Mechanisms regulating Evf2 long non-coding RNA transcriptional control

描述（由申请人提供）：虽然发育调节转录因子在模式和分化中的关键作用已经确立了一段时间，但长链非编码rna （lncRNAs）的作用才刚刚开始被理解。我的实验室发现了新的非编码rna， Evf2和Evf1，它们调节转录因子对gaba能中间神经元发育至关重要。我们对Evf2的研究表明，lncRNA具有特定的基因调控功能。Evf2是一个拼接反义转录物。Evf2在脊椎动物中是保守的（超保守的），并在发育中的前脑中被sonic hedgehog激活。我们发现Evf2与DLX2同源域转录因子形成复合体，并调节Dlx5/6增强子的活性。通过对我实验室建立的Evf2功能缺失小鼠模型的分析，我们发现Evf2不仅是Dlx5和Dlx6转录调控的必要条件，也是胚胎脑和成人海马回路中gaba能中间神经元发育的必要条件。虽然我过去的大部分工作都集中在Evf2 lncRNA在中间神经元发育中的生物学作用上，但Evf2长链非编码rna依赖性转录调控的确切机制仍然未知。拟开展的研究将利用小鼠遗传学、RNA和蛋白质生物化学、胚胎脑核RNA云可视化等方法来确定Evf2 lncRNA转录调控的机制。其中一些技术是新颖的，例如在三维空间中使用RNA云可视化，单等位基因和双等位基因分析，以及在体外

DESCRIPTION (provided by applicant): Although a critical role for developmentally regulated transcription factors in patterning and differentiation has been established for some time, the role of long non-coding RNAs (lncRNAs) is only beginning to be understood. My lab discovered novel non-coding RNAs, Evf2 and Evf1, that regulate transcription factors essential for proper GABAergic interneuron development. Our work on Evf2, an lncRNA transcribed as a spliced anti-sense transcript shows that lncRNAs play specific gene regulatory functions. Evf2 is conserved across vertebrates (ultraconserved), and activated by sonic hedgehog in the developing forebrain. We show that Evf2 forms a complex with the DLX2 homeodomain transcription factor and regulates Dlx5/6 enhancer activity. Through analysis of an Evf2 loss-of-function mouse model developed in my lab, we find that Evf2 is not only required for proper transcriptional regulation of Dlx5 and Dlx6, but also for proper GABAergic interneuron development in embryonic brain and adult hippocampal circuitry . While much of my past work has focused on the biological role of Evf2 lncRNA in interneuron development, the precise mechanisms involved in Evf2 long- non-coding RNA-dependent transcriptional regulation remain unknown. The proposed studies will use mouse genetics, RNA and protein biochemistry, and RNA cloud visualization in nuclei of embryonic brain to determine the mechanism of Evf2 lncRNA transcriptional regulation. Some of the techniques are novel, such as the use of RNA cloud visualization in 3 dimensions, mono and bi-allelic analysis, and in vitro DNA-dependent RNA-protein complex assembly; the approach as a whole is novel, as this combination of methods to study lncRNA gene regulation has not been reported. These studies will provide an understanding of a critical aspect of gene regulation with particular relevance to neural development and function, as well as growth control and cancer, where multiple ultraconserved lncRNAs have been identified. PUBLIC HEALTH RELEVANCE: This grant proposes to investigate how the ultraconserved long non-coding RNA Evf2 regulates gene expression in GABAergic interneuron precursors. Since altered GABAergic interneuron function has been linked to epilepsy, autism, schizophrenia, and mental retardation, studies on the normal development of GABAergic interneurons are critical to understanding the molecular basis for multiple neuropsychiatric disorders. In addition, the recent identification of ultraconserved long non-coding RNAs in cancer has made studies of how this class of molecules controls gene regulation relevant to growth control. Ultimately, it is hoped that a better understanding of gene regulation in neuronal subpopulations and cellular growth control will lead to the development of novel therapies for treatment of human neurological diseases and cancer.