Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes

CpG islands (CGIs) represent a unique and widespread genetic feature of vertebrate genomes, being associated with ~70% of all annotated gene promoters. CGIs have been proposed to control transcription initiation by conferring nearby promoters with unique chromatin properties. In addition, there are thousands of distal or orphan CGIs (oCGIs) whose functional relevance and mechanism of action are barely known. Here we show that oCGIs are an essential component of poised enhancers (PEs) that boost their long-range regulatory activity and dictate the responsiveness of their target genes. Using a CRISPR/Cas9 knock-in strategy in mESC, we introduced PEs with or without oCGIs within topological associating domains (TADs) harbouring genes with different types of promoters. By evaluating the chromatin, topological and regulatory properties of the engineered PEs, we uncovered that, rather than increasing their local activation, the oCGI boost the physical and functional communication between PEs and distally located developmental genes. Furthermore, we demonstrate that developmental genes with CpG rich promoters are particularly responsive to PEs and that such responsiveness depends on the presence of oCGI. Therefore, our work unveils a novel role for CGIs as genetic determinants of the compatibility between genes and enhancers, which has major implications for the current understanding of how developmental gene expression programs are deployed as well as for our ability to predict the pathological consequences of human structural variation. 4C-seq from mouse embryonic stem cells (mESC) and anterior neural progenitor cells (AntNPC)

CpG岛（CpG Islands，CGIs）是脊椎动物基因组中一类独特且广泛分布的遗传特征，与已注释的全部基因启动子中约70%相关。已有研究表明，CGIs可通过赋予邻近启动子独特的染色质特性来调控转录起始。此外，存在数千个远端孤立CpG岛（orphan CGIs，oCGIs），其功能相关性与作用机制仍有待深入解析。本研究证实，oCGIs是 poised增强子（poised enhancers，PEs）的必需组成部分，能够增强其长距离调控活性，并决定靶基因的响应性。我们在小鼠胚胎干细胞（mouse embryonic stem cells，mESC）中采用CRISPR/Cas9敲入策略，将携带或不携带oCGIs的PEs插入至携带不同类型启动子基因的拓扑关联结构域（topological associating domains，TADs）内。通过评估工程化PEs的染色质、拓扑结构与调控特性，我们发现oCGIs并未增强PEs的局部激活，而是强化了PEs与远端发育基因之间的物理与功能互作。进一步研究证明，携带CpG富集启动子的发育基因对PEs的响应性尤为显著，且该响应性依赖于oCGIs的存在。因此，本研究揭示了CGIs作为基因与增强子兼容性遗传决定因子的全新功能，这对于当前理解发育基因表达程序的构建机制，以及预测人类结构变异的病理后果均具有重要意义。本研究使用了来源于小鼠胚胎干细胞（mESC）与前脑神经前体细胞（anterior neural progenitor cells，AntNPC）的4C测序（4C-seq）数据。