Ethics Statement<\/p>\n
All animal studies were approved by the Institutional Animal Care and Use Committee of Yale University. The tumor sizes in this study did not exceed the maximal tumor size (1\u2009cm3) permitted by the institutional review board.<\/p>\n
Mouse and cell linesMouse strains<\/p>\n
Mice were housed in a specific-pathogen-free facility with controlled temperature, humidity and day\/night cycles and maintained in a mixed background. MADM11-GT (013749), MADM11-TG (013751) and Pdx1-cre (014647) were obtained from the Jackson Laboratory (JAX). KrasLSL-G12D (JAX, 008179) and Trp53KO (JAX, 002101) mice were a gift from T. Jacks. KrasLSL-G12D\/WT; MADM11-TG, Trp53KO\/MADM11-TG, Trp53WT breeder mice were generated as previously described30<\/a>. KrasLSL-G12D\/WT; MADM11-TG, Trp53KO\/MADM11-TG, Trp53WT were crossed with MADM11-GT mice (with or without Pdx1-cre) to generate K-MADM-Trp53 or Pdx1-cre; K-MADM-Trp53 experimental mice of both sexes for lung and pancreatic cancer analyses, respectively. Genotyping primers and protocols have been previously reported30<\/a>. Large-scale chromatin tracing in lung included four WT mice (91-day male, 191-day female, 283-day male and 91-day female), four mice with adenoma (225-day male, 221-day male, 232-day female and 224-day male) and five mice with LUAD (232-day male, 487-day male, 494-day male, 221-day male and 551-day male). Large-scale chromatin tracing in pancreas included a WT mouse (59-day male) and a mouse with PanIN and PDAC tumors (46-day male). Fine-scale chromatin tracing in mouse lung included a WT mouse (191-day female), a mouse with adenoma (225-day male) and a mouse with LUAD (494-day male).<\/p>\n Lentivirus production and infection<\/p>\n pPGK-Cre lentiviral backbone was amplified and transfected into 293T cells using VSV-G (Addgene, 8454) and psPAX2 (Addgene, 12260) packaging vectors and TransIT-LT1 kit (MirusBio). After 48\u201372\u2009h, virus was collected, filtered and ultracentrifuged. Lentivirus was resuspended in OptiMEM (Thermo Fisher Scientific) and administered intratracheally to K-MADM-Trp53 mice at 6\u201310 weeks of age to generate lung tumors.<\/p>\n Cell line<\/p>\n The KP LUAD cell line, 31671, was a gift from N. Joshi and was derived from an autochthonous KrasLSL-G12D\/WT; Trp53flox\/flox mouse administered with Adeno-Cre77<\/a>. The K-MADM-Trp53 LUAD cell line (SA6082inf) was derived from collagenase-based dissociation of a large GFP+ tumor dissected from a K-MADM-Trp53 mouse. Kras and Trp53 genotypes were confirmed by PCR. Both cell lines were cultured in DMEM (Corning, 10-013-CV) containing 10% (vol\/vol) FBS (Gibco, 26140-079) and 1% (vol\/vol) Penicillin-Streptomycin (Thermo Fisher Scientific, 15140-122) at 37\u2009\u00b0C with 5% CO2. Cells were passaged whenever they reached confluency. Both cell lines were seeded onto UV-sterilized coverslips (Bioptechs, 40-mm diameter; 1.5) and allowed to grow until 60\u201370% confluency before primary probe hybridization. All cell lines were tested for the presence of mycoplasma using PCR (ATCC, 30-1012K).<\/p>\n Probe design and synthesis<\/p>\n Detailed probe design and synthesis information are included in the Supplementary Note<\/a>. Template probe sequences for chromatin tracing and RNA MERFISH are provided in Supplementary Table 1<\/a>. Codebooks for chromatin tracing and RNA MERFISH, and genome coordinates and target region features in chromatin tracing are provided in Supplementary Table 2<\/a>. PCR primers, reverse transcription primers for probe synthesis and adaptors, readout and blocking probes for sequential readout imaging are provided in Supplementary Table 3<\/a>.<\/p>\n Mouse K-MADM-Trp53 lung tissue chromatin tracing experimentsTissue preparation<\/p>\n K-MADM-Trp53 mice were dissected at sign of respiratory distress through CO2 asphyxiation. Lungs were perfused with ice-cold 1\u00d7 PBS, incubated in 4% (vol\/vol) paraformaldehyde (Electron Microscopy Sciences, 15710-S) overnight, and cryoprotected with 30% sucrose. Tissue was embedded in Tissue-Tek optimal cutting temperature compound (OCT) and frozen on dry ice before storage at \u221280\u2009\u00b0C.<\/p>\n Tissue sectioning<\/p>\n Frozen mouse lung blocks were sectioned at a thickness of 10\u2009\u03bcm at \u221220\u2009\u00b0C on a cryostat. The following three consecutive tissue slices were sectioned at a time: one for hematoxylin and eosin (H&E) staining, one for whole-section fluorescence imaging and one for chromatin tracing. The sections were air-dried at room temperature for 1\u2009h and then either used directly or stored at \u221220\u2009\u00b0C for months.<\/p>\n H&E staining and whole-section fluorescence imaging<\/p>\n H&E staining was performed by Yale Pathology Tissue Services. For whole-section fluorescence imaging, the section was mounted in mounting medium with DAPI (VectorLabs, H-1800-2). Stitched fluorescence images in 353-nm, 488-nm and 592-nm illumination channels for DAPI, GFP and tdTomato, respectively, were collected with a Plan-Apochromat \u00d710\/0.45 M27 objective on a Zeiss Axio Imager M2 microscope.<\/p>\n Co-immunofluorescence and fluorescent protein imaging of the chromatin tracing section<\/p>\n Frozen sections were first balanced at room temperature for 10\u2009min, followed by hydration in Dulbecco\u2019s phosphate-buffered saline (DPBS) for 5\u2009min twice. Tissue sections were permeabilized with 0.5% (vol\/vol) Triton X-100 in DPBS for 30\u2009min at room temperature and washed twice in DPBS for 2\u2009min. Tissue sections were then blocked for 30\u2009min at room temperature in blocking buffer containing 1% (wt\/vol) BSA (Sigma-Aldrich, A9647-100G), 22.52\u2009mg\u2009ml\u22121 glycine (AmericanBio, AB00730), 10% (vol\/vol) donkey serum (MilliporeSigma, S30\u2013100\u2009ml), 5% (vol\/vol) goat serum (Invitrogen, 31873) and 0.1% (vol\/vol) Tween-20 in DPBS. Tissue sections were incubated with rabbit anti-SPC antibody (MilliporeSigma, AB3786, 1:50) and rat anti-CD45 antibody (BioLegend, 103101, 1:100) in blocking buffer at 4\u2009\u00b0C overnight. The samples were washed thrice in DPBS for 5\u2009min and then incubated with DyLight 800-labeled donkey anti-rabbit secondary antibody (Invitrogen, SA5-10044, 1:1,000) and Alexa Fluor 647-labeled goat anti-rat secondary antibody (Invitrogen, A-21247, 1:1,000) in blocking buffer for 1\u2009h at room temperature. The samples were washed thrice in DPBS for 5\u2009min and then incubated with DAPI (Thermo Fisher Scientific, 62248) at 1:1,000 dilution in 2\u00d7 saline-sodium citrate buffer (SSC) for 10\u2009min. The samples were then mounted onto a Bioptechs FCS2 flow chamber and replenished with freshly prepared imaging buffer with an oxygen scavenging system (50\u2009mM Tris\u2013HCl pH 8.0, 10% wt\/vol glucose, 2\u2009mM Trolox (Sigma-Aldrich, 238813), 0.5\u2009mg\u2009ml\u22121 glucose oxidase (Sigma-Aldrich, G2133), 40\u2009\u03bcg\u2009ml\u22121 catalase (Sigma-Aldrich, C30)). The imaging buffer was covered with a layer of mineral oil (Sigma-Aldrich, 330779) in the reservoir tube to prevent continuous oxidation. We then selected multiple fields of view (FOVs) at predefined tumor regions based on tumor grades of the adjacent whole-section fluorescence images. Tumor grades were confirmed independently by two investigators (S.S.A. and M.D.M.). At each FOV, we sequentially took z-stack images of DAPI, GFP fluorescence, tdTomato fluorescence, anti-SPC immunofluorescence and anti-CD45 immunofluorescence with 405-nm, 488-nm, 560-nm, 647-nm and 750-nm laser illuminations. The z-stacks (7\u201310\u2009\u03bcm total) had a 200-nm step size and a 0.4\u2009s exposure time per step.<\/p>\n Primary probe hybridization<\/p>\n After imaging, tissue sections were de-assembled from the chamber, treated with 1\u2009\u03bcg\u2009ml\u22121 proteinase K (Invitrogen, AM2546) in 2% (vol\/vol) sodium dodecyl sulfate in 2\u00d7 SSC at 37\u2009\u00b0C for 10\u2009min, and washed twice in DPBS for 2\u2009min. Tissue sections were then treated with 0.1\u2009M HCl for 5\u2009min and washed twice in DPBS for 2\u2009min. Tissue sections were digested with 0.1\u2009mg\u2009ml\u22121 RNase A (Thermo Fisher Scientific, EN0531) in DPBS for 45\u2009min at 37\u2009\u00b0C and washed twice with 2\u00d7 SSC for 2\u2009min. Tissue sections were treated with prehybridization buffer containing 50% (vol\/vol) formamide (Sigma-Aldrich, F7503) and 0.1% (vol\/vol) Tween-20 in 2\u00d7 SSC. Synthesized primary probes were dissolved in 25\u2009\u03bcl hybridization buffer containing 50% (vol\/vol) formamide and 20% (vol\/vol) dextran sulfate (MilliporeSigma, S4030) in 2\u00d7 SSC. The total probe concentration was 30\u201340\u2009\u03bcM. Coverslips were immersed in hybridization buffer containing probes in 60\u2009mm petri dishes, heat denatured in a 90\u2009\u00b0C water bath for 3.5\u2009min and subsequently incubated at 47\u2009\u00b0C in a humid chamber for 36\u201348\u2009h. Samples were washed with 50% (vol\/vol) formamide in 2\u00d7 SSC for 15\u2009min twice followed by 2\u00d7 SSC for 15\u2009min all at room temperature. Washed samples were then incubated with 0.1-\u03bcm yellow\u2013green beads (Invitrogen, F8803) resuspended in 2\u00d7 SSC as fiducial markers for drift correction, washed with 2\u00d7 SSC briefly, incubated with DAPI at 1:1,000 dilution in 2\u00d7 SSC for 10\u2009min for image registration and washed again with 2\u00d7 SSC for 2\u2009min twice.<\/p>\n Readout probe hybridization and imaging<\/p>\n After the primary probe hybridization, the sample was repeatedly hybridized with adaptors and readout probes, imaged and photobleached for 50 rounds. Each adaptor probe was 60 nucleotides (nt) in length, containing a 20-nt primary probe binding region and two consecutive 20-nt readout probe binding regions. Readout probes were 30-nt oligos conjugated with Alexa Fluor 647 (or Cy5) or ATTO 565 (or Cy3) fluorophores with 20-nt complementary to the readout probe binding regions of adaptors (Supplementary Table 3<\/a>). To perform automatic buffer exchange, we used a Bioptechs FCS2 flow chamber and a computer-controlled, custom-built fluidics system11<\/a>,72<\/a>. First, we used DAPI images for registration to images taken before primary probe hybridization. We selected the same FOVs as those selected during co-immunofluorescence and fluorescent protein imaging. At each FOV, we took z-stack images with 488-nm and 405-nm laser illuminations for fiducial beads and DAPI, respectively. For each round of readout probe hybridization, we flowed 2\u2009ml of readout probe hybridization buffer (20% vol\/vol ethylene carbonate (Sigma-Aldrich, E26258) in 2\u00d7 SSC) containing two adaptors each at 50\u2009nM through the chamber and incubated for 15\u2009min at room temperature. We flowed through 2\u2009ml wash buffer (20% vol\/vol ethylene carbonate in 2\u00d7 SSC) for 2\u2009min and 2\u2009ml readout probe hybridization buffer containing two dye-labeled readout probes each at 75\u2009nM, with 15\u2009min incubation at room temperature. We further flowed through 2\u2009ml wash buffer for 2\u2009min and 2\u2009ml imaging buffer. At each FOV, dye-labeled readout probes were imaged in 647-nm and 560-nm channels and fiducial beads were imaged in the 488-nm channel. The z-stacks (7\u201310 \u03bcm total) had a 200-nm step size and a 0.4\u2009s exposure time per step. After imaging, we switched buffer to readout probe hybridization buffer containing 1\u2009\u03bcM dye-free readout probes (blocking oligos) and photobleached with continuous simultaneous laser illuminations with 750-nm, 647-nm and 560-nm lasers for 40\u2009s. We then flowed 5\u2009ml 2\u00d7 SSC to wash away unbound blocking oligos before the next hybridization round. A total of 50 hybridization rounds was performed.<\/p>\n Arrayed RNAi screen of cell proliferation<\/p>\n Lentiviral infection conditions were optimized in 96-well plates for initial cell seeding number, lentiviral dosage, antibiotic concentration and assay time. KP and K-MADM-Trp53 LUAD cells were seeded in complete cell culture media at a density of 250 cells per well in a 96-well plate, incubated for 24\u2009h, and infected with lentiviral supernatant of Sigma Mission shRNAs targeting CPDs obtained from Yale Cancer Center Functional Genomics Core. A total of 200\u2009\u03bcl of media was added to each well, comprising 50\u2009\u03bcl of lentiviral supernatant and 150\u2009\u03bcl of complete cell culture medium with 10\u2009\u03bcg\u2009ml\u22121 polybrene (EMD Millipore, TR-1003-G). Each shRNA hairpin was tested in triplicate. All lentiviral infections were performed in duplicate to rule out the influence of lentiviral dosage on cell growth\u2014one replicate with 6\u2009\u03bcg\/ml puromycin selection and the other replicate with no puromycin selection. After 24\u2009h of lentiviral incubation, corresponding wells were treated with or without puromycin selection for 48\u2009h. The cells were then incubated with complete cell culture media for 4\u20135 days. Total viable cell count was determined with CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7572) using a Promega luminescence plate reader. For data analysis, we deducted luminescence readout values of blank wells from those of test wells. We then normalized luminescence readout values of each target shRNA hairpin to those of nontargeting control shRNA sequence (shNTC). All shRNA hairpin sequences are provided in Supplementary Table 6<\/a>.<\/p>\n Rnf2 targeted degradation<\/p>\n Two million cells of the KP LUAD cell line 31671 were nucleofected (Amaxa) with 2\u2009\u03bcg homology-directed repair (HDR) template and 2\u2009\u03bcM RNP (IDT Alt-R CRISPR\u2013Cas9 sgRNA) for Rnf2-dTAG. The HDR template contained a 555-nt 5\u2032 homology arm homologous to the 5\u2032 end of the Rnf2 stop codon, a 30-nt linker and two HA tags, an FKBPF36V and mScarlet insert (Supplementary Table 6;<\/a> amplified from a gift plasmid from D. Schatz at Yale University) and a 999-nt 3\u2032 homology arm homologous to the 3\u2032 end of the Rnf2 stop codon. After transfection, the cells were seeded into 6-well plates and cultured for 24\u201348\u2009h before FACS sorting. mScarlet+ cells were sorted into 96-well plates. Single colonies were manually picked, genotyped with PCR and validated with sequencing. The spacer sequence for Rnf2 sgRNA is GACTTTATTATGCACCCACCA. The dTAG-13 ligand and negative control ligand were added to the cells at a final concentration of 500\u2009nM. The cells were incubated at 37\u2009\u00b0C with 5% CO2.<\/p>\n Image analysis<\/p>\n All image analyses were performed with MATLAB R2019b.<\/p>\n The genome-wide chromatin tracing image analysis pipeline consists of the following steps: color correction, drift correction, nucleus segmentation, foci fitting, decoding and trace linking. Detailed methods are listed in the Supplementary Note<\/a>.<\/p>\n Data analysis and statisticsDecompaction<\/p>\n To compare levels of chromatin compaction, we defined a decompaction score as the mean interloci distance between a locus pair on a chromosome of a cell state.<\/p>\n Heterogeneity<\/p>\n To compare chromatin conformation heterogeneity, we defined a heterogeneity score as the coefficient of variation (COV) of interloci distances between a locus pair on a chromosome of a cell state (variation among different chromosome copies). Similarly, the heterogeneity scores for cell pairs were defined as the root mean square of interloci distance difference normalized by the mean.<\/p>\n Demixing<\/p>\n To compare levels of chromatin intermixing, we defined a demixing score as the s.d. of all normalized mean interloci distances (mean interloci distance normalized to the mean of all mean interloci distances on the chromosome) on a chromosome of a cell state. The normalization accounted for and excluded the influence of chromatin compaction on demixing measurements.<\/p>\n A and B compartment polarization analysis<\/p>\n To identify A and B compartments, we adapted a previous algorithm to determine A and B compartment scores11<\/a>,24<\/a>. In brief, we generated a mean interloci distance matrix for each chromosome, normalized the mean distance to the expected distance calculated by power-law fitting of spatial versus genomic distances and calculated the Pearson correlation coefficient among each column pair of the normalized matrix. Finally, we applied a principal component analysis (PCA) of the Pearson correlation matrix and used coefficients of the first principal component as compartment scores. We calculated correlations between compartment scores and an averaged profile of histone H3 lysine 4 monomethylation (H3K4me1), H3K4me3, DNase I hypersensitivity site and gene densities78<\/a>,79<\/a>, and flipped signs of compartment scores if necessary, so that A and B compartment genomic regions had positive and negative scores, respectively. We then used a previously described polarization index metric to quantify the polarization of A and B compartments24<\/a>. For chromosome 6 in Fig. 3d<\/a>, we used the first 30 target genomic regions to calculate the polarization index to match target region numbers in different chromosomes. We downloaded H3K4me1 and H3K4me3 chromatin immunoprecipitation sequencing peaks and DNA DNase I hypersensitivity sites from The Encyclopedia of DNA Elements (ENCODE)\u2014ENCFF536DWZ, ENCFF508WEP and ENCFF268DLZ. The gene density profile was downloaded from the UCSC table browser.<\/p>\n Radial score analysis<\/p>\n To calculate the radial score of each genomic region in each single cell, we measured the mean spatial distance among each region to the centroid of all target regions and normalized the distance to the average spatial distances from all genomic regions to the centroid.<\/p>\n The \u2018Trace2State\u2019 pipeline for single-cell chromatin conformation-based cell state visualization and classification<\/p>\n First, we constructed an input matrix where each row represented a single cell and each column represented the scA\/B score of each genomic region35<\/a>. The scA\/B score was calculated as the mean A\u2013B compartment score of all its spatially adjacent genomic regions within a 1,200-nm 3D neighborhood. Only cells with at least 10 traces were analyzed. Missing values were replaced with 0s. The matrix was used as input in a PCA, and the first 50 principal components were used for downstream visualizations. We scaled the 50 principal components with the PCA().fit-transform function in Python 3.11.12, and visualized the single-cell data with t-distributed stochastic neighbor embedding, uniform manifold approximation and projection and pairwise controlled manifold approximation37<\/a>,38<\/a>,39<\/a>. To classify different cell states with supervised machine learning, we supplied the Classification Learner App of MATLAB with the same input scA\/B score matrix and applied fivefold cross-validation to prevent overfitting. All machine learning models were trained, and the model with the highest prediction accuracy (medium Gaussian support vector machine, regularization included with box constraint level = 1) was retained to plot the confusion matrix and receiver operating characteristic curves.<\/p>\n The \u2018Trace2Biomarker\u2019 pipeline for CPD gene identification<\/p>\n To identify CPD genes, we first identified marker genomic regions with substantially changed scA\/B scores during LUAD progression. Specifically, we extracted cells with more than 325 genomic regions. We then performed rank normalization (using the tiedrank function in MATLAB) of scA\/B scores in each cell. We performed Wilcoxon rank-sum tests of rank-normalized scA\/B scores of each genomic region comparing AdenomaG and LUAD. Genomic regions with P\u2009<\u20090.1 were defined as marker genomic regions. We then defined CPD genes as genes located in marker genomic regions with increased scA\/B scores and higher expression (mean expression count\u2009>\u200910, fold change\u2009>\u20093, false discovery rate (FDR)\u2009<\u20090.05) in LUAD versus AdenomaG cells. We defined control genes as those with increased expression (mean expression count\u2009>\u200910, fold change\u2009>\u20093, FDR\u2009<\u20090.05) in regions with unchanged scA\/B scores (P\u2009\u2265\u20090.1). Genes with increased expression only are defined as those with increased expression (mean expression count\u2009>\u200910, fold change\u2009>\u20093, FDR\u2009<\u20090.05) in LUAD versus AdenomaG cells, irrespective of scA\/B score changes. Equal gene numbers (top 21 upregulated by fold change) were used for each gene list above and interrogated in TCGA patient survival analyses.<\/p>\n The \u2018Trace2Regulator\u2019 pipeline for 3D genome regulator identification<\/p>\n To identify chromatin regulators that bind to genes in marker loci, we first identified genes with increased expression (FDR\u2009<\u20090.1, fold change\u2009>\u20091) in genomic regions with increased scA\/B scores (P\u2009<\u20090.1) from AdenomaG to LUAD cells. We then input the gene list into BART55<\/a>,56<\/a> to generate chromatin regulators predicted to bind to the input genes, using default parameters. We further knocked down candidate chromatin regulators with shRNAs in the KP LUAD cell line and performed DNA MERFISH. We further compared 3D genome alterations upon the candidate chromatin regulator knockdown with those during the AdenomaG-to-LUAD progression. Candidate regulators showing concordant 3D genome alterations were identified as 3D genome regulators.<\/p>\n CUT&RUN experiment and analysisCUT&RUN protocol<\/p>\n CUT&RUN was performed using the Epicypher CUTANA ChIC\/CUT&RUN Kit (14-1048) according to the manufacturer\u2019s specifications with the following conditions and modifications: for binding to the Concanavalin beads, 500,000 cells per sample were prepared, counted twice using a hemacytometer, and averaged. Nuclei were prepared according to the Epicypher Cut and Run Manual Appendix. Nuclei were incubated with activated beads. For Escherichia coli spike-in DNA, 0.1\u2009ng was added to each sample. The following antibodies (0.5\u2009\u03bcg of antibody per reaction) were used: IgG control (CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody), Rnf2 antibody (Cell Signaling Technology, 5694), H3K4me3 antibody (EpiCypher, 13-0041), H3K27me3 antibody (Cell Signaling Technology, 9733), H2AK119ub antibody (Cell Signaling Technology, 8240), BMI1 antibody (Active Motif, 39993) and RNA Pol II p-ser5 (Abcam, ab193467).<\/p>\n Library preparation<\/p>\n Libraries were prepared using Epicypher CUTANA CUT&RUN Library Prep Kit (14-1001) according to the manufacturer\u2019s specifications with the following modification: SPRI-select beads were used after library preparation to perform 200\u2013700\u2009bp size selection to enrich for DNA fragments from CUT&RUN and remove adaptor dimers or high molecular weight DNA. Library quality was analyzed using Agilent Tapestation D1000 High Sensitivity Tapes (5067\u20135584). Sequencing was performed with 10 million reads per sample, 150\u2009bp paired-end reads, on an Illumina NovaSeq 6000.<\/p>\n CUT&RUN data analysis<\/p>\n CUT&RUN reads were analyzed for quality control using FastQC (https:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc\/<\/a>) and trimmed using Trimmomatic. Reads were then aligned to mm9 and K-12 E. coli genome U00096.3 by Bowtie2 2.3.4. Picard 2.27.4 was used to downsample the reads so that the read depths of E. coli sequences from different samples matched each other. SAMTools 1.11 was then used to convert to BAM format, index, isolate uniquely mapped paired reads and remove duplicates. MACS2 2.2.7.1 was used to call sample narrow peaks, using IgG as an input. Read counts across genomic intervals and peak visualization were performed using deepTools 3.3 (ref. 80<\/a>), and .bw files were visualized using Integrative Genomics Viewer.<\/p>\n