Control of SRF binding to CArG box chromatin regulates smooth muscle gene expression in vivo
Oliver G. McDonald, Brian R. Wamhoff, Mark H. Hoofnagle, Gary K. Owens
Oliver G. McDonald, Brian R. Wamhoff, Mark H. Hoofnagle, Gary K. Owens
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Research Article Genetics

Control of SRF binding to CArG box chromatin regulates smooth muscle gene expression in vivo

Abstract

Precise control of SMC transcription plays a major role in vascular development and pathophysiology. Serum response factor (SRF) controls SMC gene transcription via binding to CArG box DNA sequences found within genes that exhibit SMC-restricted expression. However, the mechanisms that regulate SRF association with CArG box DNA within native chromatin of these genes are unknown. Here we report that SMC-restricted binding of SRF to murine SMC gene CArG box chromatin is associated with patterns of posttranslational histone modifications within this chromatin that are specific to the SMC lineage in culture and in vivo, including methylation and acetylation to histone H3 and H4 residues. We found that the promyogenic SRF coactivator myocardin increased SRF association with methylated histones and CArG box chromatin during activation of SMC gene expression. In contrast, the myogenic repressor Kruppel-like factor 4 recruited histone H4 deacetylase activity to SMC genes and blocked SRF association with methylated histones and CArG box chromatin during repression of SMC gene expression. Finally, we observed deacetylation of histone H4 coupled with loss of SRF binding during suppression of SMC differentiation in response to vascular injury. Taken together, these findings provide novel evidence that SMC-selective epigenetic control of SRF binding to chromatin plays a key role in regulation of SMC gene expression in response to pathophysiological stimuli in vivo.

Authors

Oliver G. McDonald, Brian R. Wamhoff, Mark H. Hoofnagle, Gary K. Owens

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Figure 5

Myocardin and KLF4 exert opposing influences over SMC gene expression in transgenic mouse liver in vivo.

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Myocardin and KLF4 exert opposing influences over SMC gene expression in...
(A) mRNA was extracted from liver in mice infected with CMV-empty control viruses, CMV-KLF4, CMV-myocardin, or mice coinjected with myocardin and KLF4 viruses. Expression levels of myocardin and KLF4 were measured by real-time PCR to document that delivery of these genes was successful. Data were normalized to levels of 18S expression. (B) LacZ staining from SM-MHC-LacZ–transgenic mice injected as in A. For each panel in B, the top left image shows hearts expressing SMC-specific LacZ staining in coronary arteries (ca), and the top right image shows cross-sections taken from these mice displaying SMC-specific LacZ staining in the media (m) of aortas. The endothelial layer (e) and adventitia (adv) are labeled. The media exhibit mosaic staining, which is typical for SM-MHC–transgenic mice. The bottom image in each panel shows staining for the presence of LacZ in mouse liver. (C) mRNA levels of α-SMA and SM-MHC were measured by real-time RT-PCR, and SRF binding to CArG box chromatin of these genes was measured by ChIP, in livers of mice injected with the indicated corresponding adenoviruses.