title: Identification of singles bar as a direct transcriptional target of Drosophila Myocyte enhancer factor-2 and a regulator of adult myoblast fusion.
abstract: In Drosophila, myoblast fusion is a conserved process in which founder cells (FCs) and fusion competent myoblasts (FCMs) fuse to form a syncytial muscle fiber. Mutants for the myogenic regulator Myocyte enhancer factor-2 (MEF2) show a failure of myoblast fusion, indicating that MEF2 regulates the fusion process. Indeed, chromatin immunoprecipitation studies show that several genes involved in myoblast fusion are bound by MEF2 during embryogenesis. Of these, the MARVEL domain gene singles bar (sing), is down-regulated in MEF2 knockdown pupae, and has five consensus MEF2 binding sites within a 9000-bp region. To determine if MEF2 is an essential and direct regulator of sing during pupal muscle development, we identified a 315-bp myoblast enhancer of sing. This enhancer was active during myoblast fusion, and mutation of two MEF2 sites significantly decreased enhancer activity. We show that lack of sing expression resulted in adult lethality and muscle loss, due to a failure of fusion during the pupal stage. Additionally, we sought to determine if sing was required in either FCs or FCMs to support fusion. Interestingly, knockdown of sing in either population did not significantly affect fusion, however, knockdown in both FCs and FCMs resulted in muscles with significantly reduced nuclei numbers, provisionally indicating that sing function is required in either cell type, but not both. Finally, we found that MEF2 regulated sing expression at the embryonic stage through the same 315-bp enhancer, indicating that sing is a MEF2 target at both critical stages of myoblast fusion. Our studies define for the first time how MEF2 directly controls fusion at multiple stages of the life cycle, and provide further evidence that the mechanisms of fusion characterized in Drosophila embryos is also used in the formation of the more complex adult muscles.
authors: Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA.
authors: Tonya M
authors: TM
authors: Brunetti
authors: Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA.
authors: Brayon J
authors: BJ
authors: Fremin
authors: Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA. Electronic address: rcripps@unm.edu.
authors: Richard M
authors: RM
authors: Cripps
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002459:Cell Fusion; D002467:Cell Nucleus / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D004330:Drosophila / Q000196:embryology*; D029721:Drosophila Proteins / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D018507:Gene Expression Regulation, Developmental; D015726:Giant Cells / Q000166:cytology; D062792:MARVEL Domain-Containing Proteins / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D024510:Muscle Development / Q000235:genetics; D018485:Muscle Fibers, Skeletal; D032446:Myoblasts / Q000166:cytology*; D018006:Myogenic Regulatory Factors / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering; D015533:Transcriptional Activation / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D062792:MARVEL Domain-Containing Proteins; C499409:Mef2 protein, Drosophila; D008565:Membrane Proteins; D018006:Myogenic Regulatory Factors; D034741:RNA, Small Interfering; C000599972:sing protein, Drosophila
keywords: Drosophila; MARVEL domain; MEF2; Myoblast fusion; Singles bar; Transcriptional regulation