Paired Imaging and Sequencing of Protein-DNA Interactions in Single Human Cells Using microDamID. Paired Imaging and Sequencing of Protein-DNA Interactions in Single Human Cells Using microDamID

Genome regulation depends on carefully programmed protein-DNA interactions that maintain or alter gene expression states, often by influencing chromatin organization. Most studies of these interactions to date have relied on bulk methods, which in many systems cannot capture the dynamic single-cell nature of these interactions as they modulate cell states. One method allowing for sensitive single-cell mapping of protein-DNA interactions is DNA adenine methyltransferase identification (DamID), which records a protein’s DNA-binding history by methylating adenine bases in its vicinity, then selectively amplifies and sequences these methylated regions. These interaction sites can also be visualized using fluorescent proteins that bind to methyladenines. Here we combine these imaging and sequencing technologies in an integrated microfluidic platform (µDamID) that enables single-cell isolation, imaging, and sorting, followed by DamID. We apply this system to generate paired single-cell imaging and sequencing data from a human cell line, in which we map and validate interactions between DNA and nuclear lamina proteins, providing a measure of 3D chromatin organization and broad gene regulation patterns. µDamID provides the unique ability to compare paired imaging and sequencing data for each cell and between cells, enabling the joint analysis of the nuclear localization, sequence identity, and variability of protein-DNA interactions. Overall design: Single-cell DamID to map lamina associated domains in HEK293T cells imaged on a microfluidic device.

基因组调控依赖于精密调控的蛋白质-DNA相互作用，此类作用通常通过影响染色质构象来维持或改变基因表达状态。迄今为止，针对此类相互作用的大多数研究均依赖批量检测方法，但在诸多系统中，这类方法无法捕捉到这些相互作用在调控细胞状态时的动态单细胞特性。一种可实现蛋白质-DNA相互作用高灵敏度单细胞绘图的方法为DNA腺嘌呤甲基转移酶识别技术（DNA adenine methyltransferase identification，DamID）：该技术通过在靶蛋白邻近区域对腺嘌呤碱基进行甲基化，记录该蛋白的DNA结合历史，随后对这些甲基化区域进行选择性扩增并测序。此类相互作用位点还可借助结合甲基腺嘌呤的荧光蛋白实现可视化成像。本研究将上述成像与测序技术整合至微流控平台（µDamID）中，该平台可实现单细胞分离、成像与分选，随后开展DamID检测。我们利用该系统从人类细胞系中获取了配对的单细胞成像与测序数据，并在此基础上绘制并验证了DNA与核纤层蛋白的相互作用，以此量化三维染色质构象与广泛的基因调控模式。µDamID具备独特优势，可对单个细胞内以及不同细胞间的配对成像与测序数据进行对比分析，从而实现对蛋白质-DNA相互作用的核定位、序列同一性及其变异程度的联合分析。整体实验设计：在微流控设备上对HEK293T细胞进行成像，同时通过单细胞DamID技术绘制核纤层结合结构域图谱。