Institute of Molecular Genetics of Montpellier (IGMM), University of Montpellier, CNRS, Montpellier, France.
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris-sud and University Paris-Saclay, Gif-sur-Yvette, France.
Genome Biol. 2019 Dec 12;20(1):272. doi: 10.1186/s13059-019-1896-8.
Genomic imprinting is essential for mammalian development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. So far, the detailed allele-specific chromatin organization of imprinted gene domains has mostly been lacking. Here, we explored the chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints-the Igf2-H19 and Dlk1-Dio3 domains-and assessed the involvement of the insulator protein CTCF in mouse cells.
Both imprinted domains are located within overarching topologically associating domains (TADs) that are similar on both parental chromosomes. At each domain, a single differentially methylated region is bound by CTCF on the maternal chromosome only, in addition to multiple instances of bi-allelic CTCF binding. Combinations of allelic 4C-seq and DNA-FISH revealed that bi-allelic CTCF binding alone, on the paternal chromosome, correlates with a first level of sub-TAD structure. On the maternal chromosome, additional CTCF binding at the differentially methylated region adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal-specific sub-TAD organization. Perturbation of maternal-specific CTCF binding site at the Dlk1-Dio3 locus, using genome editing, results in perturbed sub-TAD organization and bi-allelic Dlk1 activation during differentiation.
Maternal allele-specific CTCF binding at the imprinted Igf2-H19 and the Dlk1-Dio3 domains adds an additional layer of sub-TAD organization, on top of an existing three-dimensional configuration and prior to imprinted activation of protein-coding genes. We speculate that this allele-specific sub-TAD organization provides an instructive or permissive context for imprinted gene activation during development.
基因组印记对于哺乳动物的发育至关重要，为探索染色质构象的细胞内差异提供了独特的范例。到目前为止，印迹基因区域的详细等位基因特异性染色质组织在很大程度上仍然未知。在这里，我们探索了受父源 DNA 甲基化印迹控制的两个保守印迹域的染色质结构——Igf2-H19 和 Dlk1-Dio3 域，并评估了绝缘子蛋白 CTCF 在小鼠细胞中的参与情况。
这两个印迹域都位于由父本染色体上的多个实例的双等位基因 CTCF 结合形成的拓扑关联域（TAD）内，而母本染色体上仅存在一个差异甲基化区域被 CTCF 结合。组合使用等位基因 4C-seq 和 DNA-FISH 揭示，仅在母本染色体上，双等位基因 CTCF 结合与第一级亚 TAD 结构相关。在母本染色体上，差异甲基化区域的额外 CTCF 结合增加了进一步的亚 TAD 组织层次，实质上劫持了现有的父本特异性亚 TAD 组织。使用基因组编辑破坏 Dlk1-Dio3 基因座上母本特异性 CTCF 结合位点会导致亚 TAD 组织紊乱和双等位基因 Dlk1 的激活。
印迹的 Igf2-H19 和 Dlk1-Dio3 域上母本等位基因特异性 CTCF 结合在现有的三维结构之上，并在蛋白编码基因的印迹激活之前增加了一个额外的亚 TAD 组织层次。我们推测这种等位基因特异性的亚 TAD 组织为发育过程中印迹基因的激活提供了一个指导或允许的环境。
