Epigenetic modifications in an imprinting cluster are controlled by a hierarchy of DMRs suggesting long-range chromatin interactions

Imprinted genes and their control elements occur in clusters in the mammalian genome and carry epigenetic modifications. Observations from imprinting disorders suggest that epigenetic modifications throughout the clusters could be under regional control. However, neither the elements that are responsible for regional control, nor its developmental timing, particularly whether it occurs in the germline or postzygotically, are known. Here we examine regional control of DNA methylation in the imprinted Igf2-H19 region in the mouse. Paternal germline specific methylation was reprogrammed after fertilization in two differentially methylated regions (DMRs) in Igf2, and was reestablished after implantation. Using a number of knockout strains in the region, we found that the DMRs themselves are involved in regional coordination in a hierarchical fashion. Thus the H19 DMR was needed on the maternal allele to protect the Igf2 DMRs 1 and 2 from methylation, and Igf2 DMR1 was needed to protect DMR2 from methylation. This regional coordination occurred exclusively after fertilization during somatic development, and did not involve linear spreading of DNA methylation, suggesting a model in which long-range chromatin interactions are involved in regional epigenetic coordination. These observations are likely to be relevant to other gene clusters in which epigenetic regulation plays a role, and in pathological situations in which epigenetic regulation is disrupted.