Comparative 3D Genome Architecture in Vertebrates (RNA-Seq)

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. 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基因转录等关键核过程，然而其在主要演化支中如何受演化力量影响仍未明确。本研究针对鱼类、鸡类及10种哺乳动物的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领地（CT, chromosomal territory）组织模式；而基因组架构的低层级特征，包括DNA的局部转录可及性，则通过脊椎动物演化过程被筛选保留。此外，拓扑关联结构域（TADs, topologically associating domains）的保守性，似乎与跨物种的表达谱模块化程度高度相关。另外，长散在核元件（LINE, long interspersed nuclear elements）与短散在核元件（SINE, short interspersed nuclear elements）转座因子，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调控。本研究发现可为后续基因组演化研究提供指导，拓展我们对塑造基因组架构的机制的认知。为全面探究调控三维基因组架构的演化原则，并评估基因组架构对跨物种转录调控的贡献，我们对12种脊椎动物的成纤维细胞开展了高通量染色体构象捕获（Hi-C）比较分析，涵盖两大代表性哺乳动物演化支：包括人类、恒河猴、小鼠、大鼠及兔的灵长总目（euarchontoglires）；以及包括犬、猫、猪、羊及牛的劳亚兽总目（boreoeutheria）；此外还包含两类非哺乳动物：鸟类（鸡）与鱼类（斑马鱼）。我们为11种脊椎动物的成纤维细胞完成了Hi-C实验，每个物种设置1至5个生物学重复（取自不同个体的独立细胞系或原代细胞），共获得约57.5亿个唯一比对的染色质接触片段，每个文库的平均测序深度约为2.3亿个接触片段（跨度范围从基因组大小约1.23 Gb的斑马鱼的~1.02亿，到基因组大小约2.73 Gb的小鼠的~4.42亿）。我们还将本次生成的数据集，与此前我们采用相同实验流程获取的4个猪成纤维细胞数据集（pig_DB-2、pig_DB-3、pig_RC-7及pig_RC-8，X Tian等，2020）及2个小鼠成纤维细胞数据集（mouse_3T6及mouse_MEF，M He等，2018）进行了整合。其中约63.03%为染色体内接触片段，其中约74.40%发生在10 Mb范围内。经KR归一化与分位数归一化处理后，我们为31个文库分别生成了分辨率为20-kb的染色体内接触图谱。在20-kb的分箱尺度下，约83.12%的分箱至少包含1000个染色体内接触片段。我们还利用该20-kb分辨率的矩阵，识别了拓扑关联结构域（TAD）与A/B区室（compartment A/B）。