DNA methylation analysis of gametic imprints in the mouse and search for factors involved in imprint establishment

Genomic imprinting is an epigenetic phenomenon crucial for normal mammalian development. Genes subject to imprinting (imprinted genes) are associated with differentially methylated regions (DMRs) that are methylated on either the maternal or the paternal chromosome. In mice, at least fifteen DMRs acquire such differential DNA methylation in the parental germline, and the established methylation imprints are then transmitted to the zygote and maintained throughout development. Acquisition of DNA methylation of these DMRs depends on a protein complex containing DNA methyltransferase family proteins, Dnmt3a and Dnmt3l. However, the mechanism by which specific sequences are targeted for methylation remains poorly understood. Previously in our laboratory, the extents along DNA of the allelic differential methylation of the fifteen DMRs n 12.5-days-post-coitum (12.5-dpc) embryos were determined by bisulphite sequencing. To explore the origin of the differential methylation, I have carried out a comprehensive DNA methylation analysis of the same DMRs in sperm and oocytes. I found that the extents of the differential methylation differ significantly between the gametes and 12.5-dpc embryos, suggesting that dynamic changes occur after fertilization. Both expansion and contraction of the differential methylation were observed. Small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs) were previously shown to act as guides to induce local heterochromatin or DNA methylation To test whether the small RNAs in prospermatogonia and oocytes are involved in de novo DNA methylation of the DMRs, I examined whether these small RNAs are mapped within the DMRs determined in this study. Not many of them were mapped in the DMRs, suggesting small RNA-independent targeting of the de novo DNA methylation machinery. Another factor possibly involved in the targeted methylation is a protein(s) that interacts with the DNA metlyltransferase complex. By yeast two-hybrid screening, I identified Glis 1 (a Krüppel-like zinc finger protein), Baf60a (a component of the SWI/SNF chromatin remodeling complex) and Rail (a PHD-containing transcription factor) as candidate proteins which potentially interact with Dnmt3L. Further studies are needed to explore the possibility of the involvement of these proteins in targeted methylation. These data will provide a basis for understanding the mechanism of imprint establishment