Electronic Thesis and Dissertation Repository


Doctor of Philosophy




Dr. Mellissa Mann


Genomic imprinting is an epigenetic mechanism that controls gene expression based on parental-origin of an allele. The Kcnq1ot1 imprinting cluster consists of an imprinting control region (ICR), the Kcnq1ot1 ncRNA, and maternally expressed protein-coding genes. Truncation of the Kcnq1ot1 ncRNA or deletion of the Kcnq1ot1 ICR, including the Kcnq1ot1 ncRNA promoter results in biallelic expression of normally paternally silent protein-coding genes in postimplantation, suggesting the Kcnq1ot1 ICR/ncRNA are required for bidirectional silencing. However, Kcnq1ot1 ncRNA regulation of imprinted genes during preimplantation is unknown. To address this, imprinted expression was investigated in preimplantation embryos with a paternally deleted Kcnq1ot1 ICR or truncated Kcnq1ot1 ncRNA. Kcnq1ot1 mutant embryos were capable of silencing Phlda2, Slc22a18 and Cdkn1c paternal alleles, suggesting the Kcnq1ot1 ICR and ncRNA are dispensable for repression at this stage. Imprinted expression in early postimplantation embryos carrying a paternally deleted Kcnq1ot1 ICR showed the ICR was necessary for maintenance of paternal repression at Phlda2, Slc22a18, Cdkn1c, Kcnq1 and Ascl2. However, truncation of the Kcnq1ot1 ncRNA resulted in paternal reactivation of distal genes, Phlda2, Slc22a18 and Ascl2, while genes proximal to the ICR, Kcnq1 and Cdkn1c, maintained maternal-specific or maternal-biased expression. This indicates the Kcnq1ot1 ICR and ncRNA are dispensable in early development for paternal silencing but are required later for maintenance of imprinted expression. However, epigenetic modifiers maintaining paternal silencing of adjacent protein-coding genes in coordination with the Kcnq1ot1 ICR or ncRNA are unknown. Therefore, epigenetic modifiers regulating imprinting at the Kcnq1ot1 domain were identified in embryo-derived stem cells using a positive selection, loss-of-function RNA interference (RNAi) screen. Depletion of candidates Ezh1, Smarca5 and Smarcad1 resulted in loss of imprinted expression of Cdkn1c and Kcnq1 but not Osbpl5 and Slc22a18, suggesting epigenetic modifiers identified here function at genes proximal to the Kcnq1ot1 ICR and not domain-wide. Kcnq1ot1 expression was reduced when Smarca5 and Smarcad1 but not when Ezh1 were depleted, indicating loss of imprinting can occur independently of Kcnq1ot1 ncRNA expression and epigenetic modifiers could be acting directly on imprinted genes. Further characterization of candidates will provide better understanding of imprinted gene regulation and the protein complexes responsible for maintaining repression.