1. Coactivation of MEF2 by the SAP Domain Proteins Myocardin and MASTR
Esther E. Creemers, Lillian B. Sutherland, Jiyeon Oh, Ana C. Barbosa, Eric N. Olson 
Molecular Cell 
Volume 23, Issue 1, Pages (July 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Alternative Splicing of Myocardin Transcripts Yields Proteins with Different N Termini
(A) Intron/exon organization of the mouse myocardin gene. Alternative exon 2a (shown in red) is contained in transcripts from smooth muscle but is excluded from cardiac transcripts. Inclusion of exon 2a results in a premature termination codon, but a second ATG codon in exon 4 is able to generate a protein of 856 amino acids. The cardiac-enriched transcript excludes exon 2a and generates a protein of 935 amino acids beginning with an ATG codon in exon 1.
(B) Nucleotide sequence of the 44 bp exon 2a. A termination codon (TAA) prevents translation of a complete myocardin protein from the ATG codon in exon 1.
(C) RT-PCR discriminates between myocardin transcripts in aorta and heart, based on the presence and absence of exon 2a, respectively. Positions of PCR primers in exons 2 and 5 are shown in (A). HPRT transcripts were detected as a control.
(D) Schematic diagrams of the two forms of myocardin predicted to result from translation initiation at ATG codons in exons 1 and 4 (see [A]).
(E) Detection of different myocardin isoforms by Western blot. COS cells were transfected with expression vectors encoding the full-length myocardin transcripts with and without exon 2a (left panel). In the right panel, cells were transfected with expression vectors encoding truncated forms of myocardin (C-terminal myc-tag) with mutations in the indicated ATGs. Proteins initiating translation from the indicated ATG codons are shown at the left. SM myocardin (lane 2) refers to the protein generated from the smooth muscle transcript.
(F) Schematic diagram of the NTD of myocardin and the positions of the first four ATGs.
Molecular Cell  , 83-96DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2
Selective Activation of MEF2-Dependent Transcription by the Cardiac Isoform of Myocardin
(A) Activation of SRF and MEF2-dependent reporters by 935 and 856 myocardin proteins. COS cells were transfected with the indicated reporters and expression vectors encoding the 935 and 856 amino acid myocardin proteins and MEF2C (right panel), as indicated. Both forms of myocardin activate SRF, but only myocardin-935 activates MEF2 (n = 3, mean ± SEM).
(B) COS cells were transfected with 3×MEF2-luciferase and expression vectors encoding myocardin-935 (50 ng) and increasing concentrations of MEF2C (10–100 ng) or SRF (10–100 ng), as indicated (n = 3, mean ± SEM).
(C) SRF null ES cells were transfected with 3×MEF2-luciferase and expression vectors encoding myocardin-935 (50 ng) and MEF2C (50 ng), as indicated (n = 3, mean ± SEM).
(D) COS cells were transfected with the indicated reporters and expression vectors encoding myocardin-935 (50 ng) and MEF2C (50 ng), as indicated (n = 3, mean ± SEM).
(E) 10T1/2 cells were transfected with expression vectors encoding the indicated myocardin cDNAs. RNA was isolated after 2 days, and MEF2 and SRF-dependent muscle genes were assayed by RT-PCR. HPRT was used as a loading control.
(F) Cardiomyocytes were infected with an adenovirus encoding either β-gal or myocardin-935. RT-PCR analysis was performed on RNA and harvested 48 hr after infection (left panel). ChIP assays were performed on endogenous proteins with chromatin prepared from uninfected cardiomyocytes (right panel). Chromatin was immunoprecipitated with antibodies against GFP (as a control), MEF2C, and myocardin, and precipitated genomic DNA was analyzed by PCR using primers for the Myl1 promoter and an exon of the MMP-9 gene (as a negative control). PCR amplification was performed prior to immunoprecipitation for the input control.
(G) Rat neonatal cardiomyocytes were cotransfected with 3×MEF2-luciferase reporter and myocardin siRNA or a control nontargeting siRNA in quadruplicate. Bar graphs show luciferase activity normalized for β-gal of the MEF2 reporter (∗p < 0.05).
(H) Myl1 transcripts were detected by whole-mount in situ hybridization in wild-type and myocardin KO embryos at E9.0.
(I) Expression of myocardin and MEF2 target genes in myocardin null (KO) embryos and their wild-type controls. RT-PCR analysis in myocardin knockout and wt E8.5 and E9.0 embryos is shown in the left panel. Real-time PCR was performed on embryos at E9.0 (right panel). Bar graphs show the relative mRNA levels normalized by GAPDH (∗p < 0.05).
(J) COS cells were transfected with 3×MEF2-luciferase and expression vectors encoding myocardin-935, MRTF-A, or MRTF-B (50 ng) and MEF2C (50 ng), as indicated (n = 3, mean ± SEM).
Molecular Cell  , 83-96DOI: ( /j.molcel )
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4. Figure 3 Mapping the MEF2-Sensitive Domain of Myocardin
(A) Homology of the NTD of mouse myocardin and MRTFs.
(B) N-terminal deletion and point mutants of myocardin. Dots below the top sequence indicate residues that were unchanged, and Xs designate deleted amino acids. The relative abilities of each protein to activate MEF2 (as shown in [C]) and to bind MEF2 (as shown in [D]) are indicated to the right. Nd, not determined. The shaded box represents the region that is necessary for MEF2 binding.
(C) Effects of N-terminal mutations on transcriptional activity of myocardin. COS cells were transfected with the indicated reporters and expression vectors encoding myocardin-935 or myocardin mutants (50 ng). Transfections with 3×MEF2-luciferase also included MEF2C (50 ng). Values are expressed relative to the level of expression with myocardin-935 and each reporter, which was assigned a value of 100 (n = 3, mean ± SEM).
(D) CoIP of Myc-MEF2C and FLAG-myocardin mutants. COS cells were transfected with expression plasmids encoding Myc-MEF2C and FLAG-myocardin mutants contained in residues 1–179, and extracts were prepared and immunoprecipitated with anti-Myc antibody followed by Western blot with anti-FLAG antibody (top panel). Western blots of transfected cells to detect input proteins are shown in the middle and bottom panels.
Molecular Cell  , 83-96DOI: ( /j.molcel )
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5. Figure 4
Mapping the Domains of Myocardin Required for Stimulation of MEF2
(A) The indicated myocardin mutants were tested for their abilities to activate the 3×MEF2-luciferase and SM22-luciferase reporters in transfected COS cells. Values are expressed relative to the level of expression with myocardin-935 with each reporter, which was assigned a value of 100.
(B) Schematic of the two-hybrid assay. Portions of MEF2C shown in (C) were fused to the DNA binding domain of GAL4 and tested for their ability to activate a UAS-luciferase reporter in the presence of myocardin-935.
(C) Effects on transcriptional activity of GAL4-MEF2C chimeras by myocardin. COS cells were transfected with 100 ng of UAS-luciferase reporter, 50 ng of expression vector encoding the indicated GAL4-MEF2C chimeras, and 50 ng of myocardin-935. Activities are expressed as the percentage of GAL4-MEF2C activity observed for each mutant. A schematic of MEF2C showing the positions of the MADS, MEF2-specific, and TADs is shown at the top (n = 3, mean ± SEM).
(D) GST pull-down assays. 35S-labeled MEF2C protein was translated in vitro and incubated with GST-myocardin-935 or GST-myocardin-856. The MEF2C input lanes contain 10% of the amount of MEF2 protein in the pull-down lanes. GST-myocardin fusion proteins are shown on a Coomassie gel in the bottom panel.
(E) Schematic diagram of potential interactions between myocardin, SRF, and MEF2.
Molecular Cell  , 83-96DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5 Sequence and Structure of MASTR
(A) Murine sequence of MASTR and homology of the MEF2-responsive regions and SAP domains of MASTR and myocardin. Colors of domains correspond to those in (B).
(B) Schematic diagram of MASTR.
(C) Homology of MASTR with other SAP domain proteins. The two predicted α helices of the SAP domain are shown at the top. a, acidic; b, basic; h, hydrophobic.
Molecular Cell  , 83-96DOI: ( /j.molcel )
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7. Figure 6 Coactivation and Interaction of MASTR with MEF2
(A) Immunostaining of MASTR and MEF2. COS cells were transfected with expression vectors encoding Myc-tagged MEF2C and FLAG-tagged MASTR, separately or together, and proteins were detected by immunostaining.
(B) Coactivation of MEF2 by MASTR. COS cells were transfected with 3×MEF2-luciferase and expression vectors encoding MASTR, MASTRΔ13–28, and MEF2C, as indicated (left panel). MASTR does not activate the SM22-luciferase reporter (right panel). Western blots demonstrated that MASTR did not induce MEF2 protein levels (data not shown) (n = 3, mean ± SEM).
(C) Coactivation of MEF2 by MASTR-N/myocardin, myocardin-N/MASTR, and MASTR-N/MRTF-A. Chimeric proteins of MASTR and myocardin were generated in which the N-terminal 40 amino acids were exchanged. The MASTR-MRTFA chimeric protein was generated by replacing the first 136 amino acids (all three RPELs) of MRTF-A by the first 40 amino acids of MASTR. The chimeric proteins were transfected in COS cells together with 3×MEF2 luciferase and expression vectors encoding MEF2, MASTR, and myocardin, as indicated (n = 3, mean ± SEM).
(D) GST pull-down assays. 35S-labeled MEF2C or SRF protein was translated in vitro and incubated with GST-MASTR or GST-MASTRΔ13–28. Proteins were captured on glutathione agarose beads and analyzed by SDS-PAGE. The MEF2C and SRF input lanes contain 10% of the amount of protein in the other lanes. GST-MASTR fusion proteins are shown in the bottom panel.
(E) Portions of MASTR were fused to the GAL4-DNA binding domain and tested for their ability to activate a GAL4-dependent luciferase reporter.
(F) Detection of MASTR transcripts in adult human and mouse tissues. An adult human tissue Northern blot was probed with a MASTR cDNA fragment (left panel), and MASTR expression in mouse tissues was detected by real-time PCR (right panel).
(G) MASTR enhances conversion of 10T1/2 cells to skeletal muscle by MyoD. Cells were transfected with expression vectors for MyoD and/or MASTR, and myogenic conversion was scored by staining for MHC expression. Assays were performed three independent times with comparable results (n = 3, mean ± SEM).
Molecular Cell  , 83-96DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

8. Figure 7 Potential Mechanisms of Action of Myocardin and MASTR
(Left panel) The 935 amino acid form of myocardin, which predominates in cardiac muscle, associates with SRF via the basic (+) region resulting in activation of CArG box-dependent genes. The 856 amino acid form of myocardin, which predominates in smooth muscle, acts in the same way to activate CArG box-dependent smooth muscle genes. (Right panel) The 935 amino acid form of myocardin also associates with MEF2 via the NTD to activate transcription. MASTR uses a similar NTD to stimulate MEF2-dependent genes and has a broader tissue distribution than myocardin.
Molecular Cell  , 83-96DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions