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. 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确的基因表达模式，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会在拓扑关联结构域（Topologically Associating Domains，TADs）内与基因启动子发生相互作用。然而，染色质可及性、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。

本研究介绍了Tiled-C技术——一种全新的染色体构象捕获（Chromosome Conformation Capture, 3C）技术，该技术能够以前所未有的测序深度，获取目标位点的高分辨率接触矩阵，且可针对低至2000个输入细胞进行优化。我们利用该方法，通过体内红细胞分化过程，研究了小鼠α-珠蛋白基因座的染色质架构。

对匹配的染色质可及性数据与单细胞表达数据进行整合分析后发现，α-珠蛋白基因座位于一个预先存在的拓扑关联结构域之中，该结构域在基因座激活前就已形成。在分化过程中，随着调控元件逐渐获得可及性，且增强子与启动子之间形成特异性相互作用，该拓扑关联结构域会发生进一步的亚区室化。伴随这些染色质变化的发生，基因表达水平逐步上调。

我们的研究结果表明，发育过程中染色质架构与基因激活紧密相关，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。

Tiled-C是一种基于3C的全新技术，可生成选定目标区域的高分辨率接触矩阵。该技术使用一组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，并结合经过改良的基于Capture-C的实验流程，高效富集该区域内的3C相互作用产物。这使得我们能够对目标区域内的染色质相互作用进行深度靶向测序，从而在多样本与多基因组区域中，获取类Hi-C的高分辨率、高深度数据。

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可从低至2000个细胞中生成高分辨率接触矩阵。