Functional genomic and transcriptomic analysis of amphioxus and the origin of vertebrate genomic traits [RNA-Seq]

What genomic changes led to the origin of vertebrates remains a mystery. On the one hand, animal evolution is thought to be driven mostly by changes in the cis-regulatory regions of a shared conserved and toolkit of developmental genes. On the other hand, vertebrates experienced two rounds of whole genome duplication (WGD) that increased their gene repertoire, particularly of regulatory genes controlling embryo development. To shed light into the origin and evolution of the vertebrate regulatory genome, we have generated an unprecedented transcriptomic and epigenomic resource for the non-duplicated genome of the European amphioxus, a closely related invertebrate chordate. These data include RNA-seq for more than 35 developmental stages and adult tissues, CAGE-seq, ChIP-seq, bisulphite-seq and ATAC-seq for several developmental stages and adult tissues. By comparing these data sets with equivalent novel and previously available data for various vertebrate species, especially zebrafish, we uncovered multiple conserved and vertebrate-specific regulatory landmarks. We first identify a conserved chordate phylotypic stage, a developmental period in which different chordate species show the highest gene expression similarity. We also shed light on the origin of enhancer demethylation in vertebrates, by identifying, for the first time in an invertebrate species, differentially methylated enhancers. Furthermore, we show that conserved clusters of co-expressed and tissue-specific genes display similar enrichments for cis-regulatory motifs between amphioxus and vertebrates. Finally, we study the impact of vertebrate WGDs on the evolution of gene regulation, providing the first genome-wide quantitative assessment of sub-functionalization and neo-functionalization processes after the vertebrate WGDs; changing the way in which these evolutionary mechanisms have been traditionally understood. Overall design: RNA-seq assays in different developmental stages of european amphioxus, zebrafish and medaka驱动脊椎动物起源的基因组改变至今仍是未解之谜。一方面，学界普遍认为动物演化主要由共享的保守发育基因工具箱的顺式调控区域变异所驱动。另一方面，脊椎动物经历了两轮全基因组复制（whole genome duplication, WGD），扩增了其基因库，尤其是调控胚胎发育的调控基因数量。为阐明脊椎动物调控基因组的起源与演化，我们针对亲缘关系紧密的无脊椎脊索动物——欧洲文昌鱼的未经历全基因组复制的基因组，构建了前所未有的转录组与表观基因组数据集。该数据集包含覆盖35个以上发育阶段与成体组织的RNA测序（RNA-seq）数据，以及覆盖多个发育阶段与成体组织的cap分析基因表达测序（CAGE-seq）、染色质免疫沉淀测序（ChIP-seq）、亚硫酸氢盐测序（bisulphite-seq）与转座酶可及性测序（ATAC-seq）数据。通过将上述数据集与多种脊椎动物（尤其是斑马鱼）的同类新增及已公开数据进行比对，我们发现了多个保守的及脊椎动物特有的调控特征。我们首先鉴定出一个保守的脊索动物系统型阶段——即不同脊索动物物种间基因表达相似性最高的发育时期。我们还通过在无脊椎动物中首次鉴定出差异甲基化增强子，阐明了脊椎动物增强子去甲基化的起源。此外，我们证实文昌鱼与脊椎动物中，共表达且组织特异性的基因保守簇在顺式调控基序的富集模式上具有高度相似性。最后，我们探究了脊椎动物全基因组复制对基因调控演化的影响，首次完成了脊椎动物全基因组范围内全基因组复制后亚功能化与新功能化过程的定量评估，改写了学界对这类演化机制的传统认知。实验整体设计：针对欧洲文昌鱼、斑马鱼与青鳉的不同发育阶段开展RNA测序实验。