Session: MON 389-405-Signaling Originating from Membrane Receptors
Poster Board MON-390
Damir Alzhanov, Tiffany Morrison, Peter Rotwein
Oregon Health & Science University, Department of Biochemistry and Molecular Biology.
IGF2 is highly expressed in skeletal muscle in vivo, is induced as an early event during myoblast differentiation in culture, and plays a key role in stimulating differentiation. Yet very little is known about mechanisms of IGF2 gene regulation in muscle. Until recently, no transcriptional response elements had been identified, and no transcription factors characterized. We now have defined a conserved DNA segment located ~24 kb 3’ to the H19 gene in the mouse Igf2 - H19 locus that has properties of a muscle-enriched enhancer element (D. T. Alzhanov et. al. Long-range interactions regulate Igf2 gene transcription during skeletal muscle differentiation. J Biol Chem 285, 38969-38977, 2010). The focus of this project is to elucidate the chromatin properties of this putative transcriptional enhancer. To achieve this goal we have developed an in vivo reporter system the recapitulates the characteristics of the endogenous locus. Recombinogenic engineering (‘recombineering’) is a relatively new method to modify DNA without using restriction enzymes or ligases, and has been applied to build bacterial artificial chromosomes (BACs) with specific properties for study in mammalian cells and transgenic mice. Using a 201 kb BAC (bMQ318o13) containing the mouse Igf2 - H19 locus as a template, we have substituted nuclear (n) EGFP for Igf2 coding exons 4-6 and added a selectable marker (neomycin resistance) for stable selection in mammalian cells. Transfection into the pluripotent mouse C3H10T1/2 mesenchymal stem cell line resulted in multiple stable clones harboring the entire BAC but not expressing nEGFP. Introduction of the muscle transcription factor, MyoD, into BAC-containing 10T1/2 cells, converted them to myoblasts, which upon exposure to low-serum differentiation medium progressively expressed muscle-specific genes and proteins, and formed multi-nucleated myotubes. MyoD also induced production of transgenic mRNA and nEGFP coincident with accumulation of endogenous mouse Igf2 mRNA. Additional studies using other modified BACs will test the hypothesis that the putative enhancer is necessary for induction of Igf2 gene transcription during muscle differentiation, and will examine the properties of presumptive repressor elements in controlling Igf2 gene expression in skeletal muscle.
Nothing to Disclose: DA, TM, PSR
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