Recent studies have characterized the genomic structures of many eukaryotic cells, often focusing on their relation to gene expression. However, these studies have largely investigated cells grown in 2D cultures, although the transcriptomes of 3D-cultured cells are generally closer to their in vivo phenotypes. To examine the effects of spatial constraints on chromosome conformation, we investigated the genomic architecture of mouse hepatocytes grown in 2D and 3D cultures using in situ Hi-C. Our results reveal significant differences in higher-order genomic interactions, notably in compartment identity and strength as well as in topologically associating domain (TAD)–TAD interactions, but only minor differences are found at the TAD level. Our RNA-seq analysis reveals an up-regulated expression of genes involved in physiological hepatocyte functions in the 3D-cultured cells. These genes are associated with a subset of structural changes, suggesting that differences in genomic structure are critically important for transcriptional regulation. However, there are also many structural differences that are not directly associated with changes in gene expression, whose cause remains to be determined. Overall, our results indicate that growth in 3D significantly alters higher-order genomic interactions, which may be consequential for a subset of genes that are important for the physiological functioning of the cell.

最近的研究已经描述了许多真核细胞的基因组结构，通常集中在它们与基因表达的关系上。然而，这些研究主要研究了在 2D 培养中生长的细胞，尽管 3D 培养细胞的转录组通常更接近它们的体内表型。为了检查空间限制对染色体构象的影响，我们使用原位Hi-C研究了在 2D 和3D 培养中生长的小鼠肝细胞的基因组结构。我们的结果揭示了高阶基因组相互作用的显着差异，特别是在隔室身份和强度以及拓扑关联域（TAD)–TAD 相互作用，但在 TAD 级别仅发现细微差异。我们的 RNA-seq 分析揭示了参与 3D 培养细胞中生理性肝细胞功能的基因表达上调。这些基因与结构变化的一个子集相关，表明基因组结构的差异对于转录调控至关重要。然而，也有许多与基因表达变化没有直接关系的结构差异，其原因仍有待确定。总体而言，我们的结果表明，3D 中的生长显着改变了高阶基因组相互作用，这可能对对细胞生理功能很重要的基因子集产生影响。