An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.     

Bidirectional silencing and DNA methylation-sensitive methylation-spreading properties of the Kcnq1 imprinting control region map to the same regions

The mechanisms underlying the phenomenon of genomic imprinting are poorly understood. Accumulating evidence suggests that imprinting control regions (ICR) associated with the imprinted genes play an important role in creation of imprinted expression domains by propagating parent-of-origin-specific epigenetic modifications. We have recently documented that the Kcnq1 ICR unidirectionally blocks enhancer-promoter communications in a methylation-dependent manner in Hep-3B and Jurkat cell lines. In this report we show that the Kcnq1 ICR harbors bidirectional silencing and methylation-sensitive methylation-spreading properties in a lineage-specific manner. We fine map both of these functions to two critical regions, and loss of one these regions results in loss of silencing as well as methylation spreading. The cell type-specific functions of the Kcnq1 ICR suggest binding of cell type-specific factors to various cis elements within the ICR. Fine mapping of the silencing and methylation-spreading functions to the same regions explains the fact that the silencing factors associated with this region primarily repress the neighboring genes and that methylation occurs as a consequence of silencing.     

Antisense noncoding RNA promoter regulates the timing of de novo methylation of an imprinting control region

Epigenetic marks at cis acting imprinting control regions (ICRs) regulate parent of origin-specific expression of multiple genes in imprinted gene clusters. Epigenetic marks are acquired during gametogenesis and maintained faithfully thereafter. However, the mechanism by which differential epigenetic marks are established and maintained at ICRs is currently unclear. By using Kcnq1 ICR as a model system, we have investigated the functional role of genetic signatures in the acquisition and maintenance of epigenetic marks. Kcnq1 ICR is methylated on the maternal chromosome but remains unmethylated on the paternal chromosome. Here, we show that a paternal allele of Kcnq1 ICR lacking the Kcnq1ot1 promoter remains unmethylated during spermatogenesis; however, it becomes methylated specifically during pre-implantation development. Analysis of the chromatin structure at the paternal ICR in spermatogenic cells and in E13.5 embryonic tissues revealed that the ICRs of both wild type and mutant mice are enriched with H3K4me2 in spermatiogenic cells of the testicular compartment, but the mutant ICR lost H3K4me2 specifically in epididymal sperm and an increase in repressive marks was observed in embryonic tissues. Interestingly, we also detected a decrease in nucleosomal histone levels at the mutant ICR in comparison to the wild-type ICR in epididymal sperm. Taken together, these observations suggest that the Kcnq1ot1 promoter plays a critical role in establishing an epigenetic memory in the male germline by ensuring that the paternal allele remains in an unmethylated state during pre-implantation development.     

Role of CTCF binding sites in the Igf2/H19 imprinting control region

A approximately 2.4-kb imprinting control region (ICR) regulates somatic monoallelic expression of the Igf2 and H19 genes. This is achieved through DNA methylation-dependent chromatin insulator and promoter silencing activities on the maternal and paternal chromosomes, respectively. In somatic cells, the hypomethylated maternally inherited ICR binds the insulator protein CTCF at four sites and blocks activity of the proximal Igf2 promoter by insulating it from its distal enhancers. CTCF binding is thought to play a direct role in inhibiting methylation of the ICR in female germ cells and in somatic cells and, therefore, in establishing and maintaining imprinting of the Igf2/H19 region. Here, we report on the effects of eliminating ICR CTCF binding by severely mutating all four sites in mice. We found that in the female and male germ lines, the mutant ICR remained hypomethylated and hypermethylated, respectively, showing that the CTCF binding sites are dispensable for imprinting establishment. Postfertilization, the maternal mutant ICR acquired methylation, which could be explained by loss of methylation inhibition, which is normally provided by CTCF binding. Adjacent regions in cis-the H19 promoter and gene-also acquired methylation, accompanied by downregulation of H19. This could be the result of a silencing effect of the methylated maternal ICR.