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An evolutionarily conserved Myostatin proximal promoter/enhancer confers basal levels of transcription and spatial specificity in vivo

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Abstract

Myostatin (Mstn) is a negative regulator of skeletal muscle mass, and Mstn mutations are responsible for the double muscling phenotype observed in many animal species. Moreover, Mstn is a positive regulator of adult muscle stem cell (satellite cell) quiescence, and hence, Mstn is being targeted in therapeutic approaches to muscle diseases. In order to better understand the mechanisms underlying Mstn regulation, we searched for the gene’s proximal enhancer and promoter elements, using an evolutionary approach. We identified a 260-bp-long, evolutionary conserved region upstream of tetrapod Mstn and teleost mstn b genes. This region contains binding sites for TATA binding protein, Meis1, NF-Y, and for CREB family members, suggesting the involvement of cAMP in Myostatin regulation. The conserved fragment was able to drive reporter gene expression in C2C12 cells in vitro and in chicken somites in vivo; both normally express Mstn. In contrast, the reporter construct remained silent in the avian neural tube that normally does not express Mstn. This suggests that the identified element serves as a minimal promoter, harboring some spatial specificity. Finally, using bioinformatic approaches, we identified additional genes in the human genome associated with sequences similar to the Mstn proximal promoter/enhancer. Among them are genes important for myogenesis. This suggests that Mstn and these genes may form a synexpression group, regulated by a common signaling pathway.

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Acknowledgments

We thank Dr. José Xavier-Neto for providing the pTKeGFP vector, Dr. Vera Nisaka Solferini for allowing the use of her laboratory facilities for the development of this work, and Dr. Chao Yun Irene Yan for the kind loan of equipment essential for this research. The work was a collaboration between the Alvares, Dietrich, Schubert and Salerno laboratory, supported by FAPESP (06/05375-9), CNPq (480960/07-0), and FAEPEX (1039/07) grants to LEA, by the EU grant no. LSH-CT-2004-511978 Myores to SD and by the EU Interreg IV AdMiN grant to FRS.

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Authors and Affiliations

  1. Department of Histology and Embryology, State University of Campinas—UNICAMP, Rua Charles Darwin, s/n, Cx. Postal 6109, CEP 13083-863, Campinas, SP, Brazil

    Carla Vermeulen Carvalho Grade & Lúcia Elvira Alvares

  2. Functional Muscle Genomics—AgResearch—Ruakura Research Centre, Private Bag 3123, East St., Hamilton, New Zealand

    Mônica Senna Salerno

  3. Institute of Biomedical and Biomolecular Sciences, School of Biological Sciences, University of Portsmouth, King Henry Building, Portsmouth, PO1 2DY, UK

    Frank R. Schubert

  4. School of Biomedical & Health Sciences, King’s College London, Hodgkin Building G43S/44S, Guy’s Campus, London, SE1 1UL, UK

    Susanne Dietrich

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  1. Carla Vermeulen Carvalho Grade
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  2. Mônica Senna Salerno
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  3. Frank R. Schubert
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  4. Susanne Dietrich
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  5. Lúcia Elvira Alvares
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Correspondence to Lúcia Elvira Alvares.

Additional information

Communicated by M. Hammerschmidt

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Online resource 1

Phylogenetic tree of Mstn proteins in bony vertebrates, using the Gdf8/Gdf11 sequence of the cephalochordate Branchiostoma belcheri tsingtauense as out-group. Note that the lamprey, an agnathan vertebrate, has a Gdf8/Gdf11-type protein. In contrast, the gnathostome vertebrates investigated here have distinct Mstn/Gdf8 and Gdf11 proteins, in line with the idea that gnathostome Mstn/Gdf8 and Gdf11 genes evolved from a common ancestor during the two rounds of jawed vertebrate genome duplications. Also note that tetrapod and teleost Mstn/Gdf8 sequences form two subgroups. Teleost Mstn proteins are further subdivided into distinct mstn a and mstn b type sequences, in line with the idea of a third genome duplication in this lineage. (GIF 151 kb)

High resolution image (TIFF 5967 kb)

Online resource 2

Abbreviations for the species investigated in this study. (DOC 26 kb)

Online resource 3

Genomic organization of Mstn loci in bony vertebrates (DOC 54 kb)

Online resource 4

Position and overall similarity of the conserved sequence upstream of the translation start of Mstn/mstn b genes in bony vertebrates. (DOC 42 kb)

Online resource 5

Vibratome cross section of a somite electroporated with pGgGDF8/eGFP and pCAB/RFP. Upon fixation, the embryos was eviscerated, bisected, and subjected to in situ hybridization with a GFP probe. The GFP signal is observed in the dermal precursor cells originating from the hypaxial dermomyotome (black arrow). Dorsal is to the top, NT stands for neural tube. (GIF 321 kb)

High resolution image (TIFF 6417 kb)

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Grade, C.V.C., Salerno, M.S., Schubert, F.R. et al. An evolutionarily conserved Myostatin proximal promoter/enhancer confers basal levels of transcription and spatial specificity in vivo. Dev Genes Evol 219, 497–508 (2009). https://doi.org/10.1007/s00427-009-0312-x

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