5′ CArG degeneracy in smooth muscle α-actin is required for injury-induced gene suppression in vivo
Jennifer A. Hendrix, … , Tadashi Yoshida, Gary K. Owens
Jennifer A. Hendrix, … , Tadashi Yoshida, Gary K. Owens
Published February 1, 2005
Citation Information: J Clin Invest. 2005;115(2):418-427. https://doi.org/10.1172/JCI22648.
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Article Genetics

5′ CArG degeneracy in smooth muscle α-actin is required for injury-induced gene suppression in vivo

Abstract

CC(A/T)6GG–dependent (CArG-dependent) and serum response factor–dependent (SRF-dependent) mechanisms are required for gene expression in smooth muscle cells (SMCs). However, an unusual feature of many SMC-selective promoter CArG elements is that they contain a conserved single G or C substitution in their central A/T-rich region, which reduces binding affinity for ubiquitously expressed SRF. We hypothesized that this CArG degeneracy contributes to cell-specific expression of smooth muscle α-actin in vivo, since substitution of c-fos consensus CArGs for the degenerate CArGs resulted in relaxed specificity in cultured cells. Surprisingly, our present results show that these substitutions have no effect on smooth muscle–specific transgene expression during normal development and maturation in transgenic mice. However, these substitutions significantly attenuated injury-induced downregulation of the mutant transgene under conditions where SRF expression was increased but expression of myocardin, a smooth muscle–selective SRF coactivator, was decreased. Finally, chromatin immunoprecipitation analyses, together with cell culture studies, suggested that myocardin selectively enhanced SRF binding to degenerate versus consensus CArG elements. Our results indicate that reductions in myocardin expression and the degeneracy of CArG elements within smooth muscle promoters play a key role in phenotypic switching of smooth muscle cells in vivo, as well as in mediating responses of CArG-dependent smooth muscle genes and growth regulatory genes under conditions in which these 2 classes of genes are differentially expressed.

Authors

Jennifer A. Hendrix, Brian R. Wamhoff, Oliver G. McDonald, Sanjay Sinha, Tadashi Yoshida, Gary K. Owens

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Figure 3

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Oligonucleotides containing the c-fos SRE consensus CArG substitution co...
Oligonucleotides containing the c-fos SRE consensus CArG substitution competed for SRF binding activity more effectively than did wild-type SM α-actin oligonucleotides (containing CArG-A and/or CArG-B) in EMSAs. (A) In vitro translated SRF and a 95-bp radiolabeled probe harboring the CArG-containing region of the SM α-actin promoter were used for EMSAs. Unlabeled 95-bp double-stranded oligonucleotides containing either the SM α-actin 5′ CArGs (WT) or the SRE consensus CArG substituted for CArG-A (SRE-A), CArG-B (SRE-B), or CArG-A and CArG-B (SRE-AB) in the context of the SM α-actin promoter were used as cold competitors at approximately 50-, 100-, and 200-fold excess over labeled probe. The SRE CArG in the context of the c-fos promoter (fos) was used as a cold competitor at approximately 50-fold excess over labeled probe. The SRF band was supershifted (SRF SS) by the addition of 2 μg of anti-SRF rabbit polyclonal antibody. Unprog. lysate, unprogrammed control lysate. (B) Densitometry was performed on the SRF bands (see Figure 5A) and results were plotted relative to maximal SRF binding to the radiolabeled probe in the absence of cold competitor. Results are representative of 3 independent experiments. Statistical analyses were performed using 1-way ANOVA. We found statistically significant differences between percentage of SRF binding in the presence of WT cold competitor and percentage of SRF binding in the presence of SRE-AB, SRE-A, or SRE-B cold competitor under all but 1 condition (×50 WT versus ×50 SRE-AB) across multiple experiments (data not shown).