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 1

Cell-specific histone modifications correlate with cell-selective binding of SRF to α-SMA and SM-MHC.

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Cell-specific histone modifications correlate with cell-selective bindin...
(A) Quantitative ChIP analysis for SRF enrichment at the 5′-CArG boxes of α-SMA, SM-MHC, and c-fos from chromatin isolated from rat aortic SMCs and ECs. (B) Chromatin isolated from SMCs and ECs was digested into mononucleosomal fragments by micrococcal (S7) nuclease. DNA was purified and amplified with primers flanking the 5′-CArG boxes of α-SMA, SM-MHC, and c-fos. The 5′-promoter region of the EC-specific gene VEC was also included as a control. The VEC promoter does not contain CArG boxes. See Methods for explanation. (C) Chromatin was isolated from rat aortas and blood and SRF binding measured by ChIP as in A. (D) Histone modifications that have been associated with activation of gene expression (25) were measured at α-SMA, VEC, cardiac-muscle myosin heavy chain (cardiac-specific; CM α-MHC), and c-fos promoters by ChIP in cultured ES cells (ESC), SMCs, and ECs. We observed patterns similar to α-SMA at SM-MHC (data not shown). All ChIP data for Figure 1 and other figures are plotted as fold enrichment over equivalent amounts of input DNA, and Figure 1D is scaled equally on each horizontal panel (i.e., for each modification) so that enrichments between each of the different promoters can be compared. *P < 0.05 measured by Student’s t test. Controls with beads only and without antibody consistently failed to immunoprecipitate DNA that amplified by real-time PCR (data not shown; see Supplemental Figure 2) for this and all other ChIP experiments. H3K79dMe, H3 Lys79 di-methylation; H3K9Ac, H3 Lys9 acetylation.