Understanding the molecular mechanisms that regulate cellular proliferation and differentiation is a central theme of developmental biology. MicroRNAs (miRNAs) are a class of regulatory RNAs of 22 nucleotides that post-transcriptionally regulate gene expression 1,2 . Increasing evidence points to the potential role of miRNAs in various biological processes 3-8 . Here we show that miRNA-1 (miR-1) and miRNA-133 (miR-133), which are clustered on the same chromosomal loci, are transcribed together in a tissue-specific manner during development. miR-1 and miR-133 have distinct roles in modulating skeletal muscle proliferation and differentiation in cultured myoblasts in vitro and in Xenopus laevis embryos in vivo. miR-1 promotes myogenesis by targeting histone deacetylase 4 (HDAC4), a transcriptional repressor of muscle gene expression. By contrast, miR-133 enhances myoblast proliferation by repressing serum response factor (SRF). Our results show that two mature miRNAs, derived from the same miRNA polycistron and transcribed together, can carry out distinct biological functions. Together, our studies suggest a molecular mechanism in which miRNAs participate in transcriptional circuits that control skeletal muscle gene expression and embryonic development.To understand the potential involvement of miRNAs in skeletal muscle proliferation and differentiation, we analyzed the expression of miRNAs during skeletal muscle differentiation using an established microarray analysis 9 . We used C2C12 myoblasts because these cells faithfully mimic skeletal muscle differentiation in vitro, as shown by their induction to terminally differentiated myotubes when serum is withdrawn from the culture medium 10-12 . We found that the expression of a few of the miRNAs examined was upregulated in differentiated C2C12 myoblasts or myotubes ( Fig. 1a and Supplementary Fig. 1 online). This increase in expression of miR-1 and miR-133 in differentiated myoblasts was confirmed by RNA blot analysis ( Fig. 1b and Supplementary Fig. 2 online).miR-1 and miR-133 are specifically expressed in adult cardiac and skeletal muscle tissues, but not in other tissues tested 13-15 ( Fig. 1c and Supplementary Fig. 3 online). Little is known, however, about the spatio-temporal distribution of specific miRNAs during mammalian development. We therefore examined the expression of miR-1 and miR-133 in mouse embryos and neonates. miR-1 and miR-133 are expressed in very small amounts in the developing hearts Correspondence should be addressed to D.-Z.W. (dawang@med.unc.edu).. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. and skeletal muscle of embryonic day 13.5 (E13.5) and E16.5 mice ( Fig. 1d and Supplementary Fig. 3). Increasing expression of miR-1 and miR-133 was found in neonatal hearts and skeletal muscle, although it was still substantially lower than that in adult tissues ( Fig. 1e and Supplementary Fig. 3). These data are consistent with findings in zebrafish showing that most miRNAs are expressed r...

Serum response factor (SRF) regulates transcription of numerous muscle and growth factor-inducible genes. Because SRF is not muscle specific, it has been postulated to activate muscle genes by recruiting myogenic accessory factors. Using a bioinformatics-based screen for unknown cardiac-specific genes, we identified a novel and highly potent transcription factor, named myocardin, that is expressed in cardiac and smooth muscle cells. Myocardin belongs to the SAP domain family of nuclear proteins and activates cardiac muscle promoters by associating with SRF. Expression of a dominant negative mutant of myocardin in Xenopus embryos interferes with myocardial cell differentiation. Myocardin is the founding member of a class of muscle transcription factors and provides a mechanism whereby SRF can convey myogenic activity to cardiac muscle genes.

Smooth muscle cells switch between differentiated and proliferative phenotypes in response to extracellular cues, but the transcriptional mechanisms that confer such phenotypic plasticity remain unclear. Serum response factor (SRF) activates genes involved in smooth muscle differentiation and proliferation by recruiting muscle-restricted cofactors, such as the transcriptional coactivator myocardin, and ternary complex factors (TCFs) of the ETS-domain family, respectively. Here we show that growth signals repress smooth muscle genes by triggering the displacement of myocardin from SRF by Elk-1, a TCF that acts as a myogenic repressor. The opposing influences of myocardin and Elk-1 on smooth muscle gene expression are mediated by structurally related SRF-binding motifs that compete for a common docking site on SRF. A mutant smooth muscle promoter, retaining responsiveness to myocardin and SRF but defective in TCF binding, directs ectopic transcription in the embryonic heart, demonstrating a role for TCFs in suppression of smooth muscle gene expression in vivo. We conclude that growth and developmental signals modulate smooth muscle gene expression by regulating the association of SRF with antagonistic cofactors.

Myocardin is a SAP (SAF-A͞B, Acinus, PIAS) domain transcription factor that associates with serum response factor (SRF) to potently enhance SRF-dependent transcription. Here we describe two myocardin-related transcription factors (MRTFs), A and B, that also interact with SRF and stimulate its transcriptional activity. Whereas myocardin is expressed specifically in cardiac and smooth muscle cells, MRTF-A and -B are expressed in numerous embryonic and adult tissues. In SRF-deficient embryonic stem cells, myocardin and MRTFs are unable to activate SRF-dependent reporter genes, confirming their dependence on SRF. Myocardin and MRTFs comprise a previously uncharacterized family of SRF cofactors with the potential to modulate SRF target genes in a wide range of tissues.