A systems approach reveals that the myogenesis genome network is regulated by the transcriptional repressor RP58.

We created a whole-mount in situ hybridization (WISH) database, termed EMBRYS, containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos--a highly dynamic stage of skeletal myogenesis. This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoD's ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor (MRF) inhibitors.

[1]  Yang Liu,et al.  Mouse Brain Organization Revealed Through Direct Genome-Scale TF Expression Analysis , 2004, Science.

[2]  J. Kawai,et al.  A genome-wide and nonredundant mouse transcription factor database. , 2004, Biochemical and biophysical research communications.

[3]  William H. Klein,et al.  Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene , 1993, Nature.

[4]  Barbara Gayraud-Morel,et al.  Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice , 2004, Nature.

[5]  R. Sharan,et al.  An initial blueprint for myogenic differentiation. , 2005, Genes & development.

[6]  Barbara Wold,et al.  HLH forced dimers: Tethering MyoD to E47 generates a dominant positive myogenic factor insulated from negative regulation by Id , 1993, Cell.

[7]  Lior Pachter,et al.  VISTA: computational tools for comparative genomics , 2004, Nucleic Acids Res..

[8]  H. Weintraub,et al.  Sequence-specific DNA binding by the c-Myc protein. , 1990, Science.

[9]  P. L. Puri,et al.  Regulation of muscle regulatory factors by DNA‐binding, interacting proteins, and post‐transcriptional modifications , 2000, Journal of cellular physiology.

[10]  M. Bounpheng,et al.  Differential biological activities of mammalian Id proteins in muscle cells. , 1999, Experimental cell research.

[11]  M. Campbell,et al.  PANTHER: a library of protein families and subfamilies indexed by function. , 2003, Genome research.

[12]  B. Christy,et al.  Muscle cell differentiation is inhibited by the helix-loop-helix protein Id3. , 1996, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[13]  Allan R. Jones,et al.  Genome-wide atlas of gene expression in the adult mouse brain , 2007, Nature.

[14]  H. Arnold,et al.  Pax-3 is required for the development of limb muscles: a possible role for the migration of dermomyotomal muscle progenitor cells. , 1994, Development.

[15]  L. Kedes,et al.  Acetylation of MyoD directed by PCAF is necessary for the execution of the muscle program. , 1999, Molecular cell.

[16]  A. Orth,et al.  Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[17]  H. Asahara,et al.  Dynamic gene expression of Lin-28 during embryonic development in mouse and chicken. , 2008, Gene expression patterns : GEP.

[18]  M. Rudnicki,et al.  Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death , 1992, Cell.

[19]  Eric P Hoffman,et al.  MyoD Acetylation Influences Temporal Patterns of Skeletal Muscle Gene Expression* , 2007, Journal of Biological Chemistry.

[20]  E. Prochownik,et al.  Differential Interactions of Id Proteins with Basic-Helix-Loop-Helix Transcription Factors* , 1997, The Journal of Biological Chemistry.

[21]  R. Lim,et al.  Regulation of inhibitor of differentiation gene 3 (Id3) expression by Sp2-motif binding factor in myogenic C2C12 cells: downregulation of DNA binding activity following skeletal muscle differentiation. , 2005, Biochimica et biophysica acta.

[22]  David Baltimore,et al.  A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins , 1989, Cell.

[23]  G. Martin,et al.  Deciphering skeletal patterning: clues from the limb , 2003, Nature.

[24]  Craig Nelson,et al.  Hox genes and the evolution of vertebrate axial morphology. , 1995, Development.

[25]  E. Olson,et al.  Control of muscle development by dueling HATs and HDACs. , 2001, Current opinion in genetics & development.

[26]  M. Rudnicki,et al.  Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development , 1992, Cell.

[27]  Charles Kooperberg,et al.  Global and gene‐specific analyses show distinct roles for Myod and Myog at a common set of promoters , 2006, The EMBO journal.

[28]  Harold Weintraub,et al.  The protein Id: A negative regulator of helix-loop-helix DNA binding proteins , 1990, Cell.

[29]  Scott Chapman,et al.  Using biplots to interpret gene expression patterns in plants , 2002, Bioinform..

[30]  T. Braun,et al.  Targeted inactivation of myogenic factor genes reveals their role during mouse myogenesis: a review. , 1996, The International journal of developmental biology.

[31]  Douglas A. Melton,et al.  In vivo reprogramming of adult pancreatic exocrine cells to β-cells , 2008, Nature.

[32]  M. Goulding,et al.  Pax-3 is necessary for migration but not differentiation of limb muscle precursors in the mouse. , 1996, Development.

[33]  Alexei A. Sharov,et al.  Gene expression A web-based tool for principal component and significance analysis of microarray data , 2005 .

[34]  J. C. Belmonte,et al.  MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb , 2003, Nature Cell Biology.

[35]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[36]  L. Kedes,et al.  Doxorubicin represses the function of the myogenic helix-loop-helix transcription factor MyoD. Involvement of Id gene induction. , 1994, The Journal of biological chemistry.

[37]  L. Wolpert.  Positional information and the spatial pattern of cellular differentiation. , 1969, Journal of theoretical biology.

[38]  H. Weintraub,et al.  Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. , 1990, Science.

[39]  B. Williams,et al.  Pax-3 expression in segmental mesoderm marks early stages in myogenic cell specification. , 1994, Development.

[40]  Akiyo Takahashi,et al.  The transcriptional repressor RP58 is crucial for cell-division patterning and neuronal survival in the developing cortex. , 2009, Developmental biology.

[41]  B. Han,et al.  Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function. , 1997, Nucleic acids research.

[42]  C. Tabin,et al.  Sonic hedgehog mediates the polarizing activity of the ZPA , 1993, Cell.

[43]  I. Nonaka,et al.  Myogenin gene disruption results in perinatal lethality because of severe muscle defect , 1993, Nature.

[44]  H. Kawano,et al.  Spatial and temporal expression of RP58, a novel zinc finger transcriptional repressor, in mouse brain , 2007, The Journal of comparative neurology.

[45]  T. Kouzarides,et al.  Dnmt3a binds deacetylases and is recruited by a sequence‐specific repressor to silence transcription , 2001, The EMBO journal.

[46]  W. J. Kent,et al.  BLAT--the BLAST-like alignment tool. , 2002, Genome research.

[47]  T. Jessell.  Neuronal specification in the spinal cord: inductive signals and transcriptional codes , 2000, Nature Reviews Genetics.

[48]  S. Tapscott,et al.  A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation. , 2004, Genes & development.

[49]  H. Weintraub,et al.  Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins. , 1992, Genes & development.

[50]  Li-Wei Chang,et al.  Neurogenin and NeuroD direct transcriptional targets and their regulatory enhancers , 2007, The EMBO journal.

[51]  L. Kedes,et al.  Differential roles of p300 and PCAF acetyltransferases in muscle differentiation. , 1997, Molecular cell.

[52]  D. Yaffe,et al.  Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle , 1977, Nature.

[53]  S. Tapscott,et al.  Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[54]  Wei Zhang,et al.  Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies. , 1995, Genes & development.

[55]  P. L. Puri,et al.  Chromatin: the interface between extrinsic cues and the epigenetic regulation of muscle regeneration. , 2009, Trends in cell biology.

[56]  Y. Kameoka,et al.  RP58 Associates with Condensed Chromatin and Mediates a Sequence-specific Transcriptional Repression* , 1998, The Journal of Biological Chemistry.

[57]  Helen M. Blau,et al.  Cytoplasmic activation of human nuclear genes in stable heterocaryons , 1983, Cell.

[58]  J. W. Saunders.  The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. 1948. , 1998, The Journal of experimental zoology.