated), or DM was added, and cellular pro-
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