拟南芥转录起始区反义长非编码RNA形成R-loop的调控机制及功能研究

Regulation of gene expression in multiple layers is important for organisms, of which transcription initiation and orientation determination are prerequisites. However, the knowledge of how to regulate gene expressions through these two layers is still limited. R-loop is a special chromatin structure containing one strand of RNA:DNA hybrid and another displaced strand as a single-stranded DNA. Our recent genome-wide R-loop profiling results revealed that R-loop is commonly distributed in Arabidopsis genome, and we identified a peculiar type of R-loop, which is formed by antisense lncRNAs near transcription start sites (named as asTSS_R-loops). This proposal will comprehensively investigate the asTSS_R-loops distribution in the Arabidopsis genome, explore the regulatory mechanisms of their dynamic changes, as well as their roles in regulating the adjacent gene expression. In addition, we will identify new factors that are involved in asTSS_R-loops regulation. Implementation of this project will extend our knowledge about the functions of transcription start site located antisense lncRNAs and explore the new functions of R-loops in cellular processes, such as regulation of transcription initiation. This study is also important for an in-depth understanding of organism gene transcription and its regulation in various biological processes, such as development and environmental adaption.

转录水平调节的基因表达对生物体至关重要，其中基因的转录起始和方向决定是基因表达的先决条件，但相应的调节机制还知之甚少。R-loop是由RNA:DNA杂合链和单链DNA所组成的一类特殊染色质结构。我们最新结果显示R-loop在模式植物拟南芥基因组中广泛分布，并发现一类特有的R-loop，此类R-loop是由转录起始区反义lncRNAs所形成（简称asTSS_R-loops）。本申请将深入并系统的研究asTSS_R-loops在基因组上的分布特征，动态变化调控机制，及它们在影响其临近基因转录的功能和调节机制。此外我们还将挖掘参与调控asTSS_R-loops的新因子。本项目实施将扩展我们认识植物中一类转录起始区反义lncRNAs的新功能，探究R-loop在调节基因转录起始等过程中的新功能，对深入理解生物体基因转录及其所调节的生长发育及环境适应性等各种生物学过程有重要意义。

在本研究中，我们开发了ssDRIP-seq技术，绘制了拟南芥全基因组R-loop分布图谱。通过分析ssDRIP-seq数据，我们发现R-loop是一种在拟南芥基因组中广泛存在的染色质结构。我们进一步绘制了拟南芥在不同发育时期以及各种胁迫响应下的R-loop图谱，并揭示了拟南芥R-loop特有的动态变化特征。R-loop存在模式比较稳定，并且在拟南芥世代之间会产生重排恢复。通过对不同的生理发育时期的asTSS-R-loop （转录起始区域的反义RNA形成的R-loop）研究表明，萌发幼苗和花的asTSS-R-loop与14天苗的叶子asTSS-R-loop差异显著。相反，35天植株的老叶和新叶asTSS-R-loop差异不显著。通过进一步对asTSS-R-loop进行聚类分析，以及和RNA表达水平以及染色体状态等的关联分析，发现asTSS-R-loop具有其自身的特征，其变化并未和RNA的积累量有显著的相关性。进一步的分析揭示了R-loop有可能作为一种顺式作用的基因表达调控元件。