Cell type- and transcription-independent spatial proximity between enhancers and promoters

Cell type-specific enhancers are critically important for lineage specification. The mechanisms that determine cell type-specificity of enhancer activity, however, are not fully understood. Most current models for how enhancers function invoke physical proximity between enhancer elements and their target genes. Here, we use an imaging-based approach to examine the spatial relationship of cell type-specific enhancers and their target genes with single cell resolution. Using high-throughput microscopy, we measure the spatial distance from target promoters to their cell type-specific active and inactive enhancers in individual pancreatic cells derived from distinct lineages. These images were processed to identify nuclei and the loci within them. The shared dataset is composed of spot positions for promoter and enhancer probes in individual cells. It is comprised of 14 separate files, each corresponding to an individual plate imaged on a different day. Column headers and contents are consistent between individual files.

Methods
DNA FISH

High-throughput fluorescence in-situ hybridization was performed as described previously (Finn and Misteli 2021). Probes were generated from Bacterial Artificial Chromosomes (BACs) containing target regions. Bacteria from a single colony was grown into a large-scale culture and target DNA was purified via alkaline lysis using the Nucleobond BAC 100 Maxiprep kit (from Takara). DNA was then quantified and stored at -20°C for future use. Probes were generated from BAC DNA by a nick translation reaction incubated at 16°C for 1 hr 20 min with the following mix: 40 ng/uL DNA, 0.05 M Tris-HCl pH 8.0, 5 mM MgCl2, 0.05 mg/ml BSA, 0.05 mM dNTPs with all dTTP replaced with fluorescently labeled dUTP, 1 mM β-mercaptoethanol, 0.5 U/μL E. coli DNA Polymerase and 0.5 μg/μL DNAse I. The reaction was stopped with the addition of 1 μL EDTA per 50 μL reaction volume and heat shocked to 72°C for 10 min, then stored at −20°C overnight. QC gels were run in 2% agarose to verify successful nick translation with a smear of less than 1 kb. Combinations of two probes (1 μg per probe) were mixed, ethanol precipitated, resuspended in 15 μL of hybridization buffer (50% formamide pH 7.0, 10% Dextran Sulfate, and 1% Tween-20 in 2X SSC) per well and warmed to 72°C before plating.

Cells were plated at an appropriate density and grown overnight at 37°C before fixation for 10 minutes in 4% PFA, two PBS rinses, and storage in 70% ethanol at -20°C. To perform DNA FISH, cells were warmed to room temperature and rinsed three times in PBS to remove ethanol. Cells were then permeabilized in 0.5% w/v saponin/0.5% v/v Triton X-100 in PBS at room temperature for 20 min, rinsed twice with PBS, deproteinated for 15 minutes at room temperature in 0.1 N HCl, and neutralized for 5 min at room temperature in 2X SSC before equilibration in 50% formamide/2X SSC for at least 20 min at room temperature. 13.5 μL of resuspended probe mix was added per well, pipetting to mix before adding, and plates were spun to remove air bubbles. Cells were denatured for 7.5 min at 85°C and immediately moved to a 37°C water for 72 hour hybridization. After hybridization, plates were rinsed once at room temperature with 2X SSC, and then thrice each with 1X SSC and 0.1X SSC both warmed to 45°C. Cells were stained with DAPI for 15 min, rinsed, and mounted in PBS, and imaged. All experiments were performed in twelve technical replicate wells per experiment.

Imaging

Automated imaging was performed in three channels (405, 488, and 561 nm excitation lasers) on a CV8000 dual spinning disc confocal microscope with a 60X water immersion lens (NA = 1.2) and no pixel binning for a final pixel size of 108 nm. We imaged 16 fields per well with a z-stack of 10 μm at 1 μm intervals. In the first exposure, cells were excited with the 405 nm laser and the light path included a short pass emission dichroic mirror and an sCMOS camera in front of a 445/45 nm bandpass filter. In the second exposure, cells were excited with both 488 and 561 nm lasers and emission detected through the same light path by two sCMOS cameras in front of 525/50 nm and 600/37 nm bandpass emission filters respectively. Laser power and exposure time was optimized per experiment to ensure good signal-to-noise ratios.

Image analysis

Analysis of imaging data was carried out using HiTIPS, a high-throughput image analysis software to analyze DNA FISH data (Keikhosravi et al. 2023). For each experimental plate, specific analysis parameters were selected and tailored to align with the average nucleus size, as well as the size and brightness of the DNA FISH spots observed. Within HiTIPS, the GPU-based CellPose algorithm for nuclei segmentation was used in conjunction with the Laplacian of Gaussian method for spot detection (Stringer et al. 2021). Parameter selection was guided by real-time visual feedback, enabling iterative refinement of measurement parameters.  Image processing was done on the NIH HPC Biowulf cluster (NIH Biowulf HPC Cluster). Well-specific metadata were appended to the processed data using R.

细胞类型特异性增强子（cell type-specific enhancers）对细胞谱系特化至关重要。然而，决定增强子活性细胞类型特异性的分子机制尚未完全阐明。目前主流的增强子功能模型均假定增强子元件与其靶基因之间存在物理邻近性。本研究采用基于成像的方法，以单细胞分辨率解析细胞类型特异性增强子与其靶基因的空间关联。我们借助高通量显微镜技术，在不同谱系来源的原代胰腺细胞中，定量测定靶基因启动子与其细胞类型特异性活性及非活性增强子之间的空间距离。所有成像数据均经过处理以识别细胞核及其内部的基因座。本共享数据集包含单个细胞中启动子与增强子探针的斑点位置信息，共包含14个独立文件，每个文件对应单日成像的一块检测板，各文件的列标题与内容格式保持一致。

方法

DNA荧光原位杂交（DNA FISH）
高通量荧光原位杂交实验按照既往报道的方法进行（Finn与Misteli，2021）。探针由包含靶区域的细菌人工染色体（Bacterial Artificial Chromosomes, BACs）制备。挑取单菌落的细菌进行大规模培养，采用Takara公司的Nucleobond BAC 100 大提试剂盒，通过碱裂解法纯化靶DNA。纯化后的DNA经定量后于-20℃保存备用。以BAC DNA为模板，通过切口平移反应制备探针：反应体系于16℃孵育1小时20分钟，反应组分包括40 ng/μL DNA、0.05 M Tris-HCl（pH 8.0）、5 mM MgCl₂、0.05 mg/mL 牛血清白蛋白（BSA）、0.05 mM dNTPs（所有dTTP均替换为荧光标记dUTP）、1 mM β-巯基乙醇、0.5 U/μL 大肠杆菌DNA聚合酶及0.5 μg/μL DNA酶I。反应终止时，每50 μL反应体系加入1 μL EDTA，随后于72℃热激10分钟，之后置于-20℃过夜保存。采用2%琼脂糖凝胶电泳进行质量验证（QC），以出现小于1 kb的弥散条带作为切口平移反应成功的标志。将两种探针（每探针1 μg）混合后进行乙醇沉淀，重悬于15 μL杂交缓冲液（含50%甲酰胺pH 7.0、10%硫酸葡聚糖、1% Tween-20的2×SSC溶液）中，每孔加入上述重悬液，铺板前将其预热至72℃。

细胞以适宜密度铺板，于37℃孵育过夜，随后用4%多聚甲醛（PFA）固定10分钟，经两次PBS漂洗后置于70%乙醇中，于-20℃保存。进行DNA FISH实验时，将细胞恢复至室温，用PBS漂洗三次以去除乙醇。随后将细胞置于含0.5% w/v 皂苷与0.5% v/v Triton X-100的PBS溶液中，室温通透20分钟，再用PBS漂洗两次；接着用0.1 N HCl室温处理15分钟以去除蛋白，再用2×SSC室温孵育5分钟中和酸处理，之后置于50%甲酰胺/2×SSC溶液中室温平衡至少20分钟。每孔加入13.5 μL重悬后的探针混合液，加样前通过移液充分混匀，同时离心平板以去除气泡。将细胞于85℃变性7.5分钟，随后立即转移至37℃水浴中杂交72小时。杂交结束后，平板先用室温2×SSC漂洗一次，再分别用预热至45℃的1×SSC和0.1×SSC各漂洗三次。用DAPI染色细胞15分钟，漂洗后用PBS封片，随后进行成像。本实验每个技术重复设置12个复孔。

成像流程
采用CV8000双转盘共聚焦显微镜，搭配60倍水浸物镜（数值孔径NA=1.2），不进行像素合并，最终像素尺寸为108 nm，通过三个通道（405 nm、488 nm、561 nm激发激光）完成自动化成像。每孔采集16个视野，以1 μm为步长采集10 μm厚度的Z轴堆叠图像。第一通道采用405 nm激光激发，光路配置为短通发射二向色镜，搭配前置445/45 nm带通滤光片的sCMOS相机采集信号；第二通道同时采用488 nm与561 nm激光激发，通过同一光路分别使用前置525/50 nm和600/37 nm带通发射滤光片的两台sCMOS相机采集对应荧光信号。每次实验均优化激光功率与曝光时间，以保证良好的信噪比。

图像分析
成像数据的分析采用HiTIPS软件完成，该软件是一款专为DNA FISH数据开发的高通量图像分析工具（Keikhosravi等，2023）。针对每一块实验板，均根据观测到的平均细胞核尺寸、DNA FISH斑点的大小与亮度，定制专属的分析参数。在HiTIPS软件中，我们采用基于GPU加速的CellPose算法完成细胞核分割，并结合高斯拉普拉斯方法进行斑点检测（Stringer等，2021）。参数选择通过实时可视化反馈进行优化，可迭代调整测量参数。图像处理工作在NIH HPC Biowulf集群（NIH Biowulf HPC Cluster）上完成。采用R语言为处理后的数据添加孔板特异性元数据。