br Results br Discussion In this study

Results
Discussion In this study, we identified altogether 21 novel imprinted DMRs, including a novel class of intergenic DMRs, which reside in regions with no apparent imprinted order glp-2 in PSCs. This class of imprinted DMRs either may regulate genes in a tissue-specific manner, thus adding to the complexity of parental regulation in the adult tissues, or these parental marks regulate genes that are yet to be discovered. Notably, both WHAMMP3 and LOC100289656, intergenic pDMRs in the Prader-Willi/Angelman region, are located in close proximity to a cluster of piRNAs. This class of regulatory small noncoding RNAs was previously linked with parental imprinting (Watanabe et al., 2011), and thus it is attractive to suggest that this specific cluster of genes is expressed in a monoallelic fashion. The previously identified complex three-dimensional organization of the genome (Lieberman-Aiden et al., 2009) also suggests that this class of intergenic DMRs may interact with genes located in remote regions, thus regulating them in trans. Alternatively, it is plausible that this novel class of intergenic DMRs regulates other processes beside gene expression. As it was previously suggested, parental imprinting may be involved in marking the parental genomes for recombination (Pardo-Manuel de Villena et al., 2000). It will thus be of great interest to study whether these intergenic DMRs may serve as hot spots for genetic processes such as recombination. The use of mouse model systems has greatly enhanced our understanding of parental imprinting. Yet, some genes that are imprinted in the mouse are not imprinted in the human orthologous gene (Bartolomei and Ferguson-Smith, 2011). Moreover, some mouse models fail to recapitulate phenotypes associated with human-imprinted syndromes (Mann and Bartolomei, 1999). Strikingly, our data imply that more than 50% of mouse- and human-imprinted DMRs are species specific. In addition, some of these DMRs (e.g., WHAMMP3 and LOC100289656) reside in loci, which are associated with known human diseases such as Prader-Willi and Angelman syndromes. Therefore, our results emphasize the importance of studying imprinted DMRs in human. In addition, our analysis identified that several imprinted DMRs are consistently perturbed in HESCs and HiPSCs and thus should be carefully evaluated if these cells are to be used for clinical applications. Furthermore, as loss of imprinting was correlated before with different types of cancers, it will be worthy to study the differential methylation status of both previously identified and novel imprinted DMRs in tumor cell types. The genomic coverage of RRBS is ∼10%; however, it covers the majority of CpG islands (CGIs) and promoters in the human genome (Harris et al., 2010). As the vast majority of previously identified imprinted DMRs reside in CGIs and promoters (82%, Figure S4D), our methodology is highly informative for identifying novel imprinted DMRs throughout the human genome. Future studies, using whole genome single-base resolution analysis of DNA methylation, will elucidate whether additional imprinted DMRs are also present in CpG-poor regions. In conclusion, we conducted a comprehensive analysis of imprinted DMRs in humans, identifying multiple novel DMRs, many of them not associated with gene expression. Our data shed light on the extent of the phenomenon of parental imprinting, suggesting that it may play a more extensive role than was previously thought.
Experimental Procedures
Acknowledgments