Myocardin is a master regulator of smooth muscle gene expression - PubMed (original) (raw)

Myocardin is a master regulator of smooth muscle gene expression

Zhigao Wang et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Virtually all smooth muscle genes analyzed to date contain two or more essential binding sites for serum response factor (SRF) in their control regions. Because SRF is expressed in a wide range of cell types, it alone cannot account for smooth muscle-specific gene expression. We show that myocardin, a cardiac muscle- and smooth muscle-specific transcriptional coactivator of SRF, can activate smooth muscle gene expression in a variety of nonmuscle cell types via its association with SRF. Homodimerization of myocardin is required for maximal transcriptional activity and provides a mechanism for cooperative activation of smooth muscle genes by SRF-myocardin complexes bound to different SRF binding sites. These findings identify myocardin as a master regulator of smooth muscle gene expression and explain how SRF conveys smooth muscle specificity to its target genes.

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Figures

Fig. 1.

Activation of smooth muscle cell differentiation by myocardin. (A) 10T1/2 cells were transfected with myocardin (a, b, e, f, and i_–_n) or MyoD (c, d, g, and h) expression vectors and muscle markers were assayed by immunostaining. Bright and dark field images are shown in a, c, e, and g and b, d, f, and h, respectively. FLAG-tagged myocardin was also detected within the nuclei of transfected cells in i_–_n. (B) 10T1/2 cells were transfected with expression vectors encoding lacZ (as a negative control), myocardin, myocardin-NLSΔbasic, and MyoD. Protein extracts were assayed by Western blot by using antibodies against SM22, SM-γ-actin, and SM myosin light chain kinase. α-Tubulin was detected as a loading control. (C and D) 10T1/2 cells (C) and NIH 3T3 or 3T3-L1 cells (D) were transfected with the expression vectors indicated above each lane. RNA was isolated and muscle gene expression was assayed by RT-PCR. L7 was measured as a loading control. (E) Primary rat cardiac fibroblasts were infected with adenoviruses encoding lacZ or myocardin and stained for SM-α-actin expression. Only background staining was seen with Ad-lacZ, whereas intensely stained cells were seen with Ad-myocardin. The organization of these cells can also be seen at low magnifications (Right). (Upper Right) A phase–contrast image. (Bars, 200 μm.)

Fig. 2.

Analysis of myocardin mutants. (A) Domains of myocardin required for smooth muscle gene expression. 10T1/2 cells were transfected with expression vectors encoding the indicated myocardin constructs. Values are expressed as the number of SM-MHC-positive cells with each mutant relative to the number in cultures transfected with the wild-type myocardin expression plasmid, which was assigned a value of 100. (B) Activation of smooth muscle gene expression by myocardin and MRTF-A. 10T1/2 cells were transfected with expression vectors encoding myocardin, MRTF-A, or MRTF-B and scored for SM-MHC-positive cells as in A.(C) Inhibition of smooth muscle gene expression by dominant negative myocardin. PAC1 and A10 cells were infected with adenovirus encoding lacZ or the myocardin dominant negative mutant 128–513. Smooth muscle gene expression was assayed by RT-PCR. GAPDH was measured as a loading control.

Fig. 3.

Homooligomerization of myocardin mediated by the coiled-coil domain. (A) Schematic of myocardin and myocardin deletion mutants. Deletion mutants with FLAG tags and Myc-tagged myocardin were expressed in transfected COS cells. Cell extracts were immunoprecipitated (IP) with anti-FLAG antibody and analyzed by immunoblot (IB) with anti-Myc antibody. Blots are shown (Right). LZ, leucine zipper. (B) The sequence of the leucine zipper region of myocardin and the amino acid changes in the leucine zipper mutant (LZ-mut) are shown. Wild-type and LZ-mutant myocardin were expressed in COS cells, and protein–protein interactions were detected by immunoprecipitation followed by immunoblotting, as indicated.

Fig. 4.

Functional analysis of myocardin dimerization mutant. (A) Gel mobility shift assays were performed with in vitro translated SRF, myocardin (WT), and the myocardin LZ-mutant and a radiolabeled probe corresponding to the c-fos serum response element. Only the region of the gel containing shifted probe is shown. (B) COS cells were transiently transfected with expression vectors for myocardin and the myocardin LZ-mutant and luciferase reporters linked to the SM22 promoter or three copies of the c-fos CArG box, and luciferase activity was measured. (C) 10T1/2 cells were transfected with expression vectors for myocardin or the myocardin LZ-mutant, and SM-MHC-positive cells were scored as in Fig. 2 A.(D) 10T1/2 cells were transfected with expression vectors encoding myocardin or the LZ-mutant. RNA was isolated and transcripts were assayed by RT-PCR.

Fig. 5.

A model for the regulation of smooth muscle genes by SRF. Myocardin preferentially activates smooth muscle genes controlled by pairs of CArG boxes. The c_-fos_ promoter contains a single CArG box and is not efficiently activated by myocardin. Dimerization of myocardin through the leucine zipper (LZ) may expose the TAD with resulting activation of muscle gene expression. Other promoter-specific factors (X) cooperate with SRF and myocardin.

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Myocardin is a master regulator of smooth muscle gene expression - PubMed (original) (raw)