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Muscle LIM proteins are associated with muscle sarcomeres and require dMEF2 for their expression during Drosophila myogenesis.

ABSTRACT: A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and alpha-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.

SUBMITTER: Stronach BE

PROVIDER: S-EPMC25449 | biostudies-literature | 1999 Jul

REPOSITORIES: biostudies-literature

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Muscle LIM proteins are associated with muscle sarcomeres and require dMEF2 for their expression during Drosophila myogenesis.

Stronach B E BE Renfranz P J PJ Lilly B B Beckerle M C MC

Molecular biology of the cell 19990701 7

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, w ...[more]

PMID: 10397768

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Project description:BACKGROUND: Myogenesis is an ordered process whereby mononucleated muscle precursor cells (myoblasts) fuse into multinucleated myotubes that eventually differentiate into myofibres, involving substantial changes in gene expression and the organisation of structural components of the cells. To gain further insight into the orchestration of these structural changes we have overlaid the spatial organisation of the protein components of a muscle cell with their gene expression changes during differentiation using a new 3D visualisation tool: the Virtual Muscle 3D (VMus3D). RESULTS: Sets of generic striated muscle costamere, Z-disk and filament proteins were constructed from the literature and protein-interaction databases. Expression profiles of the genes encoding these proteins were obtained from mouse C2C12 cells undergoing myogenesis in vitro, as well as a mouse tissue survey dataset. Visualisation of the expression data in VMus3D yielded novel observations with significant relationships between the spatial location and the temporal expression profiles of the structural protein products of these genes. A muscle specificity index was calculated based on muscle expression relative to the median expression in all tissues and, as expected, genes with the highest muscle specificity were also expressed most dynamically during differentiation. Interestingly, most genes encoding costamere as well as some Z-disk proteins appeared to be broadly expressed across most tissues and showed little change in expression during muscle differentiation, in line with the broader cellular role described for some of these proteins. CONCLUSION: By studying gene expression patterns from a structural perspective we have demonstrated that not all genes encoding proteins that are part of muscle specific structures are simply up-regulated during muscle cell differentiation. Indeed, a group of genes whose expression program appears to be minimally affected by the differentiation process, code for proteins participating in vital skeletal muscle structures. Expression alone is a poor metric of gene behaviour. Instead, the "connectivity model of muscle development" is proposed as a mechanism for muscle development: whereby the closer to the myofibril core of muscle cells, the greater the gene expression changes during muscle differentiation and the greater the muscle specificity.

Project description:BACKGROUND:Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, DUX4, that can encode a protein containing two homeodomains. A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how. METHODS:Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods. RESULTS:Many of the ~17,000 examined genes were differentially expressed (>2-fold, p<0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis-specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked DUX4 RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types. CONCLUSIONS:DUX4's pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated DUX4 expression at the myoblast or myotube stages. Our model could explain why DUX4's inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.

Dataset Information

Muscle LIM proteins are associated with muscle sarcomeres and require dMEF2 for their expression during Drosophila myogenesis.

Publications

Muscle LIM proteins are associated with muscle sarcomeres and require dMEF2 for their expression during Drosophila myogenesis.

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