Lecture4 Paper3 - Bch 4122 Paper 3 Skeletal Muscle...

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Unformatted text preview: Bch 4122 Paper # 3: Skeletal Muscle Specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1 Ohkawa Y., Marfella C., Imbalzano, A. (2006). EMBO Journal 25 : 490-501. (Impact Factor 8.3; 36 citations) Myogenic Regulatory Factors Control Myogenesis MyoD and Myf-5 Knockout studies suggest functional redundancy among MyoD and Myf5: Mice lacking MyoD = normal skeletal muscle Mice lacking Myf5 = normal skeletal muscle Mice lacking MyoD and Myf5 = no skeletal muscle Therefore MyoD and Myf5 thought to be involved in the early events of myogenesis (Simplified summary) Method to create knockout mice QuickTime™ and a decompressor are needed to see this picture. Myogenin Mice lacking Myogenin display normal myoblast formation but are unable to complete the later stages of skeletal muscle development What is Myogenin’s role in differentiation? MEF2D Mef2D has a skeletal muscle specific isoform (Mef2D1b) and can accelerate muscle development in synergy with MyoD. Mef2 can interact with HDACs and the components SWI/SNF chromatin remodeling enzyme. The Question What is the relationship between muscle specific transcription factors (specifically Mef2D and myogenin) and chromatin remodeling-enzymes (SWI/SNF) in the activation of late myogenic genes? The Hypothesis: Brg1 function is essential for skeletal myogenesis to occur. II: form a stably remodeled dimer , altered DNAse digestion pattern III: transfer a histone octamer to a different DNA fragment Chromatin remodeling ATPases catalyze stable alteration of the nucleosome Brg1 = SWI/SNF in this paper Early versus late myogenesis in the mouse embryo E 8.5-early First myotomal muscle Primary muscle formation (myocytes) (myotubes/myofibres) bipolar,mononucleate multinucleated elongated cells E 13-15 late E=Embryonic Day Murine Embryogenesis Times used in paper: 10.5 12.5 14.5 Is there a correlation with late myogenesis and increases in muscle structural gene expression? E10.5 E12.5 E 14.5 Took: The body (no head or organs) The limb buds The limbs myogenin Method: Isolated RNA from these time points and locations: Fig. 1A Examining the Expression of Muscle-specific genes in the developing mouse embryo by RT-PCR Found: The greatest increase in muscle-specific genes occurs from E12.5 to E14.5 MCK = Muscle creatine kinase * Fig. 1A Examining the Expression of Muscle-specific genes in the developing mouse embryo by RT-PCR Does this mean there is no MCK, Desmin, or sk -actin in E10.5 embryos? * What is desmin? Desmin is a Type III intermediate filament found in muscle How early is desmin expressed in embryogenesis? NBT/BCIP Fix embryo Cryosection Mount Pretreatment Hybridization Detection DIG-labeled RNA probe DIG Anti-DIG Antibody AP AP Red line= endogenous RNA; Blue line with flag = DIG-labeled RNA probe Orange box = Anti-DIG antibody; White circle = alkaline phosphatase Method of In Situ Hybridization In situ hybridization of desmin in E10.5 embryos Found: Staining of desmin throughout the myotome as early...
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Lecture4 Paper3 - Bch 4122 Paper 3 Skeletal Muscle...

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