Genome architecture dynamics and expression

基因组结构动态和表达

• 批准号: RGPIN-2014-06018
• 负责人: Chen, Nansheng
• 金额: $ 3.42万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=567535
• 关键词: Genome; architecture; dynamics; expression

Accelerating advances in DNA sequencing technologies is driving the creation of an unprecedented genome resource for a wide range of organisms, presenting golden opportunities for mining the genome information that instructs the generation of biological processes including development, aging, metabolism, and learning and memory. The long-term goal of my research program is to understand genome structural changes (i.e., architecture dynamics) and how this affects genome expression. The strength of this program builds on our combined experience in nematode molecular biology and in developing and applying innovative bioinformatics tools. We work with the genomes of nematodes because this phylum contains both the well-studied model organism C. elegans, and many pathogens including deadly animal (including human) and plant parasites. The proposed research will address two complementary short-term objectives over the next 5 years, aiming to understand genome expression from both the whole-genome level and a single-gene level. Our first objective is to investigate genome architecture dynamics and how such dynamics define genomic novelty and affect gene expression. We hypothesize that the formation of novel genes and isoforms is the driving force in shaping genomic and phenotypic diversity. Novel genes can form through a variety of mechanisms including duplication, extension or truncation of genes, fusion between full or partial genes, and de novo formation. In order to identify novel genes, we will analyze genomic differences between C. elegans strains (including the reference N2 strain and strains collected worldwide), as well as between C. elegans and C. briggsae (the most extensively studied sister species) and other related nematode species, followed by examining how they contribute to the formation of novel genes. Candidate novel genes will be validated through transcriptome sequencing and analysis (RNA-seq), as well as full-length cDNA analysis. In contrast, the formation of novel isoforms of a gene is not well understood. Our recent quantitative comparison RNA-seq results of C. elegans and C. briggsae revealed that orthologs in these two species can have drastically different splicing, suggesting the formation of novel isoforms. Novel isoforms (as well as de novo genes) will be uncovered via direct RNA-seq analysis of various strains and species. We will evaluate the function of the predicted novel genes through computational and experimental approaches. The second objective is to study gene expression regulation at the level of transcription, focusing on the transcriptional regulation of the ciliary gene MAK/dyf-5 in C. elegans that we have cloned and identified previously. dyf-5 is a conserved gene found in a wide range of organisms including the green algae Chlamydomonas reinhardtii and humans. Loss-of-function of dyf-5 leads to defective cilia development and function. We will carry out genetic screening to identify mutants that affect the expression of dyf-5 that is tagged with fluorescence protein tdTomato. These mutated genes will be identified through genetic mapping, genome sequencing, and bioinformatics analysis, followed by molecular studies of their functional contribution to the precise expression of dyf-5. We expect to identify co-transcription factors that function together with RFX/DAF-19, a transcription factor that is required for the expression of essentially all ciliary genes. Collectively, research of these objectives will reveal mechanisms underlying the creation of genomic novelty and phenotypic diversity, which contribute to the initiation of speciation. Bioinformatics tools developed in the course of this proposed research will greatly facilitate the field of comparative genomics.

DNA测序技术的加速发展正在推动为广泛的生物体创造前所未有的基因组资源，为挖掘基因组信息提供了黄金机会，这些信息指导生物过程的产生，包括发育，衰老，新陈代谢以及学习和记忆。我的研究计划的长期目标是了解基因组结构变化（即，架构动力学）以及这如何影响基因组表达。该计划的优势建立在我们在线虫分子生物学以及开发和应用创新生物信息学工具方面的综合经验之上。我们研究线虫的基因组，因为这个门包含了研究得很好的模式生物C。线虫和许多病原体，包括致命的动物（包括人类）和植物寄生虫。该研究将在未来5年内解决两个互补的短期目标，旨在从全基因组水平和单基因水平了解基因组表达。我们的第一个目标是研究基因组结构的动态，以及这种动态如何定义基因组的新奇和影响基因表达。我们假设新基因和亚型的形成是形成基因组和表型多样性的驱动力。新基因可以通过多种机制形成，包括基因的复制、延伸或截短、全部或部分基因之间的融合以及从头形成。为了鉴定新的基因，我们将分析C。elegans菌株（包括参考菌株N2和世界范围内收集的菌株）之间以及C. elegans和C. briggsae（研究最广泛的姐妹物种）和其他相关的线虫物种，然后研究它们如何有助于新基因的形成。候选新基因将通过转录组测序和分析（RNA-seq）以及全长cDNA分析进行验证。相比之下，新的基因异构体的形成还没有得到很好的理解。我们最近的定量比较C.和秀丽隐杆线虫C. Briggsae揭示了这两个物种中的直系同源物可以具有显著不同的剪接，这表明形成了新的同种型。新的亚型（以及从头基因）将通过各种菌株和物种的直接RNA-seq分析来发现。我们将通过计算和实验方法评估预测的新基因的功能。第二个目的是在转录水平上研究基因表达调控，重点研究纤毛基因MAK/dyf-5在C.我们以前克隆并鉴定过的线虫。dyf-5是在包括绿色藻类莱茵衣藻和人类在内的广泛生物体中发现的保守基因。dyf-5功能缺失导致纤毛发育和功能缺陷。我们将进行遗传筛选，以确定影响荧光蛋白tdTomato标记的dyf-5表达的突变体。这些突变的基因将通过遗传作图、基因组测序和生物信息学分析来鉴定，然后通过分子研究来确定它们对dyf-5精确表达的功能贡献。我们期望鉴定与RFX/RFX-19一起起作用的共转录因子，RFX/RFX-19是基本上所有纤毛基因表达所需的转录因子。总的来说，对这些目标的研究将揭示基因组新奇和表型多样性产生的机制，这有助于物种形成的启动。在这项拟议的研究过程中开发的生物信息学工具将大大促进比较基因组学领域。