Genome architecture dynamics and expression

DNA测序技术的加速进步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、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（即结构动力学）以及这如何影响基因组表达。这个项目的优势建立在我们在线虫分子生物学和开发和应用创新生物信息学工具方面的综合经验之上。我们研究的是线虫的基因组，因为这一门既包含被充分研究的模式生物秀丽隐杆线虫，也包含许多病原体，包括致命的动物（包括人类）和植物寄生虫。拟议的研究将在未来5年内实现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动力学，以及这种动力学如何定义基因组新颖性和影响基因表达。我们假设新基因和异构体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、延伸或截断、全部或部分基因之间的融合以及新生形成。为了鉴定新基因，我们将分析秀丽隐杆线虫菌株（包括参考N2菌株和全球收集的菌株）之间的基因组差异，以及秀丽隐杆线虫与briggsae（研究最广泛的姐妹种）和其他相关线虫物种之间的基因组差异，然后研究它们如何促进新基因的形成。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，一个基因的新同工异构体的形成还不是很清楚。我们最近对秀丽隐杆线虫和秀丽隐杆线虫的RNA-seq结果进行了定量比较，结果表明这两个物种的同源物可能具有截然不同的剪接，这表明形成了新的同工异构体。通过对各种菌株和物种的直接RNA-seq分析，将发现新的同种异构体（以及新生基因）。我们将通过计算和实验方法评估预测的新基因的功能。第二个目标是研究转录水平上的基因表达调控，重点研究我们之前克隆鉴定的秀丽隐杆线虫纤毛基因MAK/dyf-5的转录调控。dyf-5是一个保守的基因，广泛存在于包括绿藻莱茵衣藻和人类在内的生物中。dyf-5的功能缺失导致纤毛发育和功能缺陷。我们将进行基因筛选，以确定影响荧光蛋白tdTomato标记的dyf-5表达的突变体。这些突变基因将通过基因定位、基因组测序和生物信息学分析进行鉴定，随后进行分子研究，以确定它们对dyf-5精确表达的功能贡献。我们期望找到与RFX/DAF-19一起起作用的共转录因子，RFX/DAF-19是几乎所有纤毛基因表达所必需的转录因子。总的来说，对这些目标的研究将揭示基因组新颖性和表型多样性产生的机制，这有助于物种形成的开始。在此研究过程中开发的生物信息学工具将极大地促进比较基因组学领域的发展。

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.