DOI: 10.5061/dryad.6t1g1jx5v
title: Data from: Cell type- and transcription-independent spatial proximity between enhancers and promoters

description: 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.
description: 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.  
description: # Data from: Cell type- and transcription-independent spatial proximity
 between enhancers and promoters
 [https://doi.org/10.5061/dryad.6t1g1jx5v](https://doi.org/10.5061/dryad.6t1g1jx5v) This dataset consists of 2D spot positions generated from maximum intensity projections of images of high-throughput fluorescence in situ hybridization experiments. The data consists of identifying information (experimental date, filename, well, field, cell, and spot indices), position information (x, y, and radial positions), and metadata information (information on probes and cell types used). ## Description of the data and file structure High-throughput DNA FISH was performed in a 384-well plate format. 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. Maximum intensity projections were performed at the time of imaging and saved; all analysis was performed on maximum intensity projections. 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). Metadata for each well and channel (probes used, cell type, experimental date, etc) were appended using R and are included in these processed data files. Probes are, as specified, either targetted to enhancer-promoter pairs at three lineage-specific genes or negative control regions. Cells are, as specified, EndoC-BH1: an immortalized cell line derived from human islet beta cells, PANC-1: human pancreatic ductal carcinoma cells, and HFF: human immortalized skin fibroblasts used as a technical control and outgroup. Column definitions: * Location: Image identifier (filename glob) * Well ID: Well position within plate * FieldIndex: Field number in well * CellIndex: Cell number in field * SpotIndex: Spot number in cell * x.centroid: X position of spot centroid in microns * y.centroid: Y position of spot centroid in microns * x.cog: X position of spot center of gravity in microns * y.cog: Y position of spot center of gravity in microns * r.centroid: Radial position (normalized and inverted exact Euclidean distance to edge) of spot centroid * r.cog: Radial position (normalized and inverted exact Euclidean distance to edge) of spot center of gravity * CentroidDistanceShellNumber: Radial position shell number of spot centroid, shells calculated to be at equal distance intervals from center * CenterofGravityDistanceShellNumber: Radial position shell number of spot center of gravity, shells calculated to be at equal distance intervals from center * TotalNumberofDistanceShells: Total number of shells used to calculate evenly spaced distances from center. * CentroidAreaShellNumber: Radial position shell number of spot centroid, shells calculated to be of equal area * CenterofGravityAreaShellNumber: Radial position shell number of spot center of gravity, shells calculated to be of equal area * TotalNumberofAreaShells: Total number of shells used to calculate shells of equal area * nrow: Row number of well * ncol: Column number of well * Color: Spot color (Green: DY-488; Red: DY-549) * Green_Probe_Full: Full name of green probe (Gene + Green_Probe + Green_Concentration) * Red_Probe_Full: Full name of red probe (Gene + Red_Probe + Red_Concentration) * Gene: Gene name * Green_Concentration: Concentration of green probe * Red_Concentration: Concentration of red probe * Green_Probe: Loop anchor targeted by green probe (Enhancer or Promoter) * Red_Probe: Loop anchor targeted by red probe (Enhancer or Promoter) * Genomic_Distance: Genomic distance between probes in kilobases * Cell_Type: Cell type * Experiment_Date: Experiment date * GreenSpotCount: Number of green spots segmented in cell * RedSpotCount: Number of red spots segmented in cell