Control of gene expression by epigenetic modification of distinct DNA sequences is a fundamental biological process that impacts crucial biological features such as embryonic development, cellular differentiation and aging. Changes of the epigenetic modification of distinct genes are associated with major human diseases and in particular carcinogenesis. Methylation of HPV genomes have been analyzed in a series of recent publications. The general observation was that the degree of methylation of the HPV genomes was consistently higher at most sites in carcinomas as compared to dysplastic precancerous lesions. Further data on the influence of DNA methylation in the HPV life cycle came from the studies that focused on methylation of the E2 binding sites in the HPV 16 URR. The E2 protein is an important transcriptional regulator of the HPV genome. It mediates its transcriptional control functions through binding to four distinct E2BSs located within the URR. The capacity of the E2 protein to bind E2BSs in vitro is inhibited by methylation of CpG dinucleotides within these E2BSs and therefore inhibits transcriptional activation by the E2 protein. Methylation analysis of E2 binding sites within URR in immortalized HPV-infected epithelial cells demonstrated that these regions are hypo-methylated upon differentiation in vitro. These observations suggest that the methylation state of the viral genome, and particular that of E2BSs, may vary during the viral life cycle and thus squamous epithelial differentiation. However, the molecular impact of the methylation pattern of HPV genomes during the various stages of the HPV life cycle and also cellular transformation remained largely obscure. To fill this gap of knowledge we analyzed for the first time the methylation status of the HPV 16 URR in different phases of the viral life cycle linked to distinct phases of the squamous epithelial differentiation pattern in naturally occurring cervical lesions. Specifically, we analyzed the methylation status of CpG dinucleotides in transcription factor binding sites within the HPV 16 URR using DNA preparations isolated from microdissected squamous epithelial cell layers, reflecting basal, intermediate and superficial squamous cell differentation. An earlier report by Thain et al. suggested that E2 does not bind to methylated E2BSs. However our data revealed that the promoter activity of constructs AbMole 3,4,5-Trimethoxyphenylacetic acid encompassing methylated CpG dinucleotides in the E2BS1 was substantially enhanced if compared to the unmethylated form. This effect was depending on co-expressed E2. We therefore hypothesized that additional cellular factors may be involved in the E2-mediated regulation of the p97 promoter activity via AbMole 2,3-Dichloroacetophenone either the methylated or unmethylated E2BS1. We used EMSA analyses with nuclear cell extracts isolated from different HPV-negative squamous epithelial cell lines to test whether differential methylation of the two CpG dinucleotides.