title: DNA methylation of a GC repressor element in the smooth muscle myosin heavy chain promoter facilitates binding of the Notch-associated transcription factor, RBPJ/CSL1.
abstract: OBJECTIVE
  The goal of the present study was to identify novel mechanisms that regulate smooth muscle cell (SMC) differentiation marker gene expression.
  
  
  RESULTS
  We demonstrate that the CArG-containing regions of many SMC-specific promoters are imbedded within CpG islands. A previously identified GC repressor element in the SM myosin heavy chain (MHC) promoter was highly methylated in cultured aortic SMC but not in the aorta, and this difference was inversely correlated with SM MHC expression. Using an affinity chromatography/mass spectroscopy-based approach, we identified the multifunctional Notch transcription factor, recombination signal binding protein for immunoglobulin κ J region (RBPJ), as a methylated GC repressor-binding protein. RBPJ protein levels and binding to the endogenous SM MHC GC repressor were enhanced by platelet-derived growth factor-BB treatment. A methylation mimetic mutation to the GC repressor that facilitated RBPJ binding inhibited SM MHC promoter activity as did overexpression of RBPJ. Consistent with this, knockdown of RBPJ in phenotypically modulated human aortic SMC enhanced endogenous SMC marker gene expression, an effect likely mediated by increased recruitment of serum response factor and Pol II to the SMC-specific promoters. In contrast, the depletion of RBPJ in differentiated transforming growth factor-β-treated SMC inhibited SMC-specific gene activation, supporting the idea that the effects of RBPJ/Notch signaling are context dependent.
  
  
  CONCLUSIONS
  Our results indicate that methylation-dependent binding of RBPJ to a GC repressor element can negatively regulate SM MHC promoter activity and that RBPJ can inhibit SMC marker gene expression in phenotypically modulated SMC. These results will have important implications on the regulation of SMC phenotype and on Notch-dependent transcription.
authors.affiliation: From the Department of Pathology, University of North Carolina, Chapel Hill.
authors.forename: Julian M
authors.initials: JM
authors.lastname: Rozenberg
authors.affiliation: From the Department of Pathology, University of North Carolina, Chapel Hill.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Tesfu
authors.affiliation: From the Department of Pathology, University of North Carolina, Chapel Hill.
authors.forename: Srilaxmi
authors.initials: S
authors.lastname: Musunuri
authors.affiliation: From the Department of Pathology, University of North Carolina, Chapel Hill.
authors.forename: Joan M
authors.initials: JM
authors.lastname: Taylor
authors.affiliation: From the Department of Pathology, University of North Carolina, Chapel Hill. cmack@med.unc.edu.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Mack
mesh_terms: D000818:Animals; D001483:Base Sequence; D000077214:Becaplermin; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D020862:GC Rich Sequence; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020574:Proto-Oncogene Proteins c-sis / Q000378:metabolism; D051880:Receptors, Notch / Q000378:metabolism; D015398:Signal Transduction; D024745:Smooth Muscle Myosins / Q000235:genetics*
chemical_list: D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D020574:Proto-Oncogene Proteins c-sis; C497570:RBPJ protein, human; C497571:Rbpj protein, mouse; D051880:Receptors, Notch; D000077214:Becaplermin; D024745:Smooth Muscle Myosins; D018995:Myosin Heavy Chains
keywords: RBPJ protein, human; epigenetics; muscle, smooth; serum response factor