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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

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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 2

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SRE-substituted transgenic mice showed no loss of SMC-specific SM α-acti...
SRE-substituted transgenic mice showed no loss of SMC-specific SM α-actin expression during normal development and maturation. (A) Analysis of LacZ expression in whole adult tissues of WT and SRE-AB transgenic mice indicated that the SRE substitutions had no effect on transgene expression in adult tissues. Sm. int., small intestine. (B) Analysis of LacZ expression in E16.5 whole embryos from wild-type and SRE-AB transgenic mice indicated no loss of specificity upon replacement of SM α-actin CArG-A and CArG-B with the c-fos SRE consensus CArG. (C) Histological analyses of adult tissues indicated no loss of SMC-specific SM α-actin expression in SRE-substituted transgenic mice. Tissues were paraffin-embedded, sectioned, and stained with H&E. LacZ expression was specific to SMCs in wild-type aorta and in aortas of SRE-AB transgenic mice across multiple independent founder lines. Magnification, ×40. In SRE-substituted transgenic mice, the same LacZ expression pattern was found in all SMC-containing tissues examined, including esophagus and small intestine (data not shown). (D) Histological examination of LacZ- and eosin-stained aortas of E16.5 embryos from wild-type and SRE-substituted transgenic mice indicated no loss of specificity in the mutants. LacZ expression was restricted to the SMC-containing layers of all tissues examined, including the aorta (magnification, ×40), esophagus, bronchi, and small intestine (data not shown).

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ISSN: 0021-9738 (print), 1558-8238 (online)

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