Basic Sciences

DNA Methylation of a GC Repressor Element in the Smooth Muscle Myosin Heavy Chain Promoter Facilitates Binding of the Notch-Associated Transcription Factor, RBPJ/CSL1Significance

Julian M. Rozenberg, Daniel B. Tesfu, Srilaxmi Musunuri, Joan M. Taylor, Christopher P. Mack
https://doi.org/10.1161/ATVBAHA.114.304634
Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:2624-2631
Originally published October 16, 2014

Jump to

Abstract

Objective—The goal of the present study was to identify novel mechanisms that regulate smooth muscle cell (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 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.

Introduction

It has been well established that serum response factor (SRF) and the myocardin family of SRF cofactors mediate smooth muscle cell (SMC)–specific transcription by interacting with conserved CArG elements within the promoters of the SMC differentiation marker genes.1 However, because these transcription factors are expressed in many non-SMC subtypes,2 it is clear that additional mechanisms are also critical for the overall pattern of SMC-specific gene expression observed in vivo.

One mechanism that is likely to be important in the regulation of SMC-specific transcription is the modification of chromatin structure. Histone modifications that favor gene expression (ie, H3 and H4 acetylation, H3K4 methylation, and H3K9 demethylation/acetylation) have been observed at the SMC-specific promoters in SMC.3–6 In addition, we and others have shown that the myocardin factors can facilitate chromatin modification by recruiting histone-modifying enzymes.5,7–11

Another epigenetic mechanism that has received less attention in regard to its effects on SMC-specific transcription is DNA methylation. This epigenetic mark is predominantly associated with gene silencing and has been shown to be important for a wide variety of cellular functions, including genomic imprinting, X-inactivation, cellular differentiation, and carcinogenesis.12 DNA methylation is catalyzed by a family of DNA methyltransferases and typically occurs on cytosines at the 5 position of the pyrimidine ring and in the context of CpG dinucleotides (C followed by G). The majority of CpGs within the genome are dispersed and methylated. However, many gene promoters, especially those of highly expressed house-keeping genes, contain regions of high CpG content known as CpG islands that are typically unmethylated. Methylated cytosines are thought to suppress gene expression by sterically inhibiting transcription factor binding to cis regulatory elements or by associating with methyl binding domain-containing proteins (MBDs1–4, MeCP2, Kaiso) that recruit additional transcription repressors. Interestingly, DNA regions that are heavily methylated are also associated with high levels of trimethyl H3K9, and these negative chromatin marks act cooperatively by the reciprocal recruitment of their respective methyltransferases.13

On the basis of our previous demonstration that H3K9 methylation status at the CArG-containing regions of the SMC-specific promoters was an important determinant of SMC differentiation marker gene expression,7 we hypothesized that DNA methylation may also play a role. Our results indicate that methylation of a GC repressor in the SM myosin heavy chain (MHC) promoter inversely correlates with SM MHC expression in aortic SMC, that the methylated GC repressor recruits the multifunctional transcription factor recombination signal binding protein for immunoglobulin κ J region (RBPJ)/CSL-1, and that RBPJ can inhibit SMC marker gene expression in phenotypically modulated human aortic SMC.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

SM MHC Expression in SMC Inversely Correlates With Promoter Methylation

To begin to examine whether DNA methylation plays a role in the regulation of SMC-specific gene expression, we searched for CpG islands within the SMC differentiation marker gene promoters using the formal definition (sequence of ≥200 bp in length, a GC content of >50%, and an observed:expected CpG ratio >60%). In both human and mouse, the CArG-containing regions shown to be important for SM MHC, SM22, and calponin expression were imbedded within CpG islands, suggesting that methylation might regulate the SRF/myocardin factor–dependent activation of these genes. We used standard bisulfite sequencing to measure CpG island methylation within the SM22 and SM MHC promoters in primary mouse aortic SMC cultures and in mouse 10T1/2 cells, a pluripotent line frequently used as a SMC precursor. As shown in Figure 1A, the CpG island within the SM22 promoter was highly methylated in 10T1/2 cells but almost completely unmethylated in SMC. This result correlates well with SM22 expression levels in these cell types and suggests that promoter methylation may play a role in the regulation of SM22 expression. The CpG island within the proximal SM MHC promoter was also highly methylated in 10T1/2 cells (Figure 1B). However, we also detected significant methylation of the SM MHC promoter in SMC. Because the SM MHC gene is the first differentiation marker to be downregulated in phenotypically modulated SMC, we hypothesized that the observed methylation of the SM MHC promoter reflected phenotypic modulation of our cultured SMC. To test this idea, we measured SM MHC promoter methylation in freshly isolated aortic media after removal of the adventitial and endothelial cell layers by collagenase/elastase digestion and microdissection. The SM MHC CpG island was completely unmethylated in aortic medial SMC in vivo, and this result correlated with much stronger expression of SM MHC as measured by Western blotting of lysates prepared from the same samples (Figure 1C).

Figure 1.
Figure 1.

A GC repressor element in the mouse smooth muscle (SM) myosin heavy chain (MHC) promoter is methylated in cultured SMC. Schematic of CpG island methylation within the CArG-containing regions of the SM22 (A) and SM MHC (B) promoters as determined by bisulfite sequencing of genomic DNA isolated from 10T1/2 cells, mouse aortic SMC cultures, and mouse aorta. C, Western blot for SM MHC expression in the indicated cells and tissues. D, The indicated radiolabeled mouse SM MHC GC repressor probes were incubated with nuclear extracts prepared from mouse aortic SMC treated for 24 hours with vehicle, platelet-derived growth factor -BB (20 ng/mL), or transforming growth factor (TGF)-β (1 ng/mL), and from control (Ctl) or injured (Inj) femoral arteries. After 30 minutes, reactions were run on a 5% nondenaturing polyacrylamide gel, which was then dried and exposed to film.

Several CpGs within the SM MHC promoter were almost completely methylated in cultured SMC. One of these was present within a GC-rich sequence just downstream of CArG2 that had been previously characterized as a repressor element.14 Deletion of a similar but not identical GC repressor within the SM22 promoter prevented the downregulation of SM22 promoter activity that occurs after vessel injury.15,16 The GC repressor within the SM MHC promoter was also fully methylated in mouse endothelial cells (data not shown) perhaps suggesting that this mechanism is important for the repression of SM MHC expression in other cell types in the vessel wall.

Because the effects of promoter methylation are thought to be mediated by alterations in protein–DNA binding, we performed gel shift analyses with methylated and unmethylated mouse SM MHC GC repressor probes and nuclear extracts isolated from SMC cultures. Importantly, the major protein complex that bound to the methylated GC repressor did not bind to the unmethylated probe (Figure 1D, compare lanes 1 and 4). In addition, complex binding to the methylated repressor was increased when extracts were prepared from cells treated with platelet-derived growth factor (PDGF)-BB, an agonist known to downregulate SMC differentiation marker gene expression. In contrast, treatment of cells with transforming growth factor (TGF)-β that activates SMC differentiation marker gene expression had little effect on complex formation. To examine the formation of this methylation-dependent complex in vivo, we performed gel shift assays using nuclear extracts prepared from control and wire-injured carotid arteries. A similar methylation-dependent binding complex was detected and was significantly increased in extracts prepared from injured arteries (Figure 1D, compare lanes 7 and 8). We initially hypothesized that this complex contained one of the known methylated DNA-binding proteins, but we were unable to supershift this band with antibodies to these factors. Several additional bands were detected in some gel shift assays, but these were either not methylation-dependent or were mostly nonspecific (Figure 2, data not shown).

Figure 2.
Figure 2.

The methylated smooth muscle (SM) myosin heavy chain (MHC) GC repressor binds recombination signal binding protein for immunoglobulin κ J region (RBPJ). A, The indicated oligonucleotide probes were incubated with SM cell nuclear extracts +/− an antibody for RBPJ. After 30 minutes, reactions were run on a 5% nondenaturing polyacrylamide gel, which was then dried and exposed to film. ns, nonspecific band. B, The indicated nonradiolabeled competitor oligonucleotide was added in increasing concentrations to gel shift reactions containing the methylated GC repressor probe and SMC nuclear extracts.

RBPJ Binds the Methylated SM MHC GC Repressor

We next used agarose beads conjugated to the methylated GC repressor to affinity purify the methylation-dependent protein-binding complex from SMC nuclear extracts. Proteins that precipitated with the methylated, but not the unmethylated probe, were cut out of SDS-PAGE gels and sent for mass spectrometric analysis. Each of 3 separate experiments identified the multifunctional Notch transcription factor, RBPJ, as a methylated GC repressor–binding protein. Additional gel shifts demonstrated that the major complex that bound the methylated GC repressor was similar to that formed when using a consensus RBPJ oligonucleotide probe (Figure 2A; compare lanes 1 and 5). Moreover, addition of an RBPJ antibody to gel shift reactions resulted in a complete supershift providing conclusive evidence for the presence of RBPJ in this complex (Figure 2A; compare lanes 1 and 2). As shown in Figure 2B, RBPJ binding to the methylated GC repressor was specific in that it was completely abolished by addition of cold methylated probe (or the consensus RBPJ element) but not by cold unmethylated probe.

Notch signaling plays an important role in vascular development and maintenance by regulating cell fate decisions in both endothelial cells and SMC.17 Activation of the integral membrane Notch receptors by Delta-like or Jagged ligands results in proteolytic cleavage of the receptor by γ-secretase, release of the Notch intracellular domain (NICD), and translocation of the NICD to the nucleus where it interacts with RBPJ. In the absence of NICD, RBPJ binds a consensus site (GTGGGA) within the promoters of Notch target genes and has been shown to inhibit gene expression by recruiting histone deactylases and other transcriptional repressors. NICD binding to RBPJ displaces the repressive factors (resulting in derepression) but also aids in the recruitment of additional transcription activators. Although Notch signaling has been shown to be required for SMC differentiation of neural crest cells, epicardial cells, or Tie1-expressing progenitor cells in vivo,18–20 the direct effects of Notch/RBPJ signaling on SMC differentiation marker gene expression are relatively weak,21–23 are significantly complicated by feedback inhibition mediated by the Notch target genes, Hes and Hey,24 and are context dependent with several studies showing both positive and negative effects.21–23,25–27

Characterization of RBPJ Binding to Methylated DNA

We noted that the reverse strand of the GC-rich element (GCGGGA) differed from the consensus core RBPJ-binding site by only 1 base pair, a thymine to cytosine substitution. Additional gel shift assays with hemimethylated probes demonstrated that only methylation of the reverse strand was required for RBPJ binding (Figure 3A). Because the pyrimidine ring of thymine is constitutively methylated at the 5' position (Figure 3B), we hypothesized that methylation of cytosine conferred enough structural similarity between these nucleotides to facilitate RBPJ binding. Supporting this idea, a GC repressor containing a cytosine to thymine substitution was shown to interact with RBPJ (Figure 3C, lane 3). Conversely, RBPJ did not bind well to a consensus sequence in which the thymine was replaced by cytosine, but did bind strongly to a consensus probe in which the thymine was replaced with a methylated cytosine (Figure 3C, compare lanes 7 and 6).

Figure 3.
Figure 3.

Analysis of the recombination signal binding protein for immunoglobulin κ J region (RBPJ)–DNA binding complex. A, Gel shift analysis of RBPJ binding to fully and hemimethylated GC repressor probes. B, Comparison between consensus and GC repressor RBPJ-binding sites and thymine and methylated cytosine structures. C, Gel shift reactions using the indicated variant of the GC repressor or consensus RBPJ probes. Materials and Methods are available in the online-only Supplement for full oligonucleotide sequences. D, Wild-type (Wt) and E63D RBPJ variants were expressed in vitro and used in gel shift reactions with Wt and methylated (T to Cm) consensus RBPJ probes. Note equal expression of each RBPJ variant as measured by Western blotting for the flag epitope.

The crystal structure of RBPJ bound to its consensus DNA element has been solved.28,29 Several conserved polar amino acids in the RBPJ DNA-binding pocket (especially Arg 65) were shown to interact with the 3 central guanine residues at least partially explaining the specificity of RBPJ binding to the consensus sequence. Interestingly, no such interactions were detected for the consensus thymine supporting our contention that binding specificity at this residue is mediated by the presence of a methyl group at the pyrimidine 5' position. Although Glu63 is the nearest amino acid to this thymine residue, the 5' methyl group is positioned closer to the carbon backbone of the glutamate side chain than to the polar head group. As shown in Figure 3D, a conservative Glu63Asp mutation almost completely inhibited RBPJ binding to the consensus probe and the same probe in which the thymine was replaced by methylated cytosine. Taken together, these data strongly suggest that the positioning of the carboxyl group of Glu63 is critical for the formation of a methyl binding pocket that stabilizes the RBPJ–DNA interaction.

RBPJ Negatively Regulates SM MHC Promoter Activity

We next used several gain-of-function/loss-of-function approaches to examine the effects of RBPJ on SMC-specific promoter activity. As shown in Figure 4A, overexpression of RBPJ in 10T1/2 cells significantly inhibited the activities of multiple SMC-specific promoters in the presence of myocardin. We also established SMC cultures from RBPJflox/flox mice and used adenoviral-mediated expression of Cre recombinase to reduce RBPJ expression and DNA binding in these cells by ≈85% (Figure 4B). Importantly, SM MHC promoter activity in SMC overexpressing myocardin was significantly higher in Cre versus LacZ-infected SMC (Figure 4C). Although these results strongly suggest that RBPJ can function as an inhibitor in these contexts, the lack of reagents to the alter the methylation of specific CpGs has made it difficult to study the functional effects of this modification. However, our ability to promote RBPJ binding to the GC repressor by a thymine substitution that mimics cytosine methylation (Figure 3C) allowed us to assess the functional significance of this interaction. A cytosine to thymine substitution within the GC repressor significantly attenuated myocardin-dependent SM MHC promoter activity supporting our hypothesis that methylation-dependent binding of RBPJ to the GC repressor inhibits SM MHC promoter activity. Chromatin immunoprecipitation (ChIP) assays in our mouse SMC cultures demonstrated that RBPJ binds to the endogenous SM MHC GC repressor (Figure 5A), and in excellent agreement with our gel shift assays, that this interaction was enhanced by PDGF-BB treatment. As shown in Figure 5B, PDGF-BB treatment increased RBPJ protein levels in both SMC and 10T1/2 cells. Given that methylation of the GC repressor is already high in our cultured SMC, this result likely explains the increase in RBPJ binding to the GC repressor in PDGF-BB–treated cells.

Figure 4.
Figure 4.

Recombination signal binding protein for immunoglobulin κ J region (RBPJ) inhibits smooth muscle (SM) myosin heavy chain (MHC) promoter activity. A, 10T1/2 cells were transfected with SM22, SM α-actin, SM22 or minimal thymidine kinase (TK) reporter/luciferase constructs +/−myocardin and +/−RBPJ expression vectors. The total amount of expression vector in each well was equalized by addition of empty vector (ev). Luciferase activity was measured at 48 hours and is expressed relative to promoter activity plus empty vector only (Con). *P<0.05 vs control. **P<0.05 vs plus myocardin. B, Western blot and gel shift analyses demonstrating RBPJ knockdown in our RBPJflox/flox SMC model. C, A mutation (C to T) within the GC repressor that facilitates RBPJ binding was made in the context of the SM MHC promoter shown. SMC isolated from RBPJflox/flox mice were treated with Cre- or LacZ-expressing adenovirus for 48 hours and then transfected with the wild-type (Wt) and T mutant constructs. Luciferase activity was measured at 48 hours. *P<0.05 vs Wt. **P<0.05 vs LacZ.

Figure 5.
Figure 5.

Recombination signal binding protein for immunoglobulin κ J region (RBPJ) binds the endogenous smooth muscle (SM) myosin heavy chain (MHC) repressor. A, Chromatin immunoprecipitation assays for RBPJ were performed in control and platelet-derived growth factor (PDGF)-BB–treated SMC using primers spanning the GC repressor-containing region of the SM MHC promoter. B, Western blot for RBPJ expression in mouse aortic SMC and 10T1/2 cells treated with PDGF-BB and transforming growth factor (TGF)-β. The chromatin immunoprecipitation and Western Blot results shown are representative of ≥3 independent experiments.

RBPJ Has Dual Effects on SMC-Specific Expression

The ability of Notch to stimulate SMC differentiation has been attributed to the recruitment of the NICD to RBPJ binding sites within the SMC-specific promoters.21–23 However, our demonstration that RBPJ binds to methylated DNA, is upregulated by PDGF-BB and vessel injury, and can inhibit SMC-specific promoter activity, led us to hypothesize that RBPJ can also function as a repressor in phenotypically modulated SMC. To begin to test this idea, we used siRNA to knockdown RBPJ in a human aortic SMC line that exhibits low levels of the SMC marker genes under basal growth conditions but high levels when treated with TGF-β. As shown in Figure 6A, RBPJ bound to the CArG-containing regions of the SM MHC, SM α-actin, calponin, and SM22 genes in human aortic SMC and treatment of these cells with siRNA significantly inhibited RBPJ binding in this model. Importantly, depletion of RBPJ under growth conditions resulted in an increase in SMC marker gene mRNA levels, suggesting that RBPJ functions as a repressor in this context (Figure 6B, undiff). Given the positioning of the GC repressor and other RBPJ-binding sites to CArG elements in the SM MHC and other promoters,22,23 we postulated that RBPJ might interfere with SRF binding under these conditions. Indeed, ChIP assays demonstrated that SRF binding to the SMC promoters was increased in RBPJ-depleted cells (Figure 6C) even though SRF protein levels were not affected (Figure I in the online-only Data Supplement). Moreover, the increase in SMC marker gene mRNA in RBPJ knockdown cells was completely inhibited by codepletion of SRF (Figure 6D). As expected, the positive effects of RBPJ depletion on SMC-specific gene expression and SRF binding were accompanied by the increased recruitment of RNA polymerase II.

Figure 6.
Figure 6.

recombination signal binding protein for immunoglobulin κ J region (RBPJ) inhibits smooth muscle cell (SMC) differentiation marker gene expression in phenotypically modulated human SMC. A, Chromatin immunoprecipitation (ChIP) assays for RBPJ binding to the indicated promoters in control and RBPJ knockdown human aortic SMC. B, RNA was isolated from control and RBPJ knockdown human aortic SMC that were maintained in growth media (undiff) or treated with transforming growth factor (TGF)-β. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for the indicated SMC marker genes and GAPDH. Results of RT-PCR using 5 and 45 ng of RNA from TGF-β–treated cells are shown for quantification. C, ChIP assays for SRF and RNA Pol II binding in control and RBPJ knockdown SMC under growth or TGF-β–treated conditions. D, RT-PCR of SMC marker gene expression in RBPJ knockdown, SRF knockdown, and RBPJ/SRF double knockdown human SMC under growth conditions. Notch3 binding (E) and H3K9 acetylation (F) at the SMC-specific promoters were measured by ChIP assay in control and RBPJ knockdown human aortic SMC treated with TGF-β. The RT-PCR and ChiP results shown are representative of ≥3 independent experiments.

In contrast, depletion of RBPJ from TGF-β–treated human aortic SMC had an inhibitory effect on SMC-specific transcription. Although SRF binding in these cells (which was much stronger than that observed under growth conditions) was mostly unchanged, RBPJ depletion strongly attenuated the presence of RNA polymerase II, suggesting that recruitment of transcriptional cofactors or chromatin-modifying enzymes was affected. In support of this idea, additional ChIP assays demonstrated that Notch3 binding (Figure 6E) and H3K9 acetylation (Figure 6F) were decreased in RBPJ-depleted cells under these conditions.

Discussion

The current studies indicate that methylation-dependent recruitment of RBPJ to a GC repressor element inhibits SM MHC promoter activity and that RBPJ can inhibit SMC differentiation marker gene expression in phenotypically modulated SMC by inhibiting SRF binding to the CArG-containing regions of the SMC-specific promoters. Interestingly, RBPJ was required for full SMC marker gene activation in TGF-β–treated cells, a result consistent with the requirement of Notch signaling for SMC differentiation in vivo.18–20 It is likely that inhibition of NICD recruitment under these conditions decreased positive chromatin remodeling and Pol II binding leading to reductions in SMC marker gene expression. Taken together, our results support the current model of Notch signaling in which RBPJ inhibits gene expression under unstimulated conditions but is required for recruitment of the NICD and the positive transcriptional effects of Notch signaling.

Although DNA methylation can promote long-term and heritable gene inactivation,30 its role in the regulation of cell-type–specific gene expression is less clear30–37 and may be related to the number and methylation status of CpGs within a particular promoter.37–39 Because de novo DNA methylation is catalyzed by DNA methyltranferases 3a and 3b, it will be important to identify the mechanisms by which these enzymes are recruited to the SM MHC promoter in cultured SMC. We have previously shown that H3K9 methylation at the CArG-containing regions of the SMC-specific promoters was associated with decreased SM MHC expression,7 and we are currently investigating whether this histone modification precedes or promotes SM MHC promoter methylation. Another interesting question is whether changes in DNA methylation regulate SMC-specific gene expression more acutely. We observed modest reductions in SM MHC promoter methylation during TGF-β–induced upregulation of SM MHC expression in 10T1/2 cells (Figure II in the online-only Data Supplement). However, the known heterogeneity in these cells is a confounding factor and additional experiments in the subset of cells that upregulate SM MHC expression will likely be required to demonstrate that SM MHC promoter methylation is responsive to TGF-β. The failure to identify a robust DNA demethylation regulatory pathway has also hindered our understanding of the temporal control of DNA methylation. Enzymes that facilitate the demethylation reaction have been identified,40,41 suggesting that DNA methylation (like histone methylation) is more dynamic than previously thought.42 Several studies provide support for the idea that SMC differentiation marker gene expression is regulated by DNA methylation. For example, the decrease in SM22 expression observed in human SMCs treated with high phosphate was associated with increased SM22 promoter methylation,43 whereas SM22 methylation was decreased in multipotential adventitial cells that were induced to differentiate into SMC.44 Hu et al45 have also shown that SM α-actin expression in fibroblasts inversely correlated with DNA methylation near the SM α-actin transcription start site.

Using an unbiased SILAC-based biochemical screen, Bartels et al46 were to show that RBPJ interacted with methylated DNA. Our data strongly confirm that RBPJ binds to the sequence, GCmGGGA, and we have significantly extended this finding by providing functional significance to the interaction of RBPJ with methylated DNA and by identifying SM MHC as an in vivo target for this mechanism. It is currently unclear whether RBPJ interacts with other gene promoters through a methylation-dependent mechanism or whether RBPJ binding to a methylated versus unmethylated sequence results in different transcriptional outcomes. Our gel shift data indicate that the methylated GC repressor can support the formation of a RBPJ/Notch complex in vitro (Figure III in the online-only Data Supplement), but it is unknown whether the interaction of RBPJ with repressive cofactors is affected. Defining the relationship between multiple RBPJ binding sites within a single promoter will also be important. As shown in Figure IV in the online-only Data Supplement, we did not detect significant binding of RBPJ to the SM22 GC repressor in gel shift assays most likely because it does not conform to the consensus RBPJ-binding site even when methylated. Recent studies indicated that the effects of the SM22 GC repressor are mediated by the pluripotency factor, KLF4,16 and it may be interesting to test whether KLF4 binding to this element is affected by DNA methylation status.

Structural analyses of several zinc finger transcription factors that bind to methylated DNA identified a 5mCytosine-Arg-Guanine triad that mediates this interaction,47 and our data strongly support this mechanism. In this model, hydrogen bonding of Arg65 with the guanine residue at position 3 of the consensus RBPJ-binding sequence promotes van der Waals contacts between the Arg65 guanidino carbon moiety and the methyl group of the nucleotide at position 2 (whether 5mC or T). Our data also suggest that Glu63 is important for RBPJ binding to DNA, a result in excellent agreement with a recent human genetic study that identified a Glu63Gly mutation in RBPJ that was casual for Adams–Oliver syndrome, a disease that affects limb and cranium formation.48 Interestingly, the methylated DNA-binding proteins, Kaiso and Zfp57, have similarly positioned Glu residues47 further supporting the idea that Glu63 is critical for RBPJ binding to the methylated pyrimidine ring.

Several recent studies have used ChIP seq analyses to identify RBPJ and Notch-binding sites on a genome-wide level in T-lymphoblastic leukemia cells, mouse E13.5 neural cortices, and C2C12 cells.49–51 Although Notch/RBPJ binding to the SMC marker gene promoters was not detected in these assays, several interesting findings are worth noting. Many RBPJ-only and Notch-only binding sites were identified, suggesting that these transcription factors have independent effects and perhaps that Notch can be recruited to DNA by transcription factors other than RBPJ. In addition, Notch/RBPJ binding was a poor predictor of gene activation. In fact, only 3% of the genes that were shown to bind Notch exhibited significant expression changes on Notch activation strongly suggesting that additional transcription mechanisms are important for regulating Notch/RBPJ-dependent gene expression. Finally, bioinformatic analysis of over-represented sequences in the RBPJ ChIP seq data set from neuronal cells failed to identify the consensus RBPJ element but did identify a GC-rich sequence similar to the GC repressor. Additional studies that also incorporate methylation status and changes in gene expression in knockout cells will be required to characterize the extent to which promoter methylation regulates Notch/RBPJ-dependent gene activation or repression.

In summary, our results indicate that RBPJ can inhibit SMC marker gene expression in phenotypically modulated SMC and that methylation-dependent recruitment of RBPJ may facilitate this repressive mechanism. These results have significant implications on our understanding of RBPJ/Notch-dependent regulation of cardiovascular development and disease and support further characterization of this mechanism and its consequences.

Sources of Funding

This work was supported by National Institutes of Health grants HL070953 and HL109607 (C.P. Mack).

Disclosures

None.

Footnotes

  • The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304634/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    ChIP
    chromatin immunoprecipitation
    MHC
    myosin heavy chain
    NICD
    Notch intracellular domain
    PDGF
    platelet-derived growth factor
    RBPJ
    recombination signal binding protein for immunoglobulin kappa J region
    SMC
    smooth muscle cell
    SRF
    serum response factor
    TGF
    transforming growth factor

  • Received February 10, 2014.
  • Accepted October 5, 2014.

References

  1. 1.↵
    1. Mack CP
    . Signaling mechanisms that regulate smooth muscle cell differentiation. Arterioscler Thromb Vasc Biol. 2011;31:1495–1505.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    1. Wang DZ,
    2. Li S,
    3. Hockemeyer D,
    4. Sutherland L,
    5. Wang Z,
    6. Schratt G,
    7. Richardson JA,
    8. Nordheim A,
    9. Olson EN
    . Potentiation of serum response factor activity by a family of myocardin-related transcription factors. Proc Natl Acad Sci U S A. 2002;99:14855–14860.
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Qiu P,
    2. Li L
    . Histone acetylation and recruitment of serum responsive factor and CREB-binding protein onto SM22 promoter during SM22 gene expression. Circ Res. 2002;90:858–865.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    1. Manabe I,
    2. Owens GK
    . CArG elements control smooth muscle subtype-specific expression of smooth muscle myosin in vivo. J Clin Invest. 2001;107:823–834.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. McDonald OG,
    2. Wamhoff BR,
    3. Hoofnagle MH,
    4. Owens GK
    . Control of SRF binding to CArG box chromatin regulates smooth muscle gene expression in vivo. J Clin Invest. 2006;116:36–48.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Manabe I,
    2. Owens GK
    . Recruitment of serum response factor and hyperacetylation of histones at smooth muscle-specific regulatory regions during differentiation of a novel P19-derived in vitro smooth muscle differentiation system. Circ Res. 2001;88:1127–1134.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Lockman K,
    2. Taylor JM,
    3. Mack CP
    . The histone demethylase, Jmjd1a, interacts with the myocardin factors to regulate SMC differentiation marker gene expression. Circ Res. 2007;101:e115–e123.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Cao D,
    2. Wang Z,
    3. Zhang CL,
    4. Oh J,
    5. Xing W,
    6. Li S,
    7. Richardson JA,
    8. Wang DZ,
    9. Olson EN
    . Modulation of smooth muscle gene expression by association of histone acetyltransferases and deacetylases with myocardin. Mol Cell Biol. 2005;25:364–376.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Nissen LJ,
    2. Gelly JC,
    3. Hipskind RA
    . Induction-independent recruitment of CREB-binding protein to the c-fos serum response element through interactions between the bromodomain and Elk-1. J Biol Chem. 2001;276:5213–5221.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Zhou J,
    2. Zhang M,
    3. Fang H,
    4. El-Mounayri O,
    5. Rodenberg JM,
    6. Imbalzano AN,
    7. Herring BP
    . The SWI/SNF chromatin remodeling complex regulates myocardin-induced smooth muscle-specific gene expression. Arterioscler Thromb Vasc Biol. 2009;29:921–928.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Zhang M,
    2. Fang H,
    3. Zhou J,
    4. Herring BP
    . A novel role of Brg1 in the regulation of SRF/MRTFA-dependent smooth muscle-specific gene expression. J Biol Chem. 2007;282:25708–25716.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. Klose RJ,
    2. Bird AP
    . Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci. 2006;31:89–97.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Rottach A,
    2. Leonhardt H,
    3. Spada F
    . DNA methylation-mediated epigenetic control. J Cell Biochem. 2009;108:43–51.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Madsen CS,
    2. Hershey JC,
    3. Hautmann MB,
    4. White SL,
    5. Owens GK
    . Expression of the smooth muscle myosin heavy chain gene is regulated by a negative-acting GC-rich element located between two positive-acting serum response factor-binding elements. J Biol Chem. 1997;272:6332–6340.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Wamhoff BR,
    2. Hoofnagle MH,
    3. Burns A,
    4. Sinha S,
    5. McDonald OG,
    6. Owens GK
    . A G/C element mediates repression of the SM22alpha promoter within phenotypically modulated smooth muscle cells in experimental atherosclerosis. Circ Res. 2004;95:981–988.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Salmon M,
    2. Gomez D,
    3. Greene E,
    4. Shankman L,
    5. Owens GK
    . Cooperative binding of KLF4, pELK-1, and HDAC2 to a G/C repressor element in the SM22α promoter mediates transcriptional silencing during SMC phenotypic switching in vivo. Circ Res. 2012;111:685–696.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Gridley T
    . Notch signaling in the vasculature. Curr Top Dev Biol. 2010;92:277–309.
    OpenUrlCrossRefPubMed
  18. 18.↵
    1. Grieskamp T,
    2. Rudat C,
    3. Lüdtke TH,
    4. Norden J,
    5. Kispert A
    . Notch signaling regulates smooth muscle differentiation of epicardium-derived cells. Circ Res. 2011;108:813–823.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. High FA,
    2. Zhang M,
    3. Proweller A,
    4. Tu L,
    5. Parmacek MS,
    6. Pear WS,
    7. Epstein JA
    . An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation. J Clin Invest. 2007;117:353–363.
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Chang L,
    2. Noseda M,
    3. Higginson M,
    4. Ly M,
    5. Patenaude A,
    6. Fuller M,
    7. Kyle AH,
    8. Minchinton AI,
    9. Puri MC,
    10. Dumont DJ,
    11. Karsan A
    . Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling. Proc Natl Acad Sci U S A. 2012;109:6993–6998.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Tang Y,
    2. Urs S,
    3. Liaw L
    . Hairy-related transcription factors inhibit Notch-induced smooth muscle alpha-actin expression by interfering with Notch intracellular domain/CBF-1 complex interaction with the CBF-1-binding site. Circ Res. 2008;102:661–668.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Noseda M,
    2. Fu Y,
    3. Niessen K,
    4. Wong F,
    5. Chang L,
    6. McLean G,
    7. Karsan A
    . Smooth Muscle alpha-actin is a direct target of Notch/CSL. Circ Res. 2006;98:1468–1470.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    1. Doi H,
    2. Iso T,
    3. Sato H,
    4. Yamazaki M,
    5. Matsui H,
    6. Tanaka T,
    7. Manabe I,
    8. Arai M,
    9. Nagai R,
    10. Kurabayashi M
    . Jagged1-selective notch signaling induces smooth muscle differentiation via a RBP-Jkappa-dependent pathway. J Biol Chem. 2006;281:28555–28564.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Fouillade C,
    2. Monet-Leprêtre M,
    3. Baron-Menguy C,
    4. Joutel A
    . Notch signalling in smooth muscle cells during development and disease. Cardiovasc Res. 2012;95:138–146.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. Doi H,
    2. Iso T,
    3. Yamazaki M,
    4. Akiyama H,
    5. Kanai H,
    6. Sato H,
    7. Kawai-Kowase K,
    8. Tanaka T,
    9. Maeno T,
    10. Okamoto E,
    11. Arai M,
    12. Kedes L,
    13. Kurabayashi M
    . HERP1 inhibits myocardin-induced vascular smooth muscle cell differentiation by interfering with SRF binding to CArG box. Arterioscler Thromb Vasc Biol. 2005;25:2328–2334.
    OpenUrlAbstract/FREE Full Text
  26. 26.
    1. Proweller A,
    2. Pear WS,
    3. Parmacek MS
    . Notch signaling represses myocardin-induced smooth muscle cell differentiation. J Biol Chem. 2005;280:8994–9004.
    OpenUrlAbstract/FREE Full Text
  27. 27.
    1. Morrow D,
    2. Scheller A,
    3. Birney YA,
    4. Sweeney C,
    5. Guha S,
    6. Cummins PM,
    7. Murphy R,
    8. Walls D,
    9. Redmond EM,
    10. Cahill PA
    . Notch-mediated CBF-1/RBP-J{kappa}-dependent regulation of human vascular smooth muscle cell phenotype in vitro. Am J Physiol Cell Physiol. 2005;289:C1188–C1196.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Nam Y,
    2. Sliz P,
    3. Song L,
    4. Aster JC,
    5. Blacklow SC
    . Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes. Cell. 2006;124:973–983.
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Kovall RA,
    2. Hendrickson WA
    . Crystal structure of the nuclear effector of Notch signaling, CSL, bound to DNA. EMBO J. 2004;23:3441–3451.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Walsh CP,
    2. Bestor TH
    . Cytosine methylation and mammalian development. Genes Dev. 1999;13:26–34.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Futscher BW,
    2. Oshiro MM,
    3. Wozniak RJ,
    4. Holtan N,
    5. Hanigan CL,
    6. Duan H,
    7. Domann FE
    . Role for DNA methylation in the control of cell type specific maspin expression. Nat Genet. 2002;31:175–179.
    OpenUrlCrossRefPubMed
  32. 32.↵
    1. Palacios D,
    2. Summerbell D,
    3. Rigby PW,
    4. Boyes J
    . Interplay between DNA methylation and transcription factor availability: implications for developmental activation of the mouse Myogenin gene. Mol Cell Biol. 2010;30:3805–3815.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    1. Lister R,
    2. Pelizzola M,
    3. Dowen RH,
    4. Hawkins RD,
    5. Hon G,
    6. Tonti-Filippini J,
    7. Nery JR,
    8. Lee L,
    9. Ye Z,
    10. Ngo QM,
    11. Edsall L,
    12. Antosiewicz-Bourget J,
    13. Stewart R,
    14. Ruotti V,
    15. Millar AH,
    16. Thomson JA,
    17. Ren B,
    18. Ecker JR
    . Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 2009;462:315–322.
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Brunner AL,
    2. Johnson DS,
    3. Kim SW,
    4. Valouev A,
    5. Reddy TE,
    6. Neff NF,
    7. Anton E,
    8. Medina C,
    9. Nguyen L,
    10. Chiao E,
    11. Oyolu CB,
    12. Schroth GP,
    13. Absher DM,
    14. Baker JC,
    15. Myers RM
    . Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver. Genome Res. 2009;19:1044–1056.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Illingworth R,
    2. Kerr A,
    3. Desousa D,
    4. Jørgensen H,
    5. Ellis P,
    6. Stalker J,
    7. Jackson D,
    8. Clee C,
    9. Plumb R,
    10. Rogers J,
    11. Humphray S,
    12. Cox T,
    13. Langford C,
    14. Bird A
    . A novel CpG island set identifies tissue-specific methylation at developmental gene loci. PLoS Biol. 2008;6:e22.
    OpenUrlCrossRefPubMed
  36. 36.↵
    1. Doi A,
    2. Park IH,
    3. Wen B,
    4. Murakami P,
    5. Aryee MJ,
    6. Irizarry R,
    7. Herb B,
    8. Ladd-Acosta C,
    9. Rho J,
    10. Loewer S,
    11. Miller J,
    12. Schlaeger T,
    13. Daley GQ,
    14. Feinberg AP
    . Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nat Genet. 2009;41:1350–1353.
    OpenUrlCrossRefPubMed
  37. 37.↵
    1. Weber M,
    2. Hellmann I,
    3. Stadler MB,
    4. Ramos L,
    5. Pääbo S,
    6. Rebhan M,
    7. Schübeler D
    . Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet. 2007;39:457–466.
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. Meissner A,
    2. Mikkelsen TS,
    3. Gu H,
    4. Wernig M,
    5. Hanna J,
    6. Sivachenko A,
    7. Zhang X,
    8. Bernstein BE,
    9. Nusbaum C,
    10. Jaffe DB,
    11. Gnirke A,
    12. Jaenisch R,
    13. Lander ES
    . Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature. 2008;454:766–770.
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. Saxonov S,
    2. Berg P,
    3. Brutlag DL
    . A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc Natl Acad Sci U S A. 2006;103:1412–1417.
    OpenUrlAbstract/FREE Full Text
  40. 40.↵
    1. Wu SC,
    2. Zhang Y
    . Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol. 2010;11:607–620.
    OpenUrlCrossRefPubMed
  41. 41.↵
    1. Okada Y,
    2. Yamagata K,
    3. Hong K,
    4. Wakayama T,
    5. Zhang Y
    . A role for the elongator complex in zygotic paternal genome demethylation. Nature. 2010;463:554–558.
    OpenUrlCrossRefPubMed
  42. 42.↵
    1. Métivier R,
    2. Gallais R,
    3. Tiffoche C,
    4. Le Péron C,
    5. Jurkowska RZ,
    6. Carmouche RP,
    7. Ibberson D,
    8. Barath P,
    9. Demay F,
    10. Reid G,
    11. Benes V,
    12. Jeltsch A,
    13. Gannon F,
    14. Salbert G
    . Cyclical DNA methylation of a transcriptionally active promoter. Nature. 2008;452:45–50.
    OpenUrlCrossRefPubMed
  43. 43.↵
    1. Montes de Oca A,
    2. Madueno JA,
    3. Martinez-Moreno JM,
    4. Guerrero F,
    5. Munoz-Castaneda J,
    6. Rodriguez-Ortiz ME,
    7. Mendoza FJ,
    8. Almaden Y,
    9. Lopez I,
    10. Rodriguez M,
    11. Aguilera-Tejero E
    . High-phosphate-induced calcification is related to SM22alpha promoter methylation in vascular smooth muscle cells. J Bone Miner Res. 2010;25:1996–2005.
    OpenUrlCrossRefPubMed
  44. 44.↵
    1. Klein D,
    2. Benchellal M,
    3. Kleff V,
    4. Jakob HG,
    5. Ergün S
    . Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells. Sci Rep. 2013;3:2178.
    OpenUrlPubMed
  45. 45.↵
    1. Hu B,
    2. Gharaee-Kermani M,
    3. Wu Z,
    4. Phan SH
    . Epigenetic regulation of myofibroblast differentiation by DNA methylation. Am J Pathol. 2010;177:21–28.
    OpenUrlCrossRefPubMed
  46. 46.↵
    1. Bartels SJ,
    2. Spruijt CG,
    3. Brinkman AB,
    4. Jansen PW,
    5. Vermeulen M,
    6. Stunnenberg HG
    . A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein. PLoS One. 2011;6:e25884.
    OpenUrlCrossRefPubMed
  47. 47.↵
    1. Liu Y,
    2. Zhang X,
    3. Blumenthal RM,
    4. Cheng X
    . A common mode of recognition for methylated CpG. Trends Biochem Sci. 2013;38:177–183.
    OpenUrlCrossRefPubMed
  48. 48.↵
    1. Hassed SJ,
    2. Wiley GB,
    3. Wang S,
    4. Lee JY,
    5. Li S,
    6. Xu W,
    7. Zhao ZJ,
    8. Mulvihill JJ,
    9. Robertson J,
    10. Warner J,
    11. Gaffney PM
    . RBPJ mutations identified in two families affected by Adams-Oliver syndrome. Am J Hum Genet. 2012;91:391–395.
    OpenUrlCrossRefPubMed
  49. 49.↵
    1. Wang H,
    2. Zou J,
    3. Zhao B,
    4. Johannsen E,
    5. Ashworth T,
    6. Wong H,
    7. Pear WS,
    8. Schug J,
    9. Blacklow SC,
    10. Arnett KL,
    11. Bernstein BE,
    12. Kieff E,
    13. Aster JC
    . Genome-wide analysis reveals conserved and divergent features of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells. Proc Natl Acad Sci U S A. 2011;108:14908–14913.
    OpenUrlAbstract/FREE Full Text
  50. 50.↵
    1. Li Y,
    2. Hibbs MA,
    3. Gard AL,
    4. Shylo NA,
    5. Yun K
    . Genome-wide analysis of N1ICD/RBPJ targets in vivo reveals direct transcriptional regulation of Wnt, SHH, and hippo pathway effectors by Notch1. Stem Cells. 2012;30:741–752.
    OpenUrlCrossRefPubMed
  51. 51.↵
    1. Castel D,
    2. Mourikis P,
    3. Bartels SJ,
    4. Brinkman AB,
    5. Tajbakhsh S,
    6. Stunnenberg HG
    . Dynamic binding of RBPJ is determined by Notch signaling status. Genes Dev. 2013;27:1059–1071.
    OpenUrlAbstract/FREE Full Text

Significance

We show that methylation of a GC repressor in the smooth muscle (SM) myosin heavy chain promoter inversely correlates with SM MHC expression in aortic SMC, that the methylated GC repressor recruits the multifunctional transcription factor recombination signal binding protein for immunoglobulin κ J region (RBPJ) and that RBPJ can inhibit SMC marker gene expression in phenotypically modulated human aortic SMC. These results have important implications on the regulation of SMC-specific and Notch/RBPJ–dependent gene expression.

View Abstract

This Issue

Jump to

Article Tools

Share this Article

  • Email
  • DNA Methylation of a GC Repressor Element in the Smooth Muscle Myosin Heavy Chain Promoter Facilitates Binding of the Notch-Associated Transcription Factor, RBPJ/CSL1Significance
    Julian M. Rozenberg, Daniel B. Tesfu, Srilaxmi Musunuri, Joan M. Taylor and Christopher P. Mack
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:2624-2631, originally published October 16, 2014
    https://doi.org/10.1161/ATVBAHA.114.304634
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...

Subjects