Extended supplementary data for manuscript: Extensive folding variability between homologous chromosomes in mammalian cells

Genetic variation and 3D chromatin structure have major roles in gene regulation. Due to challenges in mapping chromatin conformation with haplotype-specific resolution, the effects of genetic sequence variation on 3D genome structure and gene expression imbalance remain understudied. Here, we applied Genome Architecture Mapping (GAM) to a hybrid mouse embryonic stem cell (mESC) line with high density of single nucleotide polymorphisms (SNPs). GAM resolved haplotype-specific 3D genome structures with high sensitivity, revealing extensive allelic differences in chromatin compartments, topologically associating domains (TADs), long-range enhancer-promoter contacts, and CTCF loops. Architectural differences often coincide with allele-specific differences in gene expression, mediated by Polycomb repression. We show that histone genes are expressed with allelic imbalance in mESCs, are involved in haplotype-specific chromatin contact marked by H3K27me3, and are targets of Polycomb repression through conditional knockouts of Ezh2 or Ring1b. Our work reveals highly distinct 3D folding structures between homologous chromosomes, and highlights their intricate connections with allelic gene expression.遗传变异与三维染色质结构在基因调控中发挥关键作用。由于受限于单倍型分辨率下的染色质构象图谱绘制难题，遗传序列变异对三维基因组结构及基因表达失衡的影响迄今仍未得到充分研究。本研究将基因组架构图谱（Genome Architecture Mapping, GAM）技术应用于携带高密度单核苷酸多态性（single nucleotide polymorphisms, SNPs）的杂交小鼠胚胎干细胞（mouse embryonic stem cell, mESC）系。GAM技术以高灵敏度解析出单倍型特异性三维基因组结构，揭示了染色质区室、拓扑关联结构域（topologically associating domains, TADs）、远程增强子-启动子互作及CTCF环中广泛存在的等位基因差异。这类架构差异通常与多梳蛋白介导的等位基因特异性基因表达差异相契合。本研究发现，组蛋白基因在小鼠胚胎干细胞中呈现等位基因失衡表达，且参与以组蛋白H3赖氨酸27三甲基化（H3K27me3）为标记的单倍型特异性染色质互作；同时通过Ezh2或Ring1b条件性敲除实验证实，这类基因是多梳蛋白抑制通路的靶标。本研究揭示了同源染色体之间截然不同的三维折叠模式，并突显了其与等位基因表达之间的复杂关联。