Expression patterns of myocardin and MRTFs in adult and embryonic tissues. (A and B) Northern blot analyses of MRTF-A and -B, respectively, in adult mouse tissues. (C) Expression of myocardin (a–e), MRTF-A (f–j), and MRTF-B (k–o) in mouse embryos as detected by in situ hybridization. Myocardin is expressed in cardiac and smooth muscle cells of an E13.5 embryo (a). A higher magnification of the same embryo showing myocardin expression in heart, smooth muscles of esophagus, and dorsal aorta (b), lung (c), bladder and small intestine (d), and stomach (e) is shown. MRTF-A is expressed in the tongue (f and g), lung and diaphragm (h), kidney (i), bladder (j), and colon of E13.5 (f) and E15.5 (g–j) mouse embryos. Expression of MRTF-B in E13.5 (k) and E15.5 (l–o) mouse embryos. Note higher-level expression present in the lung (m), kidney (n), and olfactory epithelium (o). Arrowheads in h and m point to the lung and in i and n to the kidney.

Lack of transcriptional activity of myocardin and MRTFs in Srf(−/−) ES cells. Wild-type (A) and Srf(−/−) (B) ES cells were transiently transfected with the SM22-luciferase and (mSm)₂-luciferase reporters and expression plasmids encoding myocardin, MRTFs, and SRF as described in Materials and Methods. (A) Myocardin lacking the N-terminal domain (NTD) was used to transactivate (mSm)₂-luciferase. Values are presented as the fold activation of expression above the background level of expression of vector alone. All transfection assays were performed at least three times, and representative data are shown.

Structure of the myocardin family of transcription factors. (A) Schematic diagrams of myocardin, MRTF-A, and MRTF-B proteins. ++, basic region; NTD, N-terminal domain; Q, glutamine-rich region. The number of amino acids in each protein is shown to the right, and percent identity between the indicated domains of each MRTF and myocardin is shown. (B) Amino acid sequence homology between myocardin and MRTFs. Colored bars correspond to the conserved regions shown in A. (C) Gene organization of mouse myocardin, MRTF-A, and MRTF-B. The colors of exons correspond to the regions shown in A. Dashed lines designate an alternative exon. Kilobases of genomic DNA are shown above the gene structures. Translation initiation (ATG) and termination codons (TAA and TGA) are indicated.

Transcriptional activity of MRTFs. The complete ORFs (A) or TADs (B) of myocardin and MRTFs were fused to the GAL4 DNA-binding domain and tested for transcriptional activity by using a GAL4-dependent luciferase reporter (UAS-luc) in transfected COS cells. (C) Transactivation of luciferase reporters linked to SM22 or ANF promoters or four tandem copies of CArG-near from the SM22 promoter by myocardin and MRTFs, as indicated. WT refers to the wild-type promoter, and Mut refers to the promoters with mutations in the two CArG boxes. Values are presented as the fold activation of expression above the background level of expression of vector alone. All transfection assays were performed at least three times, and representative data are shown.

Interaction of SRF and MRTFs. (A) Gel-mobility shift assays were performed with a ³²P-labeled oligonucleotide probe for SM22 CArG-far and in vitro translation products of myocardin, MRTF-A, or MRTF-B in the presence and absence of SRF. Asterisks designate the position of the ternary complexes formed between SRF and myocardin or MRTF-A. The ternary complex between SRF and MRTF-A was very weak and between SRF and MRTF-B was undetectable. (B) GST-SRF protein interaction. Myocardin, MRTF-A and -B, translated in vitro with [³⁵S]methionine, were incubated with either GST-SRF-agarose beads or GST-agarose beads as indicated. After washing, proteins associated with beads were separated on 10% SDS/PAGE and analyzed by autoradiography. One-tenth of the in vitro-translated proteins were also separated directly on the gel as a loading control.