TRIC: A novel method for genome-wide identification and functional characterization of ncRNA:DNA triplexes

许多非编码RNA(NcRNAs)通过影响转录因子和染色质修饰物的关联，在不同的基因组位置发挥表观遗传调节作用。然而，关于ncRNA如何识别其靶点并与其相互作用，人们知之甚少。序列特异的DNA识别可以通过形成一个三螺旋(Triplex)来介导，在这个三螺旋中，ncRNA的一部分插入到DNA双链中，形成氢键。事实上，最近的一些研究提供了ncRNA的证据：在人类和小鼠中存在DNA三联体，但到目前为止，在细胞中直接和公正地检测这些结构是不可能的。为了克服这一障碍，我开发了一种名为TIRE(细胞中的三链识别)的方法，它使用三链结合蛋白Stm1TBD从细胞中分离出ncRNA：DNA三链。在建立这种方法的过程中，我在小鼠Kras基因的启动子上发现了一个ncRNA：DNA三联体。这个项目的目的之一是使用TIC来剖析Kras三联体的调节功能。在这方面，我们还将跟进初步结果，表明ncRNA：DNA三联体的形成与另一种非规范核酸结构--G-四联体竞争。这两种结构对Kras表达的相反作用与转录因子hnRNP K、Miz1和Myc的不同结合有关。通过阐明三链、G-四链和三个转录因子之间的相互作用，我们将获得关于Kras原癌基因如何调控的重要见解。鉴于许多原癌基因启动子包含有可能形成三链或G-四链的序列，Kras启动子很可能作为这种“构象转换”的范例。该项目的第二部分旨在通过将TIC与下一代测序相结合来系统地鉴定ncRNA：DNA三联体。初步实验证实了Kras三联体的存在，并发现了另一种新的ncRNA：Rab5c启动子上的DNA三联体。在拟议的项目中，我们将增加RNA测序深度，并在TRIC之后进行额外的DNA测序，以全面检测人和小鼠细胞中形成三链的RNA和DNA序列。然后，根据定义的三链碱基配对规则，以生物信息学的方式推导出ncRNA：DNA三链的基因组分布。这些结果将建立关于ncRNA：DNA三联体在基因调控中作用的基础知识，从而启动关于ncRNA与表观遗传学之间联系的新研究。

Many non-coding RNAs (ncRNAs) act as epigenetic regulators at distinct genomic sites by impacting the association of transcription factors and chromatin modifiers. However, little is known about how ncRNAs recognize and interact with their target sites. Sequence-specific DNA-recognition can be mediated by formation of a triple helix (triplex), in which a part of a ncRNA intercalates into the DNA duplex and forms hydrogen bonds. Indeed, a few recent studies provide evidence for ncRNA:DNA triplexes in humans and mice, but so far the direct and unbiased detection of these structures in cells was not possible. To overcome this obstacle, I have developed a method termed "TRIC" (triplex recognition in cells), which uses the triplex-binding protein Stm1TBD to isolate ncRNA:DNA triplexes from cells. In the course of establishing this approach, I have discovered a ncRNA:DNA triplex at the promoter of the mouse Kras gene.One aim of this project is to use TRIC for dissecting the regulatory function of the Kras triplex. In this regard we will also follow up preliminary results showing that formation of the ncRNA:DNA triplex competes with another non-canonical nucleic acid structure, a G-quadruplex. The opposing effects of both structures on Kras expression correlate with differential binding of the transcription factors hnRNP K, Miz1 and Myc. By elucidating the interplay between triplex, G-quadruplex and the three transcription factors we will gain important insights into how the Kras proto-oncogene is regulated. Given that many proto-oncogene promoters contain sequences with the potential to form either triplexes or G-quadruplexes, the Kras promoter is likely to serve as a paradigm for such a "conformational switch". The second part of the project aims at the systematic identification of ncRNA:DNA triplexes by combining TRIC with next-generation sequencing. Pilot experiments in which TRIC was followed by RNA-sequencing confirmed the existence of the Kras triplex and uncovered another novel ncRNA:DNA triplex at the Rab5c promoter. In the proposed project, we will increase the RNA sequencing depth and will conduct additional DNA sequencing after TRIC to comprehensively detect triplex-forming RNA- and DNA-sequences in the human and mouse cells. The genomic distribution of ncRNA:DNA triplexes will then be bioinformatically deduced according to the defined triplex base pairing rules. The results will establish fundamental knowledge about the role of ncRNA:DNA triplexes in gene regulation, thereby initiating new research on the connection between ncRNAs and epigenetics.