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 SMC differentiation marker gene expression. Approach and 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 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, RBPJ/CSL1, as a methylated GC-repressor binding protein. RBPJ protein levels and binding to the endogenous SM MHC GC repressor were enhanced by PDGF-BB treatment. A methylation mimetic mutation to the GC repressor that facilitated RBPJ binding inhibited SM MHC promoter activity as did over-expression 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 SRF and Pol II to the SMC-specific promoters. In contrast, depletion of RBPJ in differentiated TGF-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.

RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

“…In contrast, RBPJ did bind to the CGGGA sequence, but only when this cytosine was methylated. We and others have shown that thymine and methylated cytosine can function interchangeably within an RBPJ binding site ( 14 , 32 ) most likely because both of these pyrimidines are methylated at the 5′ position. However, this was not the case for RBPJ binding to the MCR (Figure 1C compare lanes 4 and 6).…”

“…Our recent demonstration that RBPJ binds a methylated repressor element within the SM MHC promoter ( 14 ) suggested a novel mechanism for RBPJ-dependent gene regulation. To begin to examine this possibility, we identified RBPJ binding sites in HuAoSMCs using ChIP-seq approaches.…”

“…It is well known that SMCs are not terminally differentiated and under various stresses (including cell culturing) modulate their phenotype by repressing SMC differentiation marker gene expression. We have recently shown that down regulation of SM MHC expression in cultured SMCs was associated with progressive methylation of a GC rich repressor element (GCGGGA) within the SM MHC promoter ( 14 ). Importantly, using a non-biased affinity chromatography/mass spectroscopy-based approach, we identified RBPJ as a methylated GC-repressor binding protein and went on to show that structural similarities between methylated cytosine and thymine allow for RBPJ binding to this sequence.…”

Epigenetic Control of Smooth Muscle Cell Identity and Lineage Memory

“…Nevertheless, this study does not provide direct evidence that the hypomethylation phenomenon occurs in SMC and the full spectrum of genes impacted by the hypomethylation is unknown. Second , SMC marker genes including Tagln and Myh11 promoters are hypomethylated in SMC compared with 10T1/2 cells 67 . In addition, the Myh11 promoter in cultured SMC contains significantly more 5mC as compared to freshly isolated aortic SMC 67 .…”

“…Second , SMC marker genes including Tagln and Myh11 promoters are hypomethylated in SMC compared with 10T1/2 cells 67 . In addition, the Myh11 promoter in cultured SMC contains significantly more 5mC as compared to freshly isolated aortic SMC 67 . These latter observations suggest that dynamic modifications of DNA methylation patterns of SMC marker genes may contribute to changes in expression of these genes during phenotypic transitions, at least during adaptation of cells to culture.…”