Dissection of the 4D chromatin structure of the alpha-globin locus through in vivo erythroid differentiation with extreme spatial and temporal resolution [Tiled-C]

Precise gene expression patterns during mammalian development are controlled by regulatory elements in the non-coding genome. Active enhancer elements interact with gene promoters within Topologically Associating Domains (TADs). However, the precise relationships between chromatin accessibility, nuclear architecture and gene activation are not completely understood. Here, we present Tiled-C, a new Chromosome Conformation Capture (3C) technology, which allows for the generation of high-resolution contact matrices of loci of interest at unprecedented depth, and which can be optimized for as few as 2,000 cells of input material. We have used this approach to study the chromatin architecture of the mouse alpha-globin locus through in vivo erythroid differentiation. Integrated analysis of matched chromatin accessibility and single-cell expression data shows that the alpha-globin locus lies within a pre-existing TAD, which is established prior to activation of the domain. During differentiation, this TAD undergoes further sub-compartmentalization as regulatory elements gradually become accessible and specific interactions between enhancers and promoters are formed. As these chromatin changes develop, gene expression is progressively upregulated. Our findings demonstrate that chromatin architecture and gene activation are tightly linked during development and provide insights into the distinct mechanisms contributing to the establishment of tissue-specific chromatin structures. Overall design: Tiled-C is a new 3C-based approach which generates high-resolution contact matrices of selected regions of interest. Tiled-C uses a panel of capture oligonucleotides tiled across all restriction fragments of specified genomic regions, combined with an adapted Capture-C based protocol, to efficiently enrich for 3C contacts within this region. This allows for deep, targeted sequencing of chromatin interactions within regions of interest and thus for the generation of high-resolution, high-depth Hi-C-like data, across multiplexed samples and genomic regions. The combination of the efficient tiled enrichment strategy with previous Capture-C library preparation optimizations allows Tiled-C to generate high-resolution contact matrices from as few as 2,000 cells.

哺乳动物发育过程中精准的基因表达模式，由非编码基因组中的调控元件所调控。具有活性的增强子元件可在拓扑关联结构域（Topologically Associating Domains, TADs）内与基因启动子发生相互作用。然而，染色质开放性、细胞核架构与基因激活之间的确切关联，目前尚未完全阐明。本研究介绍Tiled-C技术——一种全新的染色体构象捕获（Chromosome Conformation Capture, 3C）技术，可以前所未有的测序深度获取目标基因座的高分辨率接触矩阵，且仅需低至2000个细胞的起始材料即可实现实验优化。我们利用该技术，通过体内红系分化模型，对小鼠α-珠蛋白基因座的染色质架构展开研究。对匹配的染色质开放性数据与单细胞表达数据开展整合分析后发现，α-珠蛋白基因座处于一个预先形成的TAD之中，该结构域在该基因座激活前便已建立。在分化进程中，随着调控元件逐步开放染色质并形成增强子与启动子间的特异性相互作用，该TAD会发生进一步的亚区室化重构。伴随这些染色质动态变化的发生，基因表达也逐步被上调。本研究结果证实，发育过程中染色质架构与基因激活紧密关联，并为解析组织特异性染色质结构建立的不同机制提供了新见解。

实验整体设计：Tiled-C是一种基于3C技术的全新方法，可针对选定的目标区域生成高分辨率接触矩阵。该技术通过设计覆盖指定基因组区域所有限制性酶切片段的捕获寡核苷酸探针池，并结合优化后的Capture-C实验流程，高效富集该区域内的3C互作产物。这一策略可实现对目标区域内染色质互作的深度靶向测序，从而在多重样本与多基因组区域中获得高分辨率、高深度的类Hi-C数据。结合高效的平铺式富集策略与此前优化的Capture-C文库制备流程，Tiled-C仅需低至2000个细胞即可生成高分辨率接触矩阵。