The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes
Figure 1
Experimental design of MeDIP analysis.
A: Schematic representation of the experimental setup for the analysis of CpG methylation patterns. The KSHV episome in infected cells is expected to be partially methylated, as indicated by black and white circles which symbolize methylated or unmethylated CpG dinucleotides, respectively. Genomic DNA was isolated from such cells and the samples were subjected to immunoprecipitation using a methylcytidine specific antibody (MeDIP procedure), followed by hybridization of the precipitated samples versus the input on tiling microarrays. For each probe, an enrichment score ES was calculated, which represents the ratio of MeDIP over input fluorescence signals. The efficiency of the immunoprecipitation depends on the total number of methylated CpG motifs in a given fragment and ES is thus a function of the extend of methylation as well as local CpG frequencies. Therefore, to obtain reference values which signify maximum methylation for each probe, we generated a positive control by subjecting KSHV bacmids to CpG methylation in vitro. The bacmid was mixed with cellular DNA to simulate the host background and subjected to the same MeDIP procedure as samples from infected cells. Similarly, a negative control of unmethylated bacmid was prepared to control for cross-hybridization of unspecific background. After normalization of the array data using a spike-in control (see Material & Methods for details), background-corrected methylation values MS and MP were calculated for each probe by subtraction of the corresponding negative control value. B: Confirmation of successful in vitro methylation of KSHV bacmids used as a positive control. A bacmid carrying the complete KSHV genome (BAC36 [36]) was methylated using M.SssI, a methyltransferase specific for CpG dinucleotides. Methylated or unmethylated bacmids were subjected to restriction digestion using the methylation sensitive enzyme HpaII and its isoschizomer MspI, which cuts regardless of methylation. Methylated bacmids were resistant to HpaII digestion, signifying complete methylation.