Dnmt3b is an essential methyltransferase, a protein that transfers methyl groups to DNA. Expression of the gene that encodes Dnmt3b is tightly controlled by unknown mechanisms during early mammalian development and later in somatic cells. The controlled expression of Dnmt3b is critical for cellular differentiation and for maintenance of cell identity. One mechanism by which Dnmt3b expression is regulated potentially involves a non-coding RNA, named Dnmt3bas, and its interaction with regulatory switches near the Dnmt3b gene. The goal of this study is to characterize this mechanism in detail. This study will make a significant contribution to current understanding of unconventional roles of RNA thus advancing the field of study. The research plan will provide opportunities for both undergraduate and graduate students, including women and minorities, to undergo intensive training in biochemistry, molecular biology, genomics, and bioinformatics. The appreciation of non-coding RNAs as functional genomic elements that contradicts the traditional view of how the genetic information in DNA is converted from RNA to protein will be introduced in the form of interactive lessons to local middle or high school students as a part of Purdue's GK-12 program. Given that the field of non-coding RNA is mature and is now found in college textbooks, introducing these concepts at the middle/high school level will prepare students for molecular biology and biochemistry courses in college and increase student retention in STEM careers.
Using mouse embryonic stem cell (ESC) differentiation as a developmental model system, our objective in this application is to elucidate the mechanism by which the Dnmt3b enhancer and the promoter-associated divergent lncRNA (Dnmt3bas) coordinate to regulate the transcription of Dnmt3b gene. By using methods that include CRISPR/Cas9 mediated manipulations of the Dnmt3bas, crosslinking immunoprecipitation (CLIP), and chromatin isolation by RNA purification (ChIRP-Seq) analysis, the investigators will determine the role of Dnmt3bas in maintaining a transcriptionally competent state of the inactive Dnmt3b promoter in naive pluripotent ESCs and in stabilizing the interaction of the Dnmt3b promoter with the upstream enhancer upon induction of differentiation. At the completion of this project, the investigators expect to have identified a novel role for Dnmt3bas in maintaining a state that facilitates rapid activation of the Dnmt3b promoter and enhanced expression of Dnmt3b gene during the transition from the pluripotent to differentiated state. By using 3C and ChIP assays, they will further determine the role of chromatin conformation and of the repressive complex containing Dnmt3a in establishing a stably repressed state at Dnmt3b regulatory regions in differentiated cells.
