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 4

KLF4 promotes loss of SRF binding and H4Ac at α-SMA and SM-MHC.

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Characterization of transcription factor and histone modification distri...
(A) SMCs were infected with CMV-KLF4 (KLF4) or CMV-empty adenoviruses and SRF, H4Ac, and H3K4dMe measured by ChIP. *P < 0.05 by Student’s t test. Accessibility to micrococcal nuclease digestion was measured as in Figure 1B. (B) Cultured SMCs were infected with equivalent amounts of CMV-empty, CMV-myocardin, or CMV-myocardin with CMV-KLF4 (myo+KLF4) adenoviruses and SRF binding to CArG boxes measured by ChIP as above. (C, D, and F) ChIP and real-time RT-PCR were measured from cultured SMCs infected with the corresponding adenoviruses with TSA dissolved in DMSO at 1 ng/ml or 5 ng/ml or in DMSO only. *P < 0.05 for samples when compared with CMV-empty control cells by Student’s t test. (E) SRF was immunoprecipitated from SMCs infected with CMV-KLF4 or control (CMV) viruses, and immunoprecipitates were subjected to Western blotting for H3K4dMe and SRF (IP and IP control).