Date of Award
Master of Science
Dr. Chris Pin
For a small group of important developmental genes, there is preferential silencing of either the maternal or paternal allele. This process of preferential expression is called 'genomic imprinting'. Imprinted genes often cluster in imprinted domains. Interestingly, many imprinting domains are associated with a non-coding RNA (ncRNA) that may regulate imprinted gene expression across the entire domain. Disruptions in imprinting can have severe consequences for growth and development. To understand the complex regulation of genomic imprinting, studies are required to determine how early embryos set up a hierarchy of events that will establish imprinting across large chromosomal domains. The goal of this project is to characterize the mouse Kcnq1ot1 ncRNA and its role in imprinted gene regulation. My findings indicate that the Kcnq1ot1 ncRNA terminates at 463 kb from the transcriptional start site, and that this length is conserved in various tissues and developmental stages. This extends the boundary between the Kcnq1ot1 and H19 domains, downstream of Th, between 464 and 617 kb. shRNA and siRNA depletion of the Kcnq1ot1 transcript at 43 kb and 460 kb, respectively, indicates that Kcnq1ot1 is one continuous transcript as opposed to having multiple start sites along the length. In addition, the Kcnq1ot1 transcript originates from the imprinting control region (ICR), as deletion of the paternal ICR, which contains the Kcnq1ot1 promoter, results in loss of amplification along the entire length of the transcript. To determine whether the Kcnq1ot1 ncRNA regulates domain-wide imprinting during early embryogenesis, RNA interference was employed to deplete Kcnq1ot1 in embryonic and extraembryonic stem cells. Loss of >90% of the Kcnq1ot1 transcript had no effect on imprinted expression in the domain, nor on imprinted methylation at the Kcnq1ot1 ICR, suggesting that transcription itself may play a more important role than the transcript per se. Results from this study will lead to a better understanding of the role that long ncRNAs play in establishing and/or maintaining imprinted gene regulation across imprinting domains, as well as their role in human imprinted disorders.
Lalone, Sarah Allen, "Characterization of the mouse KCNQ1OT1 noncoding RNA" (2009). Digitized Theses. 3219.