原生动物嗜热四膜虫新月小核的三维空间转录调控分子机制

Three-dimensional transcriptional regulation, which consists of chromosome interactions and noncoding RNAs，has been gradually accepted. However, the mechanism of this genome-wide regulation remains to be studied. This application will use the protozoan model Tetrahymena Thermophila, and 3D-DSL technology (mainly developed by the applicant) combined with other genomic techniques such as transcriptome sequencing (RNA-Seq), co-immunoprecipitation coupled with deep sequencing (ChIP-Seq) to study the three-dimensional transcriptioanl regulation in the transcriptional active crescent micronucleus. To uncover the molecular mechanism of three-dimensional transcriptional regulation during this activation period, we will identify transcriptome,cis-acting elements and chromosome interactions between different regions of the crescent micronucleus. Since Tetrahymena Thermophile is a model organism which shares a higher degree of conservation on evolution and function with humans than yeast, the results of this application will provide both experimental evidence and theoretical basis for understanding the molecular mechanism of three-dimensional transcriptional regulation in other organisms.

由染色体相互作用和非编码RNA构成的三维空间基因转录调控逐渐得到人们的认可。然而，在全基因组水平上实现这种三维空间基因转录调控的机制有待于进一步的研究。本项目拟以模式原生动物嗜热四膜虫特异转录活化的新月期小核为研究对象，运用项目申请人为主开发的3D-DSL技术结合转录组测序（RNA-Seq）、免疫共沉淀耦合深度测序（ChIP-Seq）等基因组学方法，检测新月期小核的转录情况，鉴定小核基因组中的顺式作用元件，并观察这一特殊时期小核染色体各区域间的相互作用，从而阐述新月期小核短暂转录活跃的三维空间基因调控作用和分子机制。鉴于四膜虫相对酵母等模式生物在进化上和人类具有更高程度上的功能保守性，本项目的研究成果将为理解其他物种中的三维空间转录调控分子机制提供实验依据和理论基础。

模式原生动物嗜热四膜虫一个细胞中同时存在两个细胞核，其基因组发育来源相同，但基因组大小、功能方面差异极大。本项目基于自行研发的分离经甲醛交联的四膜虫小核的方法，获得高纯度的嗜热四膜虫小核，并在此基础上建立和完善了嗜热四膜虫中的Hi-C和HiChIP实验体系，通过Hi-C等技术研究了嗜热四膜虫生殖核（小核）和营养核（大核）的染色质高级结构。结果表明嗜热四膜虫中不存在A/B compartment 结构，此外小核中存在TAD 结构而大核中则不存在TAD 结构。小核TAD boundaries 与染色质断裂位点高度吻合，提示小核TAD 结构与大核的染色体发育密切相关。此外，小核新月期特异的染色质相互作用也与小核在新月期的形态改变相吻合。本项目的研究结果首次提供了嗜热四膜虫的三维基因组结构图谱，并发现四膜虫中的染色质高级结构与大核染色体发育相关，同时也在整个三维基因组领域（不仅仅是四膜虫）揭示了TADs形成的新的生物学见解。