Lecture_4Paper_31 - Bch 4122 Paper # 3: Skeletal Muscle...

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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)
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Myogenic Regulatory Factors Control Myogenesis
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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)
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Method to create knockout mice QuickTimeª and a decompressor are needed to see this picture.
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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?
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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.
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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?
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The Hypothesis: Brg1 function is essential for skeletal myogenesis to occur.
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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
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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
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Murine Embryogenesis Times used in paper: 10.5 12.5 14.5
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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:
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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 *
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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 α -actin in E10.5 embryos? *
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What is desmin? Desmin is a Type III intermediate filament found in muscle
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How early is desmin expressed in embryogenesis?
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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
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In situ hybridization of desmin in E10.5 embryos Found: Staining of desmin throughout the myotome as early
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This note was uploaded on 12/18/2011 for the course BCH 4122 taught by Professor Ilonaskerjanc during the Spring '11 term at University of Ottawa.

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Lecture_4Paper_31 - Bch 4122 Paper # 3: Skeletal Muscle...

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