Comparative 3D Genome Architecture in Vertebrates (Hi-C)

Three-dimensional genome architecture influences the regulation of essential nuclear processes, such as gene transcription. However, how 3D genome architecture is affected by evolutionary forces within major lineages remains unclear. Here, we report a comprehensive comparison of 3D genomes, using high resolution Hi-C data in fibroblast cells of fish, chickens, and 10 mammalian species. This analysis shows a correlation between genome size and chromosome length that affects chromosomal territory (CT) organization in the upper hierarchy of genome architecture, whereas lower hierarchical features, including local transcriptional availability of DNA, are selected through vertebrate's evolution. Further, conservation of topologically associating domains (TADs) appears strongly associated with the modularity of expression profiles across species. Additionally, LINE and SINE transposable elements likely contribute to heterochromatin and euchromatin organization, respectively, during the evolution of genome architecture. These findings can guide ongoing investigations of genome evolution by extending our understanding of the mechanisms shaping genome architecture. Overall design: To comprehensively explore the evolutionary principles governing 3D genome architecture and to assess the contributions of genome architecture to transcriptional regulation across species, we performed comparative analyses of high-throughput chromosome conformation capture (Hi-C) in fibroblast cells of 12 vertebrates, including two representative mammalian lineages: euarchontoglires including humans, rhesus macaques, mice, rats, and rabbits; boreoeutheria including dogs, cats, pigs, sheep, and cows; as well as two non-mammals including birds (chickens) and fish (zebrafish). We performed the Hi-C experiments in 25 fibroblasts from 11 vertebrates with 1 to 5 biological replicates (distinct cell lines or primary cells derived from different individuals) for each species, produced a total of ~ 5.75-billion uniquely aligned contacts with an average depth of ~230 million (M) contacts per library (range from ~102 M for zebrafish with a relatively small genome size of ~ 1.23 Gb to ~442 M for mouse with a genome size of ~2.73 Gb). We also combined the resulting datasets with those previously generated by us for four pig fibroblasts (pig_DB-2, pig_DB-3, pig_RC-7, and pig_RC-8) (X Tian et al.,2020) and two mouse fibroblasts (mouse_3T6 and mouse_MEF) (M He et al.,2018) using the same experimental protocol. Among these, ~ 63.03% are intra-chromosomal contacts, of which ~74.40% were occurred within 10 Mb. After KR-normalization and quantile-normalization, we generated 31 intra-chromosomal contact maps at 20-kb resolution for each of 31 libraries. With the bin size at 20-kb, there are about 83.12% of bins have at least 1,000 intra-chromosomal contacts. TAD and compartment A/B were also identified by using the 20-kb matrix.

三维基因组架构可调控诸多关键核过程，例如基因转录。然而，主要演化支内的演化力量如何影响三维基因组架构，目前仍不明确。本研究基于鱼类、鸡类及10种哺乳动物成纤维细胞的高分辨率高通量染色体构象捕获（Hi-C）数据，开展了全面的三维基因组比较分析。分析结果显示，基因组大小与染色体长度的相关性，会影响基因组架构上层级的染色体区域（chromosomal territory, CT）组织；而基因组架构的低层级特征，包括DNA的局部转录可及性，则是通过脊椎动物演化过程被选择保留的。进一步研究发现，拓扑关联结构域（topologically associating domains, TADs）的保守性，与不同物种间表达谱的模块化特征显著相关。此外，长散在核元件（long interspersed nuclear elements, LINE）与短散在核元件（short interspersed nuclear elements, SINE）可能分别在基因组架构演化过程中，参与异染色质与常染色质的组织构建。本研究结果拓展了我们对塑造基因组架构的分子机制的认知，可为后续基因组演化相关研究提供指导。整体实验设计：为全面解析调控三维基因组架构的演化规律，并评估跨物种基因组架构对转录调控的贡献，我们对12种脊椎动物成纤维细胞的Hi-C数据开展了比较分析。该12种脊椎动物涵盖两大代表性哺乳动物演化支：灵长总目（euarchontoglires），包含人类、恒河猴、小鼠、大鼠与家兔；以及劳亚兽总目（boreoeutheria），包含犬、猫、猪、羊与牛；另外还包括2种非哺乳动物：鸟类（鸡）与鱼类（斑马鱼）。我们为11种脊椎动物的成纤维细胞构建了Hi-C文库，每个物种设置1至5个生物学重复（即源自不同个体的独立细胞系或原代细胞），最终获得约57.5亿条唯一比对的接触读段，每个文库的平均测序深度约为2.3亿（M）个接触对，测序深度范围从基因组较小（约1.23 Gb）的斑马鱼的约1.02亿，到基因组约2.73 Gb的小鼠的约4.42亿不等。我们还将本次生成的数据集与此前本团队采用相同实验流程获得的4个猪成纤维细胞（pig_DB-2、pig_DB-3、pig_RC-7及pig_RC-8，X Tian等，2020）及2个小鼠成纤维细胞（mouse_3T6及mouse_MEF，M He等，2018）的数据集进行了合并。其中约63.03%为染色体内接触对，此类接触对中约74.40%发生在10 Mb范围内。经KR标准化与分位数标准化处理后，我们为31个文库分别生成了分辨率为20 kb的染色体内接触矩阵。以20 kb作为分箱区间时，约83.12%的分箱至少包含1000个染色体内接触对。我们还利用该20 kb分辨率的矩阵，预测了TAD与染色质区室A/B（compartment A/B）。