Lecture_5Paper_43 - Pax7 activates myogenic Paper#4 Marks...

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Paper #4: Pax7 activates myogenic genes by recruitment of a histone methyltransferase complex I.W. McKinnel, J. Ishibashi, F. Le Grand, V.G. J. Punch, G.C. Addicks, J.F. Greenblatt, F.J. Dillworth, and M.A. Rudnicki, Nature Cell Biology (2008) 10, 77-84. (IF=19.5, 45 citations) Marks the important slides *
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Where does methylation fit into the regulation of transcription?
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(Ac= Acetyl; Me= Methyl; MeCP2= Methyl-CpG binding Protein 2; HDAC= Histone De-Acetylase) and prevents transcription
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SUVAR39H is a methyltransferase which methylates the Lysine 9 of histone H3, creating a binding site for the Heterochromatin Protein HP1, which recruits a DNA methyltransferase, which methylates CpG in DNA. (Me= Methyl; Methyl H3-K9= Methyl on Lysine 9 of Histone H3; HP1=Heterochromatin Protein 1; DNMT=DNA Methyltransferase).
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Does methylation always mean inhibition?
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a) RNA polymerase II (Pol II) binds the promoter region of the gene but cannot proceed without specific methylation marks on histone. b) RNA transcription may be initiated when a promoter region (I) carries histone H3 lysine 4 (H3K4) methylation, and extend when an open-reading frame region (II) carries H3K36 methylation. H3K79 methylation has a broad distribution across promoter and open-reading frame regions (III). Emerging roles for histone methylation c) Mixed lineage leukaemia (MLL) is a member of a multiprotein complex that mediates methylation of H3K4 within the promoter region of genes occupied by RNA polymerase II.
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The MLL complex WDR5 interacts with methylated H3K4, presenting it for further methylation by linking the catalytic MT(methyltransferase) domain of MLL1 with its substrate. This interaction is necessary for H3K4 trimethylation and subsequent transcription of MLL1 target genes. Nomenclature: H3K4 H3K4-me H3K4-2me H3K4-3me
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What targets the MLL complex to specific genes? What is the role of Pax7 transcription factors?
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Robson EJD et al. (2006) A PANorama of PAX genes in cancer and development Nat. Rev. Cancer. 6: 52–62 doi:10.1038/nrc1778 The structure of Pax family members
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Structure of the Pax6 paired domain with DNA
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What is the role of Pax3/7 in myogenesis?
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Mice lacking Pax3/7 have defects in muscle development Pax3 -/- mice: Do not form limb muscle Pax7 -/- mice: Do not form satellite cells Pax3-/-,Pax7-/- mice: Do not form most muscle or satellite cells
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Annual Reviews The Role of Pax3/7 in Embryonic Myogenesis
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Annual Reviews The Role of Pax3/7 in Satellite Cell Repair of muscle
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Hypothesis: Pax7 regulates muscle development by recruiting methyltransferases to the Myf5 locus (Remember: Myf5 is one of the 4 MRFs)
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What genes are regulated by Pax7? Pax7-flag retrovirus C2C12 myoblasts Microarray analysis Validation of genes
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Production of retrovirus for overexpression of Gene X Packaging cell line Viral proteins Neo GeneX Transfection Viral proteins Producer Cell line Packaged retroviral vector RNA Ψ= encapsidation sequence Ψ Retrovirus vector DNA ∆Ψ gag ∆Ψ pol env Full Length Packageable vector RNA Transfection of the retrovirus vector into the packaging cell line results in packaged retroviral vector RNA, which
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