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

背景：我的研究计划的长期目标是了解芳香烃受体(AhR)的生物学作用，这是一种几乎在我们所有细胞中都能找到的蛋白质。这是因为--尽管经过了几十年的研究--AhR的细胞功能(S)仍然未知。AhR是一种普遍表达的受体/转录因子，历史上已知可以介导人造环境污染物二恶英的毒性效应。作为对二恶英的反应，AhR易位到细胞核，并与二恶英反应元件(DRE)结合，从而引起毒性效应。使用广泛的生化、分子和遗传工具，在过去的5年里，我们已经证明AhR控制基本的细胞过程，独立于它对二恶英的反应。我们已经展示的生物过程是由AhR调节的，包括增殖和细胞存活。我们还发现，AhR调控这些细胞过程的新机制之一是通过非转录控制microRNA(MiRNA)的表达。MiRNAs是越来越多的非编码RNA(NcRNA)的一部分，其中还包括长的非编码RNA(LncRNAs)。LncRNA是长度超过200个核苷酸的非蛋白质编码转录本。虽然最初被认为是转录噪音(即“垃圾DNA”)，但现在人们认识到，lncRNA控制着许多与AhR相同的基本生物学功能--包括细胞生存和增殖。关于AhR和lncRNA几乎一无所知。假设：我们假设AhR控制在细胞生存中重要的lncRNA的表达。目的：本研究计划的总体目标是确定AhR对lncRNA的调控是其控制细胞凋亡的基础。目的1：确定受AhR调控的lncRNA-我们将评估lncRNA是否受AhR的存在控制，以及这些lncRNA是否因AhR的激活而进一步增加/减少。这些lncRNA(指示其生物学功能)的细胞定位将通过荧光原位杂交(FISH)进行鉴定。目的：评估选择的lncRNA对AhR依赖的细胞存活的影响：我们将利用慢病毒/shRNA分别在AhrWT和AhrKO细胞中过表达/敲除已鉴定的lncRNA，并利用我们已发表的技术评估细胞死亡机制。目的3：确定AhR是否独立于其转录能力调节lncRNA的表达：我们将使用来自具有二恶英反应元件(DRE)突变的AhR的小鼠的细胞来测试AhR控制lncRNA的能力是否归因于DNA结合。结果将包括lncRNA表达(qRT-PCR)和定位(FISH)。意义：自1976年发现以来，AhR的生物学作用一直难以捉摸。我们认为，AhR作为lncRNA的主要调节者在细胞生物学中发挥着基础性的作用，lncRNA是自然科学中一个基本的新概念。

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.