Abstract Genomic imprinting is an epigenetic process that restricts expression of specific genes to predominantly one parental allele. Disruptions in imprinting cause aberrant growth and development, as well as imprinting disorders, such as Beckwith-Wiedemann Syndrome (BWS). Most imprinted genes reside in domains, which are thought to be coordinately regulated by an imprinting control region (ICR). ICRs harbor an imprinting mark, which includes differential DNA methylation acquired during oogenesis or spermatogenesis. Although the ICR could be equated with a simple on/off switch, the regulation of imprinted domains is considerably more complex. Genetic and epigenetic defects at the KCNQ1OT1 domain, including those arising from assisted reproduction, cause the overgrowth disorder BWS. The importance of the Kcnq1ot1 imprinting control region (ICR) and the Kcnq1ot1 noncoding RNA (ncRNA) in repressing adjacent imprinted genes on the paternal allele have been firmly established. However, since these analyses were performed during midgestation, they do not distinguish between failure to establish and failure to maintain Kcnq1ot1 domain imprinting. In this proposal, we will determine the role of the Kcnq1ot1 ICR and the Kcnq1ot1 ncRNA in establishing paternal allelic silencing across the domain in preimplantation and early postimplantation embryos. Surprisingly, in preliminary data, we found that blastocysts with a paternal Kcnq1ot1 ICR deletion or Kcnq1ot1 ncRNA truncation were capable of paternal allelic silencing. Furthermore, day 6.5 embryos with paternal Kcnq1ot1 ICR deletion lost paternal allelic silencing, while embryos with a Kcnq1ot1 ncRNA truncation had activated the silent paternal Phlda2 allele, had partially reactivated paternal Cdkn1c expression, and maintained paternal Kcnq1 silencing. Based on this data, we hypothesize that the Kcnq1ot1 ICR and the Kcnq1ot1 ncRNA are dispensable for establishment of domain-wide paternal allelic silencing, and that additional genetic elements outside of the Kcnq1ot1 ICR regulate paternal allelic repression during preimplantation development. We also hypothesize that the Kcnq1ot1 ncRNA is required for paternal allelic repression at genes distal to, but not those proximal to the Kcnq1ot1 ICR during early postimplantation development. However, it should be kept in mind that these analyses were carried out with conventional PCR followed by strain-specific restriction digestion for quantification of allele-specific expression, a method that can be problematic in samples with low starting material. Caveats are insufficient quantities of mRNA for detection, or biased amplification that skews allelic ratios of parental expression. To bypass these issues, we have developed allelic assays for Droplet Digital PCR, which will allow higher sensitivity and greater precision in measures of parental allelic expression. If we confirm our preliminary data, these results will represent a paradigm shift in the field, by showing that the Kcnq1ot1 ICR and the Kcnq1ot1 ncRNA are dispensable for establishment of paternal allelic silencing, and have distinct functions in early postimplantation embryos.
NIH ExPorter
NIH ExPorter