| DNA methylation is an important epigenetic mark in all mammalian genomes studied to date. Strongly associated with gene silencing, DNA methylation is an important component of gene regulation at many loci throughout the genome. Because of the variability in DNA methylation patterns between individuals and across tissue types, disease states, stages of development, and time points in cancer progression, there is huge demand for comprehensive profiling methods that can be performed with ease on multiple samples in parallel. Here I describe a number of profiling projects and demonstrate the use of three different methods for large-scale DNA methylation profiling. First I describe the development of a new method, Methyl-seq, that uses a methyl-sensitive restriction enzyme and ultra high-throughput sequencing performed on Illumina's Genome Analyzer to profile 90,612 regions in the human genome. This method uses small HpaII fragments to cover a range of genomic elements, including thousands of CpG islands, promoters, exons, introns, UTRs, and other non-repetitive intergenic regions. I then describe the application of Methyl-seq to profile several developmentally interesting samples, including human embryonic stem cells (hESCs), differentiated hESCs, and tissue samples. We observed many methylation differences, but not nearly the number expected given the extent of gene expression differences. Our results suggest first, that in vitro differentiation of hESCs does not properly establish tissue-like methylation patterns, and second, that DNA methylation alone is not responsible for the numbers of gene expression changes that occur during in vitro differentiation. This dissertation continues with a comparison of DNA methylation profiles for forty-five different human samples comprising normal tissues, cancer tissues, and cell lines using the Illumina Infinium Methylation27 BeadChips. The final data chapter describes DNA methylation profiling of HCT-116 cells (a human colorectal cancer cell line) as well as the previously described HCT-116 DNMT1-/-, HCT-116 DNMT3b-/- and DKO (HCT-116 DNMT1-/-; DNMT3b-/-) cells using three different profiling methods. I describe a previously unpublished method that uses several methyl-sensitive restriction enzymes, fragment size selection, and custom Agilent microarrays to assay for depletion of unmethylated fragments. I also profile these samples with Methyl-seq and Infinium Methylation27 BeadChips to provide the most comprehensive methylation study of these cells to date. The final chapter discusses pros and cons for several profiling methods and proposes what is needed to improve DNA methylation profiling efforts in the future. |
