Mechanisms regulating Evf2 long non-coding RNA transcriptional control

描述（由申请人提供）：尽管发育调节转录因子在模式化和分化中的关键作用已经确定了一段时间，但长非编码RNA（lncRNA）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，Evf 2和Evf 1，它们调节GABA能中间神经元发育所必需的转录因子。我们对转录为剪接反义转录本的lncRNA Evf 2的研究表明，lncRNA具有特异的基因调控功能。evf 2在脊椎动物中是保守的（超保守的），并在发育中的前脑中被音速刺猬激活。 我们表明，Evf 2形成一个复杂的DLX 2同源结构域转录因子和调节Dlx 5/6增强子活性。通过对本实验室建立的Evf 2功能缺失小鼠模型的分析，我们发现Evf 2不仅是Dlx 5和Dlx 6转录调控所必需的，而且也是胚胎脑和成年海马回路中GABA能中间神经元发育所必需的。虽然我过去的大部分工作都集中在Evf 2 lncRNA在中间神经元发育中的生物学作用上，但涉及Evf 2长非编码RNA依赖性转录调控的精确机制仍然未知。该研究将利用小鼠遗传学、RNA和蛋白质生物化学以及胚胎脑细胞核中的RNA云可视化来确定Evf 2 lncRNA转录调控的机制。其中一些技术是新颖的，如使用RNA云可视化在3维，单和双等位基因分析，并在体外 DNA依赖的RNA-蛋白质复合物组装;该方法作为一个整体是新颖的，因为这种方法的组合来研究lncRNA基因调控还没有报道。这些研究将提供对基因调控的一个关键方面的理解，该基因调控与神经发育和功能以及生长控制和癌症特别相关，其中已经鉴定出多种超保守lncRNA。 公共卫生相关性：这项资助旨在研究超保守的长非编码RNA Evf 2如何调节GABA能中间神经元前体的基因表达。由于GABA能中间神经元功能的改变与癫痫、自闭症、精神分裂症和精神发育迟滞有关，因此对GABA能中间神经元正常发育的研究对于理解多种神经精神疾病的分子基础至关重要。此外，最近在癌症中鉴定出的超保守的长非编码RNA已经研究了这类分子如何控制与生长控制相关的基因调控。最终，希望更好地理解神经元亚群和细胞生长控制中的基因调控将导致开发用于治疗人类神经系统疾病和癌症的新疗法。

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.