Control of SRF binding to CArG box chromatin regulates smooth muscle gene expression in vivo
Oliver G. McDonald, … , Mark H. Hoofnagle, Gary K. Owens
Oliver G. McDonald, … , Mark H. Hoofnagle, Gary K. Owens
Published January 4, 2006
Citation Information: J Clin Invest. 2006;116(1):36-48. https://doi.org/10.1172/JCI26505.
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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 3

Identification modifications that contribute to myocardin/SRF binding to CArG boxes.

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Identification modifications that contribute to myocardin/SRF binding to...
(A) α-SMA transgenes were stably transfected into rat aortic SMCs by the integrase system as described in Methods, and ChIP was performed with primers specific for the transgene and the endogenous gene. *P < 0.05 by Student’s t test. (B) SMCs were infected with adenovirus harboring CMV-myocardin (Myo) or CMV-empty (CMV) expression vectors, and ChIP was performed for SRF, H4Ac, and H3K4dMe. (C) SMCs were infected with adenovirus as in B. Elk-1, SRF, and FLAG-myocardin immunoprecipitates were subjected to Western blotting for H3K4dMe and SRF. Nonimmune IgG antisera failed to immunoprecipitate SRF and H3K4dMe from SMC extracts in these and all other protein IP experiments (data not shown and Supplemental Figure 2). (D) SMCs were infected as in B, and SRF immunoprecipitates were subjected to Western blotting for H3K4dMe and SRF. (E) SMCs were infected with adenoviruses expressing siRNAs to myocardin (siMyo) or GFP (siGFP; control). Chromatin was isolated, and ChIP measured levels of SRF binding to 5′-CArG boxes. (F) SMCs were infected as in E, and nuclear extracts were treated as in D. (G) Peptide binding assay with FLAG-myocardin as described in Methods. FLAG-myocardin immunoprecipitates collected from SMC extracts containing the corresponding biotinylated peptides were subjected to Western blotting using HRP-streptavidin. H3unmod, unmodified H3 peptide. (H) Myocardin peptide binding assay as in G, comparing the ability of myocardin to immunoprecipitate 2 μg of H3 peptides di-methylated at Lys4 (H3K4dMe), di-methylated at Lys9 (H3K9dMe), acetylated at Lys9, or phosphorylated at serine 10 (H3S10P).