Regulation of long-noncoding RNA (lncRNA) by the aryl hydrocarbon receptor

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Background: The long-term goal of my research program is to understand the biological role of the aryl hydrocarbon receptor (AhR), a protein that is found in almost all of our cells. This is because- despite decades of research- the cellular function(s) of the AhR is still unknown. The AhR is a ubiquitously-expressed receptor/transcription factor historically known to mediate the toxic effects of the man-made environmental contaminant dioxin. In response to dioxin, the AhR translocations to the nucleus and binds to the dioxin response element (DRE) to elicit toxic effects. Using a broad range of biochemical, molecular and genetic tools, over the past 5 years we have shown that the AhR controls fundamental cellular processes independent of its response to dioxin. The biological processes that we have shown are regulated by the AhR include proliferation and cell survival. We have also discovered that one of novel mechanisms through which the AhR regulates these cellular processes is via non-transcriptional control over microRNA (miRNA) expression. miRNAs are part of a growing list of non-coding RNAs (ncRNA) that also includes long non-coding RNA (lncRNAs). lncRNA are non-protein coding transcripts longer than 200 nucleotides. Although originally thought to be transcriptional noise (i.e. “junk DNA”), lncRNA are now recognized to control many of the same basic biological functions as the AhR- including cell survival and proliferation. There is almost nothing known about the AhR and lncRNA. Hypothesis: We hypothesize that the AhR controls the expression of lncRNA important in cell survival. Objectives: The overall goal of this research program is to establish that regulation of lncRNA by the AhR underlies its control over apoptosis. Objective 1: Identify lncRNA that are regulated by the AhR- We will assess if lncRNA are controlled by AhR presence and if these lncRNA are further increased/decreased by AhR activation. Cellular localization of these lncRNA (which is indicative of their biological function) will be identified by fluorescence in situ hybridization (FISH). Objective 2: Assess the capacity of select lncRNA to affect AhR-dependent cell survival:We will over-expression/knockdown of identified lncRNA in AhrWT and AhrKO cells using lentivirus/ shRNA, respectively, and cell death mechanisms assessed using techniques we have published. Objective 3: Determine if the AhR regulates lncRNA expression independent of its transcriptional ability: We will test whether the ability of the AhR to control lncRNA is due to DNA binding using cells derived from mice that have an AhR with mutations in the dioxin response element (DRE). Outcomes will include lncRNA expression (qRT-PCR) and localization (FISH). Significance: Since its discovery in 1976, the biological role of the AhR has remained elusive. We believe that the AhR plays a fundamental role in cell biology as a master regulator of lncRNA, a fundamental new concept in the natural sciences.