Epigenetic modifications, such as DNA methylation and histone modifications, play pivotal roles in gene expression and regulation, and are highly involved in cellular processes such as stem cell pluripotency/differentiation and tumorigenesis. In mammals, DNA methylation is the best studied epigenetic modification. It refers to the addition of a methyl group at the carbon-5 position of cytosine residues within CpG dinucleotides, and it is heavily associated with a large number of human diseases. For example, transcriptional silencing of tumor-suppressor genes by CpG-promoter hypermethylation plays an important role in cancer development. Therefore, understanding of epigenetic regulations will help to improve various aspects of biomedicine. For instance, personalized medicine can be tailored based on epigenetic profile of certain patient to specifically control gene expression in the disease treatment. However, the technology for profiling epigenetic modifications, i.e. methylated DNA immunoprecipitation (MeDIP), suffers from serious limitations. The key limitation is the sensitivity of the assay. Conventional assay requires a large number of cells (>10
6 cells per MeDIP). This is feasible when using cell lines. However, such requirement has become a major challenge when primary cells are used because very limited amounts of samples can be generated from lab animals or patients. Population heterogeneity information may also be lost when a large cell number is used.
In this project, we developed an automated ultrasensitive microfluidic methylated DNA immunoprecipitation followed by next-generation sequencing (MeDIP-Seq) technology for profiling DNA methylomes. We extensively optimized design parameters for each and every step of MeDIP (e.g. sonication/crosslinking time, antibody concentration, washing conditions) in order to reach highest sensitivity of 0.1 ng DNA (or ~50-100 cells) as starting material for IP, which is roughly 4-5 orders of magnitude higher than the prevailing protocol and 2-3 orders of magnitude higher than the-state-of-the-art(~50 ng). With such sensitivity, we were able to study temporal dynamics in the DNA methylomes during the various stages of mammary cancer development from a transgenic mouse model.
