DNA methylation is an epigenetic mark that has a crucial role in many biological processes. To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input in order to minimize the drain on precious samples. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) fulfils these criteria, combining MeDIP with massively parallel DNA sequencing. Here we report an improved protocol using 100-fold less genomic DNA than that commonly used. We show comparable results for specificity (>97%) and enrichment (>100-fold) over a wide range of DNA concentrations (5,000-50 ng) and demonstrate the utility of the protocol for the generation of methylomes from rare bone marrow cells using 160-300 ng of starting DNA. The protocol described here, i.e., DNA extraction to generation of MeDIP-seq library, can be completed within 3-5 d.

Stem cells have been found in most tissues/organs. These somatic stem cells produce replacements for lost and damaged cells, and it is not completely understood how this regenerative capacity becomes diminished during aging. To study the possible involvement of epigenetic changes in somatic stem cell aging, we used murine hematopoiesis as a model system. Hematopoietic stem cells (HSCs) were enriched for via Hoechst exclusion activity (SP-HSC) from young, medium-aged and old mice and subjected to comprehensive, global methylome (MeDIP-seq) analysis. With age, we observed a global loss of DNA methylation of approximately 5%, but an increase in methylation at some CpG islands. Just over 100 significant (FDR < 0.2) aging-specific differentially methylated regions (aDMRs) were identified, which are surprisingly few considering the profound age-based changes that occur in HSC biology. Interestingly, the polycomb repressive complex -2 (PCRC2) target genes Kiss1r, Nav2 and Hsf4 were hypermethylated with age. The promoter for the Sdpr gene was determined to be progressively hypomethylated with age. This occurred concurrently with an increase in gene expression with age. To explore this relationship further, we cultured isolated SP-HSC in the presence of 5-aza-deoxycytdine and demonstrated a negative correlation between Sdpr promoter methylation and gene expression. We report that DNA methylation patterns are well preserved during hematopoietic stem cell aging, confirm that PCRC2 targets are increasingly methylated with age, and suggest that SDPR expression changes with age in HSCs may be regulated via age-based alterations in DNA methylation.