The DNA methylome of HPV16. (A) Unsupervised clustering analysis of the HPV16 DNA methylome in asymptomatic carriers of the virus, pre-malignant lesions (CIN), primary tumors (SCCs), and cancer cell lines (CaSki, SiHA, CCL-879, and CCL-866). (Red) Methylated, (green) unmethylated, and (black) deleted sequences. (Top) The HPV16 genome. (B) Example of bisulfite genomic sequencing analysis of multiple clones for the HPV16 genome. (Black squares) Methylated and (white squares) unmethylated CpG dinucleotides; (gray squares) deleted genome sequences. (C) Bisulfite sequencing showing how the mostly unmethylated HPV16 DNA methylome from pre-immortal keratinocytes undergo hypermethylation in immortalized cells. (D) Confocal studies for fluorescence in situ hybridization for HPV16 and 5-methylcytosine DNA staining demonstrate the colocalization of the integrated viral genome in a human chromosome and the DNA methylation signal in CaSki cells. (E) The use of a DNA demethylating agent in CaSki cells causes DNA hypomethylation of the HPV16 genome. (F) The expression of HPV16 E6 and E7 oncoproteins is associated with a loss of binding of the E2 repressor to the URR methylated sites. (Top left) The expression of E2 and the methylated status of the URR binding site in CaSki. (Top right) DNA demethylating treatment reduces E6 and E7 expression in Western blot and (bottom left) qRT-PCT in association with a recruitment of the E2 protein to the demethylated URR E2-binding site, (bottom right) shown by q-ChIP. (G) Methylation-specific PCR analysis of the HPV16 L2, L1, and URR sequences in cervical tumorigenesis. The presence of a band under the U or M lanes indicates unmethylated or methylated sequences. In vitro methylated DNA (IVD) is shown as a positive control.
