3D ATAC-PALM: Super-resolution Imaging of the Accessible Genome

Spatial genome organization is essential to direct fundamental DNA-templated biological processes (e.g. transcription, replication, and repair), but the 3D in situ nanometer-scale structure of accessible cis-regulatory DNA elements within the crowded nuclear environment remains elusive. Here, we combined the recently developed Assay for Transposase-Accessible Chromatin with visualization (ATAC-see), PALM super-resolution imaging and lattice light-sheet microscope (a method termed 3D ATAC-PALM) to selectively image and quantitatively analyze key features of the 3D accessible genome in single cells. 3D ATAC-PALM reveals that accessible chromatin are non-homogeneously organized into spatially segregated clusters or accessible chromatin domains (ACDs). To directly link imaging and genomic data, we optimized multiplexed imaging of 3D ATAC-PALM with Oligopaint DNA-FISH, RNA-FISH and protein fluorescence. We found that ACDs colocalize with active chromatin and enclose transcribed genes. By applying these methods to analyze genetically purterbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight the 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.

空间基因组组织（Spatial genome organization）对于调控以DNA为模板的基础生物学过程（例如转录、复制与修复）至关重要，但在拥挤的细胞核微环境中，可及顺式调控DNA元件的原位纳米级三维结构仍难以探明。本研究将新近开发的转座酶可及染色质可视化检测技术（Assay for Transposase-Accessible Chromatin with visualization，ATAC-see）、光激活定位显微镜（photoactivated localization microscopy，PALM）超分辨率成像技术与晶格层光显微镜相结合，构建了名为3D ATAC-PALM的实验方法，实现对单细胞中三维可及基因组关键特征的选择性成像与定量分析。3D ATAC-PALM技术揭示，可及染色质以非均一方式组装为空间分隔的簇状结构，即可及染色质结构域（accessible chromatin domains，ACDs）。为实现成像数据与基因组学数据的直接关联，本研究对3D ATAC-PALM与Oligopaint DNA荧光原位杂交、RNA荧光原位杂交及蛋白质荧光成像的多重成像方案进行了优化。研究发现，可及染色质结构域（ACDs）与活性染色质共定位，并包裹转录中的基因。借助上述方法分析遗传扰动后的细胞，本研究证实基因组架构蛋白CTCF可抑制可及染色质的过度聚集，并松解可及染色质结构域。上述研究结果表明，3D ATAC-PALM是探究基因组三维结构及其组装机制的有效工具。