5′ CArG degeneracy in smooth muscle α-actin is required for injury-induced gene suppression in vivo
Jennifer A. Hendrix, Brian R. Wamhoff, Oliver G. McDonald, Sanjay Sinha, Tadashi Yoshida, Gary K. Owens
Jennifer A. Hendrix, Brian R. Wamhoff, Oliver G. McDonald, Sanjay Sinha, Tadashi Yoshida, Gary K. Owens
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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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Myocardin can differentially regulate SRF binding to degenerate versus c...
Myocardin can differentially regulate SRF binding to degenerate versus consensus CArGs and is decreased following vascular injury. (A) Results of ChIP assays in cultured rat aortic SMCs indicate that SRF binding is enhanced at the CArG-containing region of the SM α-actin promoter but not the c-fos promoter in response to myocardin overexpression. Quantitative PCR was used to detect CArG-containing regions of the SM α-actin and c-fos promoters in chromatin fragments immunoprecipitated with an SRF antibody. Data represent the mean ± SE of the fold increase in SRF association in cells overexpressing myocardin versus control cells in 3 independent experiments. A fold increase value of 1 indicates no change in SRF association in cells overexpressing myocardin versus control cells. *P < 0.05 compared with SRF association at the c-fos CArG region under the same conditions. (B) Temporal expression analysis by real-time RT-PCR of endogenous myocardin in balloon-injured rat carotid arteries showed decreased myocardin expression following injury. Myocardin expression was normalized to 18S rRNA expression in the injured and uninjured contralateral control vessel. Each time point represents the mean ± SE of the injured vessel (myocardin:18S) normalized to that of the uninjured (myocardin:18S) vessel (n = 4 animals per time point). *P < 0.05 compared with myocardin expression prior to injury. (C) Effect of substitution of CArG-A and CArG-B with the c-fos SRE CArG on myocardin responsiveness of SM α-actin promoter activity. SM α-actin/luciferase and SRE-AB/luciferase promoter constructs were cotransfected with myocardin into rat aortic SMCs and assayed for luciferase activity. The activity was normalized for protein content. Normalized promoter activities of SM α-actin/luciferase and SRE-AB/luciferase in the absence of myocardin were set to 1. Fold induction over basal promoter activity in response to myocardin was calculated. Values represent the mean ± SE of 3 independent experiments. *P < 0.05 compared with WT fold induction under the same conditions.