Methylated and unmethylated DNA probes spanning the regions at nt 7444-7468 were 39 end-labeled with biotin. The methylated probes showed three shifted bands. In contrast, unmethylated probes were not shifted by any of the nuclear extracts. Competition experiments were AbMole Sibutramine HCl performed with full-length, non-labeled probes as illustrated in Figure 7F. Binding to the methylated probe was competed by pre-incubation with a 100-fold excess of a methylated oligonucleotide, suggesting that a yet uncharacterized protein complex binds specifically to this methylated sequence. The EMSA experiments indicate that a complex of not yet in detail characterized proteins apparently bind to the region of methylated E2BS1, but fails to bind to the unmethylated E2BS1. This experiment supports the hypothesis that binding of cellular factor might be substantially influenced by the methylation status of respective CpG dinucleotides. Therefore, methylation of the E2BS1 of the HPV 16 URR observed in the transforming mode of HPV-infection may have a direct influence on the transcriptional activity of the HPV 16 URR by binding of a yet uncharacterized complex of transcription factors. Previous reports suggested that the HPV genome is differentially methylated during progression from simple infected to transformed cells, suggesting that differential methylation of the viral genome may somehow be involved in the AbMole Sarafloxacin HCl regulation of viral gene expression and possibly also replication control. Alterations of the HPV-methylome were observed particularly in the URR and L2 and L1 gene in high grade precancer and invasive cancer suggesting that the lack of expression of these genes may be attributed at least in part to increasing methylation of the respective parts of the viral genomes. The E2BS2 to 4 were also found to be increasingly methylated in more advanced dysplasia or invasive carcinomas. Although it has not been analyzed in detail, the increased methylation pattern in some of those studies might well be explained by integration of the viral genomes into the host cell chromosomes. Genomic integration of viral genomes has been repeatedly shown to be associated with hypermethylation of the viral genomes in the integrated context. The permissive life cycle of HPV is restricted to preneoplastic lesions and essentially coupled to squamous epithelial differentiation. In this report we for the first time used DNA isolated from microdissected squamous epithelial cells reflecting various differentiation conditions of HPV-infected squamous epithelial cells of the uterine cervix. This was surprising, since the E2BS1 is known to activate the HPV URR. Methylation of this site and reduced binding of E2 to this site was thus expected to suppress the activity of the HPV 16 URR. This in turn should have resulted in decreased but not increased expression of the downstream early genes E6 and E7 as it is consistently observed in the transforming HPV 16 transcription mode.