Specific aim 1: correlation of DNA methylation with clinical traits age stage gradesurvivalProgression free interval Preliminary analyses didn’t show any significant association. Need to Re-do it by including days to the last Follow up. Association is not significant > 2000 loci identified Do these loci belong to one or two comethylation modules ?

Specific aim 2: correlation of DNA methylation with other molecular information miRNACNVSNPRNA Many significant pairs (cis-) Mostly positive, rather than neg. corr. Specific GO categories associated with neg. and pos. How can we find out the status of CpGs associated with expression? (see some ideas on the p. with aim3) Are there particular enrichment of TF binding sites in “+” and “-” correl. groups? Many CpGs map to the same nearby gene. Do they have the same correlation sign? Is there a correlation between sign of correl. with expression and distance to TSS ? Do genes correlated with methylation have any association with survival, PFI, age, stage, grade?

Specific aim 3: comethylation networks Build the networks Correlation with RNA < 10% of correlated genes map to CpGs in the same modules Module is correlated with multiple genes Each gene correlated with only one module Are genes correlated with module in the same pathway as CpG within that module ? Get a list of epigenes to calculate their enrichment in the list of genes correlated With modules If enrichment for epigenes is weak can other genes be tied to regulation of methylation? Are those genes correlated with survival (in single locus analysis)? -stage -grade -age Causes for modules formation

Specific aim 3: comethylation networks Build the networks Correlation with RNA Causes for modules formation Chromosomal enrichment Loci cluster within a module GO categories meaningful Correlation with survival weak Correlation with other traits Stage, grade, age Combination of modules and survival Explanation Do we know any proteins/miRNA that define meth. boundaries? K.In Doesn’t seem to be any particular pattern in the cluster Loci mapped to the same gene End up in different modules How often does it happen? Dependant on distance to TSS Specific TF binding sites? Within each chromosome, do we see any overlap between groupings of CpGs from different modules? Can we “paint” a chr into different modules? How can we identify hypo- and hypermeth. modules? One idea: process unmeth. loci similar to meth. Combine to make M value, draw a cutoff. Classify modules according to that. Any association with CNV ?

Specific aim 3: coexpression networks Causes for modules formation Chromosomal location Gene Ontology DNA methylation PCs correlation ~28 correlated Need to calculate P values for the overlap Correlation with survivalCorrelation with stage, grade, age Will combining of coexpression and comethylation improve prediction of survival