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.

HIV-associated Salivary Gland Disease (HIVSGD) is among the most common salivary gland-associated complications in HIV positive individuals and was associated with the small DNA tumorvirus BK polyomavirus (BKPyV). The BKPyV non-coding control region (NCCR) is the main determinant of viral replication and rearranges readily. This study analyzed the BKPyV NCCR architecture and viral loads of 35 immunosuppressed individuals. Throatwash samples from subjects diagnosed with HIVSGD and urine samples from transplant patients were BKPyV positive and yielded BKPyV NCCR sequences. 94.7% of the BKPyV HIVSGD NCCRs carried a rearranged OPQPQQS block arrangement, suggesting a distinct architecture among this sample set. BKPyV from HIV positive individuals without HIVSGD harbored NCCR block sequences that were distinct from OPQPQQS. Cloned HIVSGD BKPyV isolates displayed active promoters and efficient replication capability in human salivary gland cells. The unique HIVSGD NCCR architecture may represent a potentially significant oral-tropic BKPyV substrain.