SARS-CoV-2 restructures host chromatin architecture

Cell culture

Human lung adenocarcinoma cells A549 expressing human ACE2 (A549-ACE2, NR-53821) were acquired from BEI Resources. They were maintained in DMEM/F-12 (1:1, Corning) medium supplemented with 10% FBS (GeneDepot) and blasticidin (100 μM). Normal A549 cells were purchased from ATCC (CCL-185) and cultured in DMEM/F-12 (1:1, Corning) supplemented with 10% FBS. 293T cells were from ATCC and were cultured in DMEM with 10% FBS. HCT-8 cells were purchased from ATCC (CCL-224) and cultured in RPMI 1640 with 10% FBS. Vero-E6 cells were acquired from ATCC (CRL-1586). Mouse embryonic stem cells (ESCs) (F121-9) were a gift from the David Gilbert lab (San Diego Biomedical Research Institute, CA) and were cultured following standard procedure of the 4D nucleome consortium (data.4dnucleome.org/biosources/4DNSRMG5APUM/). RAD21-mAID2-mClover HCT116 cells were a gift from Masato Kanemakei lab (NIG, Japan) and were cultured in McCoy’s 5A medium with 10% FBS. All these cells were cultured at 37 °C with 5% CO2. Transfection of plasmids or small interfering RNAs was performed using Lipofectamine 3000 or RNAiMAX (Life Technologies) following the manufacturer’s instructions. For CRISPRi experiments, to examine enhancer functions during cell responses to RNA virus, we introduced polyinosinic:polycytidylic acid (poly (I:C), 333 ng ml−1, Sigma, P9582) into A549 cells using lipofectamine 2000 and collected the total cellular RNAs for gene expression experiments after 4 h. For some experiments, fluorescein-labelled poly (I:C) was acquired from Invivogen (tlrl-picf). For the Hi-C 3.0 experiment, we introduced 66 ng ml−1 of poly (I:C) for 6 h to mimic RNA virus infection in the A549-ACE2 cell line. For interferon-beta (IFN-β) treatment, we treated A549-ACE2 cells with 1,000 U ml−1 recombinant human interferon beta (8499-IF-010/CF, R&D systems) for 6 h. For RAD21-mAID2-mClover cells, we first pre-treated cells wth 1 μM 5-ph-IAA for 2 h and then administered 1,000 U ml−1 interferon beta together with 1 μM 5-ph-IAA for 4 h.

SARS-CoV-2 and HCoV-OC43 infections in A549-ACE2 cells

SARS-CoV-2 isolate USA-WA1/2020 (NR-52281, BEI Resources) was used to infect human A549-ACE2 cells (NR-53821, BEI Resources). For viral infections, serum/antibiotics-free Eagle’s MEM medium supplemented with 1 mM HEPES was used. Briefly, cells grown in 10-cm culture dishes at about 70–80% confluency were washed with the serum-free medium, and viral inoculum was added at 0.1 MOI for 1 h. Subsequently, non-adsorbed viral particles were gently aspirated out and the monolayers were replenished with 10% FBS containing MEM supplemented with 1 mM HEPES. Infected cells were incubated at 37 °C with 5% CO2 for 6 h or 24 h post-infection for experiments. For heat-inactivated SARS-CoV-2 treatment, we treated the equivalent number of heat-inactivated virus (BEI resources, NR-52286) to 0.1 MOI on the basis of genomic equivalence (GE). Heat-inactivated SARS-CoV-2 (HI-WA1) was obtained from BEI Resources (NR-52286) and had 5.36 × 108 genome equivalents per ml. Heat-inactivated SARS-CoV-2 was given to cells using the same protocol as described above for SARS-CoV-2 isolate USA-WA1/2020 (NR-52281, BEI Resources), with the equivalent amount of 0.1 MOI based on GE.

HCoV-OC43 (VR-1558) was purchased from ATCC. For viral infections, serum/antibiotics-free RPMI 1640 medium was used. Briefly, A549-ACE2 cells grown in 10-cm culture dishes at about 80–90% confluency were washed two times with phosphate-buffered saline (PBS). Viruses were inoculated onto A549-ACE2 cells and allowed to adsorb for 2 h at 33 °C and 0.5 MOI. Then, non-adsorbed viral particles were gently aspirated out and the monolayers were replenished with 2% horse serum containing RPMI 1640. Infected cells were incubated at 33 °C with 5% CO2 for 24 h post-infection for experiments.

Preparation of SARS-CoV-2 and HCoV-OC43 stock

The stock SARS-CoV-2 was propagated in Vero-E6 cells. Briefly, Vero-E6 cells were grown to 80% confluence in 10% FBS containing MEM medium supplemented with 1 mM HEPES and 1X antibiotics and antimycotics. Before infection, Vero-E6 cells were washed once with PBS, the viral inoculum was added to the flask in the presence of 3 ml of serum-free and antibiotics-free MEM medium supplemented with 1 mM HEPES, and the cells incubated for 1 h at 37 °C with 5% CO2. At the end of incubation, non-adsorbed virus was aspirated out and cells were replenished with 25 ml of MEM supplemented with 10% FBS and 1 mM HEPES. Infected cells were incubated for 48 h at 37 °C with 5% CO2. At 80% of cell lysis, SARS-CoV-2 was collected by detaching all the cells with a cell scraper and centrifuging at 300 g for 3 min. Viral aliquots were stored in screw-cap vials at −80 °C.

The stock of HCoV-OC43 was propagated in HCT-8 cells. Briefly, HCT-8 cells were grown to 80% confluence in RPMI 1640 medium containing 10% FBS. Before infection, HCT-8 cells were washed two times with PBS, the viral inoculum was added to the flask in the presence of 3 ml of serum-free and antibiotics-free RPMI 1640 and the cells incubated for 2 h at 33 °C with 5% CO2. At the end of incubation, non-adsorbed virus was aspirated out and cells were replenished with 25 ml of RPMI 1640 supplemented with 2% horse serum. Infected cells were incubated for 5 d at 33 °C with 5% CO2. At 80% of cell lysis, HCoV-OC43 was collected by detaching all the cells with a cell scraper and centrifuging at 300 g for 3 min. Viral aliquots were stored in screw-cap vials at −80 °C.

Determination of plaque forming units (p.f.u. ml−1 stock)

For the determination of infectious viral titres, plaque assays were performed using Vero-E6 cells. Briefly, Vero-E6 cells grown in 6-well plates were infected with 12 serial dilutions (1:10) of the SARS-CoV-2 stock in serum/BSA/antibiotic-free MEM medium with 1 mM HEPES for 1 h at 37 °C with 5% CO2. At the end of incubation, non-adsorbed viral particles were aspirated and the infected cells were layered on MEM medium containing 0.5% agarose, 2% BSA and 1 mM HEPES, and incubated for 48 h at 37 °C with 5% CO2. Fixation was carried out using 3.75% buffered formaldehyde (in PBS) for 10 min. After aspirating formaldehyde, the agarose layers were gently removed. Infected cells were stained with 0.3% crystal violet for 5 min, followed by washing once with PBS. Plates were air-dried and visible infectious plaques were counted in each dilution to determine the plaque forming units per millilitre of the stock.

To determine infectious viral particles for HCoV-OC43, the median tissue culture infectious dose (TCID50) was determined using HCT-8 cells as follows: HCT-8 cells were seeded into 96-well plates at a concentration of 5 × 104 cells per well and incubated for 24 h to reach a confluence of 90%. Viral stock was serially diluted in RPMI 1640 containing 10% FBS in the range 10−1 to 10−8.

Diluted viral samples were incubated at 33 °C for 5 d. Next, the medium was removed and replaced with 0.3% crystal violet for 5 min, followed by washing once with PBS. Wells were scored for the presence or absence of visual cytopathic effect in each dilution. The TCID50 per millilitre was calculated by the Karber method40. For comparison to plaque assay results, TCID50 per millilitre values were converted to p.f.u. per millilitre by multiplying by 0.7.

Lentiviral transduction and CRISPRi

We in-house generated a lentiviral construct expressing dCas9-KRAB-MeCP2 by PCR amplification of the dCas9-KRAB-MeCP2 (contains a domain of MeCP2) from pB-CAGGS-dCas9-KRAB-MeCP2 (Addgene, 110824), and then inserted it to the pLenti-EF1a-dCas9-VP64-2A-Blast backbone (Addgene, 61425) to replace the dCas9-VP64. The guide RNAs (gRNAs) used in CRISPRi were cloned into the Addgene 61427 backbone using BsmBI enzyme. To generate lentivirus, 293T cells were transfected with the lentiviral transfer vector DNA, psPAX2 packaging and pMD2.G envelope plasmid DNA at a ratio of 4:3:1 by lipofectamine 2000. After 16 h, the culturing medium was changed to a fresh one, and the supernatants were collected twice at 48 h and 72 h post-transfection. The collected lentiviral supernatants were filtered using 0.45 μm syringe filter (Thermo Fisher) and used to infect target A549 cells (polybrene (Sigma) was added at a final concentration of 8 μg ml−1). To infect A549 cells for CRISPRi, cells were first infected by a lentivirus expressing dCas9-KRAB-MeCP2 for 24 h and selected with appropriate antibiotics (10 μg ml−1 blasticidin) for 7 d to generate a stable cell line. The stable cell line was then subjected to viral infection by individual gRNAs targeting each enhancer, and they were further selected with 100 μg ml−1 Zeocin for 4–7 d. These stable cells were then used for experiments. The gRNA cloning oligos are shown in Supplementary Table 2.

Promoter reporter assay

An IL6 promoter-driven mCherry reporter construct was acquired from GeneCopoeia (HPRM30562-LvPM02). This lentiviral plasmid was introduced into the target cell line using lentiviral transduction similar to the steps described above in the lentivirus section. After 24 h of infection, the infected A549-ACE2 cells were selected under 2 μg ml−1 puromycin. The established stable cell line was maintained in DMEM/F-12 (1:1, Corning) medium supplemented with 10% FBS (GeneDepot), 100 μg ml−1 blasticidin and 2 μg ml−1 puromycin. It was then used for Mock or SARS-CoV-2 infection (0.1 MOI, 24 hpi), and mCherry expression was examined by confocal imaging.

RNA extraction and RT–qPCR

RNA extraction of SARS-CoV-2-infected A549-ACE2 cells was performed by TRIzol (Thermo Fisher, 15596-026) following the manufacturer’s instructions. In some other cases, RNA extraction from cells expressing CRISPRi or other transfection were performed using Quick-RNA Miniprep kit (Zymo Research, 11-328). Reverse transcription was conducted by using Superscript IV first strand synthesis kit (Thermo Fisher, 18091050), and the random hexamer primer was often used to test the expression levels of target genes. Real-time qPCR (RT–qPCR) was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 172-5274). Primer sequences used in this study can be found in Supplementary Table 2. Relative gene expression was normalized to the internal control (18S RNA).

Western blots

Cells were lysed in RIPA (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) with cOmplete Mini Protease Inhibitor Cocktail (Roche, 11836153001) on ice for 30 min. Lysates were sonicated in Qsonica 800R (25% amplitude, 3 min, 10 s on 20 s off interval) and centrifuged at 18,407 g. The supernatants were mixed with 2x Laemmli sample buffer (Bio-Rad) and boiled at 95 °C for 10 min. The boiled proteins were separated on 4%–15% SDS–PAGE gradient gels and transferred to LF PVDF membrane (Bio-Rad, 1620260). The membranes were blocked in 5% skim milk in TBST (20 mM Tris, 150 mM NaCl and 0.2% Tween-20 (w/v)) for 1 h and then briefly washed in TBST twice. Then, the membranes were incubated in TBST with primary antibodies (GAPDH (Proteintech, 60004-1, 1:2,000 dilution), alpha-tubulin (Sigma, T5168, 1:1,000 dilution), RAD21 (Abcam, Ab992, GR214359-10, 1:1,000 dilution), CTCF (Millipore, 07-729, 1:1,000 dilution), SMC3 (Abcam, Ab9263, GR466-7, 1:1,000 dilution), total histone H3 (Abcam, Ab1791, GR206754-1,1:2,000 dilution), H3K4me3 (Abcam, Ab8580, GR3264490-1, 1:2,000 dilution), H3K9me3 (Abcam, Ab8898, GR164977-4, 1:2,000 dilution), H3K27ac (Abcam, Ab4729, GR3357415-1, 1:2,000 dilution) and H3K27me3 (Cell Signaling Technology, 9733S, 19, 1:2,000 dilution)) at 4 °C overnight. After washing 3 times in TBST, the blots were incubated in TBST with secondary antibody (horseradish peroxidase (HRP)-conjugated antibody) for 1 h. After 6 times of washing in TBST, the blots were developed in a Bio-Rad ChemiDoc gel imaging system. The intensity of the bands was quantified using ImageJ. To quantify relative expression,

protein expression normalized by the intensity of the control protein was compared between infected and the Mock-treated samples. As described in the figure legends, loading controls were run on the same membrane, and sample processing controls were run on a separate membrane with the same amount of protein loaded from the same samples.

Immunofluorescence microscopy and terminal deoxynucleotidyl transferase dUTP nick end labelling assay

Expression of spike protein of SARS-CoV-2 was measured by immunofluorescence microscopy. A549-ACE2 cells seeded on glass slides were infected with SARS-CoV-2 at an MOI of 0.1. At 24 hpi, cells were fixed with 4% paraformaldehyde in PBS for 1 h at room temperature. The coverslips were washed with 0.1% BSA in 1x PBS (wash buffer) and blocked with 1% BSA with 0.3% Triton-X-100 in PBST (blocking buffer) for 45 min at room temperature. Cells were incubated with an antibody targeting the SARS-CoV-2 spike glycoprotein (1:500, Abcam, ab272504) or a monoclonal antibody targeting the HCoV-OC43 (1:200, EMD Millipore, MAB9012) diluted in the blocking buffer overnight at 4 °C. Subsequently after washes, cells were incubated with secondary antibody diluted in blocking buffer for 1 h at room temperature, followed by incubation with 4,6-diamidino-2-phenylindole (Invitrogen, D1306) for 5 min at room temperature. Coverslips were mounted in antifade mounting medium (Thermo Fisher, TA-030-FM) and fluorescence images were recorded using a Leica confocal microscope or a Nikon A1R. To assess apoptotic dead cells in the virus-infected condition, we used Click-iT TUNEL Alexa Fluor Imaging assay (Invitrogen, C10245) following the manufacturer’s instructions. As a positive control of the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, one sample was incubated in DNase I solution for 30 min at room temperature, again following the manufacturer’s instructions.

Hi-C 3.0

Hi-C 3.0 was performed on the basis of a recent protocol14, which was largely modified on the basis of in situ Hi-C19. Briefly, ~5 million SARS-CoV-2-infected A549-ACE2 cells were washed once with PBS to remove debris and dead cells, trypsinized off the culture plates, cross-linked using 1% formaldehyde for 10 min at room temperature and quenched with 0.75 M Tris–HCl pH 7.5 for 5 min. These cells were further cross-linked with 3 mM disuccinimidyl glutarate for 50 min and again quenched with 0.75 M Tris–HCl pH 7.5 for 5 min. Cross-linked cell pellets were washed with cold PBS, resuspended in 0.5 ml ice-cold Hi-C lysis buffer (10 mM Tris–HCl, pH 8.0; 10 mM NaCl, 0.2% NP-40 and protease inhibitor cocktail) and rotated at 4 °C for 30 min. Nuclei were washed once with 0.5 ml ice-cold Hi-C lysis buffer. After pelleting down the nuclei, 100 μl 0.5% SDS was used to resuspend and permeabilize the nuclei at 62 °C for 10 min. Then, 260 μl H20 and 50 μl 10% Triton-X-100 were added to quench the SDS at 37 °C for 15 min. Subsequently, enzyme digestion of chromatin was performed at 37 °C overnight by adding an additional 50 μl of 10X NEB buffer 2, MboI (NEB, R0147M, 100U) and DdeI (NEB, R0175L, 100 U). After overnight incubation, the restriction enzyme was inactivated at 62 °C for 20 min. To fill in the DNA overhangs and add biotin, 35 U DNA polymerase I (Klenow, NEB, M0210) together with 10 μl 1 mM biotin-dATP (Jeana Bioscience) and 1 μl 10 mM dCTP/dGTP/dTTP were added and incubated at 37 °C for 1 h with rotation. Blunt-end Hi-C DNA ligation was performed using 5000 U NEB T4 DNA ligase with 10X NEB T4 ligase buffer (10 mM ATP, 90 μl 10% Triton X-100 and 2.2 μl 50 mg ml−1 BSA) at room temperature for 4 h with rotation. After ligation, nuclei were pelleted down, resuspended with 440 μl Hi-C nuclear lysis buffer (50 mM Tris–HCl, pH 7.5, 10 mM EDTA, 1% SDS and protease inhibitor cocktail) and further sheared by a QSonica 800R sonicator using the parameters 10/20 s ON/OFF cycle, 25% Amp and 4 min. Around 10% of the sonicated chromatin was subjected to overnight decrosslinking at 65 °C, proteinase K treatment and DNA extraction. After DNA extraction, biotin labelled Hi-C 3.0 DNAs were purified using 20 μl Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher, 65002). The biotinylated DNA on C1 beads was used to perform on-beads library making with NEBNext Ultra II DNA Library Prep kit for Illumina (NEB, E7645L) following the manufacturer’s instructions. The sequencing was done on a NextSeq 550 platform with PE40 mode and demultiplexed with bcl2fastq v2.2.

ChIP-seq and spike-in calibrated ChIP-seq

ChIP-seq was performed as previously described with minor modifications41. For most ChIP-seqs in A549-ACE2 cells with Mock treatment or 24 h SARS-CoV-2 infection, ~5–10% of mouse ESCs (F121-9, a gift from David Gilbert) were added as spike-in controls before sonication with equal proportions to the two human cell samples (Extended Data Fig. 6a). For cell cross-linking for ChIP-seq, briefly, the cells were trypsinized in trypsin-EDTA (or Accutase for mESCs). After centrifugation, the cells were cross-linked with 1% formaldehyde in PBS for 10 min. The fixation steps were stopped in a quenching solution (0.75 M Tris–HCl pH 7.5) for 10 min. After centrifugation of the cells, we extracted the nuclei first using buffer LB1 (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA (pH 8.0), 10% (v/v) glycerol, 0.5% NP-40, 0.25% Triton X-100 and 1× cocktail protease inhibitor), and then LB2 (10 mM Tris–HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 1× cocktail protease inhibitor). After centrifugation, the nuclei were suspended in buffer LB3 (10 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), 0.1% Na-deoxycholate, 0.5% N-lauroyl sarcosine and 1× cocktail protease inhibitor) and the chromatin was fragmented with the Q800R3 sonicator (QSonica) using conditions of 10 s ON, 20 s OFF for 7–9 min (at 20% amplitude). Sheared chromatins were collected by centrifugation and incubated with appropriate antibodies (often 2–3 μg) at 4 °C overnight. The next morning, the antibody-protein-chromatin complex was retrieved by adding 25 μl pre-washed Protein G Dynabeads (Thermo Fisher, 10004D). Immunoprecipitated chromatin DNA was decrosslinked by heating at 65 °C overnight using elution buffer (1% SDS, 0.1 M NaHCO3), treated with RNase A and proteinase K, and finally purified with phenol chloroform. The DNAs were subjected to sequencing library construction using NEBNext Ultra II DNA Library Prep kit for Illumina (NEB, E7645L) and deep sequenced on a NextSeq 550 platform using 40 nt/40 nt pair-ended mode. The antibodies used for ChIP-seq include RNA Polymerase II (RPB1 N terminus, Cell Signaling Technology, 14958S, 4), RAD21 (Abcam, Ab992, GR214359-10), SMC3 (Abcam, Ab9263, GR466-7), CTCF (Millipore, 07-729), NIPBL (Bethyl, A301-779A, 4), H3K4me3 (Abcam, Ab8580, GR3264490-1), H3K9me3 (Abcam, Ab8898, GR164977-4), H3K27ac (Abcam, Ab4729, GR3357415-1), H3K27me3 (Cell Signaling Technology, 9733S, 19) and HA (Abcam, Ab9110, GR3231414-3).

Ribo-depleted total RNA-seq

Total RNAs from mock or virus-infected A549-ACE2 cells were extracted with TRIzol, and 100–200 ng of total RNAs were used for making strand-specific ribosome-RNA-depleted sequencing library with the NEB Ultra II Directional RNA Library kit (E7760L) following the manufacturer’s instructions. Libraries were sequenced on a NextSeq 550 using 40 nt/40 nt pair-ended mode.

Bioinformatic analyses

Calibrated ChIP-seq analyses

Sequencing reads were aligned to a concatenated genome of hg19 human genome assembly and mm9 mouse genome assembly with STAR (v2.7.0)42. Duplicated reads were removed, and only unique aligned reads were considered for later visualization and quantification. The scaling factor was calculated as the ratio of the number of reads uniquely aligned to human chromosomes versus the number of reads aligned to mouse chromosomes (Extended Data Fig. 6a). Uniquely aligned human reads were extracted with samtools (v1.9)43 and normalized by the corresponding scaling factor with deeptools (v3.1.3)44. For RPB1 ChIP-seq gene transcription quantification, hg19 RefSeq gene annotation coordinates were used. The peak calling of most ChIP-seq was performed with the parameters -f BAM -q 0.01 in MACS2 (v2.1.4)45. Peaks with log2 fold change of normalized ChIP-seq reads ratio smaller than −1 or greater than 1 were considered as reduced or gained peaks, respectively. ChIP-seq reads are summarized in Supplementary Table 1. A public NIPBL ChIP-seq dataset was obtained from SRR3102878.

RNA-seq analysis

RNA-seq reads were aligned to the hg19 reference human genome or SARS-CoV-2 viral genome (NC_045512.2) with STAR (v2.7.0)42. The percentage of reads uniquely aligned to SARS-CoV-2 genome versus total reads was calculated to verify a high viral infection rate. For human gene quantification, only uniquely aligned reads mapped to the hg19 genome were kept for further analysis.

Hi-C 3.0 data processing

Hi-C 3.0 raw data were primarily processed with Hi-C-Pro (v2.11.4)46. The pairs of reads were mapped to the human reference genome assembly hg19, and multimapped pairs, duplicated pairs and other unvalid 3C pairs were filtered out following the standard procedure of Hi-C-Pro. All valid Hi-C pairs were merged between replicates (unless specifically noted), and were further converted to Juicebox format47 or cooler format48 for visualization and further analyses. Hi-C contact matrices were normalized with ‘cooler’ (v0.8.11) ‘balance’ function. Read numbers of Hi-C are listed in Supplementary Table 1. The stratum-adjusted correlation coefficients (SCC) between two replicates were calculated to assess the reproducibility of Hi-C experiments49. The P(s) curve was calculated as a function of contact frequency (P) and genomic distances (s) (Fig. 1e). Only intra-chromosomal pairs (cis) were used to calculate the P(s) curve.

A/B compartment analyses

A/B nuclear compartments were identified on the basis of decomposed eigenvectors (E1) from 20 kb or 100 kb Hi-C contact matrices using cooltools (v0.4.1). A/B compartmental scores (E1) were corrected by GC densities in each bin. Saddle plot analyses were performed to measure the compartmentalization strength on a genome-wide scale using cooltools compute-saddle (similar to previous work10,13). Briefly, we first sorted the rows and columns in order of increasing compartmental scores within observed/expected (O/E) contact maps on the basis of data in Mock cells. Then we aggregated the rows and columns of the resulting matrix into 50 equally sized aggregate bins, and plotted the aggregated observed/expected Hi-C matrices as the ‘saddle’ plots (Fig. 3a). In Fig. 1c and elsewhere, Pearson correlation Hi-C matrices were used to emphasize the compartmental checkerboard pattern. We first calculated the observed/expected Hi-C maps as O/E matrices (bin size, 80,000 bp). Each value (i,j) in Pearson matrices indicates the Pearson correlation coefficient between the i-th column and the j-th column of O/E matrices (bin size, 80,000 bp). For changes in compartmental strength (Fig. 2c, and Extended Data Figs. 4a and 5a–c), the changes for each genomic region between Mock and SARS-CoV-2 samples were identified on the basis of 100-kb-binned compartmental scores (E1) of two Hi-C 3.0 replicates, largely following a previous study20. For each 100 kb, a Student’s t-test was first performed on Mock and SARS-CoV-2 compartmental scores (E1). Only the 100 kb bins that have |delta E1| > 0.2 and P-value < 0.05 were considered as bins with changed compartmental strength. Different categories of compartment changes (in Fig. 2c) were defined as (similar to ref. 20): A to stronger A: (Mock E1 – SARS-CoV-2 E1) < −0.2, Mock E1 > 0.2; B to A: (Mock E1 – SARS-CoV-2 E1) < −0.2, Mock E1 < −0.2, SARS-CoV-2 E1 > 0; B to weaker B: (Mock E1 – SARS-CoV-2 E1) < −0.2, Mock E1 < −0.2, SARS-CoV-2 E1 < 0; B to stronger B: (Mock E1 – SARS-CoV-2 E1) > 0.2, Mock E1 < −0.2; A to B: (Mock E1 – SARS-CoV-2 E1) < −0.2, Mock E1 > 0.2, SARS-CoV-2 E1 < 0; A to weaker A: (Mock E1 – SARS-CoV-2 E1) < −0.2, Mock E1 > 0.2, SARS-CoV-2 E1 > 0. For enrichment of epigenetic features on affected compartments (for example, see Fig. 2f), we ranked all 100 kb bins into six categories (top 5%, top 5–10%, 10–50%, 50–90%, bottom 5–10%, bottom 5%) and then calculated epigenomic features at each 100 k bin. For histone modifications or chromatin regulatory factors that have sharp peaks in ChIP-seq (such as H3K27ac and H3K4me3), we quantified the numbers of peaks in each 100 kb bin. For modifications or factors that have broad ChIP-seq patterns (such as H3K9me3 and H3K27me3), we quantified the calibrated ChIP-seq reads throughout the entire 100 kb bin. The enrichment of these ChIP-seq signals was calculated by dividing the median quantification inside these six categories by the genome-wide median quantification.

TADs and insulation scores

Hi-C 3.0 data were used to identify TADs in A549-ACE2 cells following standard 4D Nucleome consortium protocol (github.com/4dn-dcic/docker-4dn-insulation-scores-and-boundaries-caller). First, insulation scores22 and boundary strengths of each 10 kb bin with a 200 kb window size were measured to quantify the TAD insulation using cooltools (github.com/open2c/cooltools/blob/master/cooltools/cli/diamond_insulation.py). Then, we identified TAD boundaries in Mock and SARS-CoV-2-infected samples by using a boundary score cut-off of 0.5. We further merged TAD boundaries identified in these two conditions and compared insulation scores at merged TAD boundaries (Fig. 4d). Merged TAD coordinates were used to perform downstream analyses. For each TAD, we quantified its mean Hi-C contacts throughout the domain (excluding very short distant interactions <15 kb), which we considered intra-TAD interaction in this paper. On the basis of the log2 fold changes of intra-TAD mean Hi-C contacts (SARS-CoV-2/Mock), we ranked all TADs into six categories (top 5%, top 5–10%, 10–50%, 50–90%, bottom 5–10%, bottom 5%) and calculated different epigenomic features of these six categories. For histone modifications or chromatin regulatory factors that have sharp peaks in ChIP-seq (such as H3K27ac, H3K4me3, CTCF or cohesin subunits), we quantified the numbers of peaks as well as the numbers of gained or lost peaks in different TADs. For modifications or factors that have broad ChIP-seq patterns (such as H3K9me3 and H3K27me3), we quantified the calibrated ChIP-seq reads throughout the TADs. The enrichment of these ChIP-seq signals was calculated by dividing the median quantification inside these six categories by the genome-wide median quantification.

Chromatin loop calling and enhancer–promoter contacts

For loop calling, we largely followed a recent 4DN benchmarking paper14. In brief, we used a reimplement of HICCUPS loop-calling tool, call-dots function inside cooltools (github.com/open2c/cooltools/blob/master/cooltools/cli/call_dots.py), to identify structural chromatin loops in different samples. We first called loops at 5 kb and 10 kb resolution separately, then used the following strategy to merge 5 kb and 10 kb loops. The 5 kb loops called at both 10 kb and 5 kb resolutions were first kept, all unique 10 kb resolution loops were kept, and only unique 5 kb loops smaller than 100 kb were kept. Differential loops were identified by first quantifying the Hi-C raw contacts at 40 kb resolution of each called loop, and then performing DESeq2 (v1.26.0)50 differential analyses on these raw counts. We considered loops with a DESeq2 FDR < 0.1 and a log2FC > 0 or <0 as virus-strengthened or weakened chromatin loops. The APA was performed by superimposing observed/expected Hi-C matrices on merged loops with the coolpuppy tool (v0.9.2)51.

ABC score

ABC score calculation largely followed a previous study26 with modifications. For the A score (enhancer activity) of a gene, we first identified all putative enhancers of this gene by selecting H3K27ac ChIP-seq peaks located within 1 Mb of the promoter. Then we quantified the calibrated H3K27ac ChIP-seq signals on these putative enhancers (extended 150 bp from MACS2 peaks) as A scores. The A-only quantification of enhancer activity for this gene would be the sum of the A scores for all putative enhancers. For the C score (enhancer–promoter contact) between a gene and putative enhancers, we quantified the normalized Hi-C contacts formed in between the 5 kb bins harbouring the gene promoter and the putative enhancer. For the ABC score, we multiplied the A score of each enhancer by the C score, and generated the summation of these if multiple putative enhancers exist for a gene. The P score of any gene was calculated as the calibrated H3K4me3 ChIP-seq signal at its promoter region (±2.5 kb from transcription start site). For ABC-P or ABC-P2 scores, we multiplied the summed ABC score of a gene by its P score (promoter H3K4me3 signal) or by the square of its P score. The transcriptional changes of any gene were calculated on the basis of the log2 fold change of RPB1 ChIP-seq reads over the whole gene body (average of three ChIP-seq replicates). Pearson correlation coefficient was used to measure the correlation between ABC score change and transcriptional change. The list of IFN response genes was obtained from GSEA molecular signature databases (Interfero_Alpha_Response), and the list of PIF genes was manually curated on the basis of recent literature52 studying immuno-pathology of SARS-CoV-2 infection (see Supplementary Table 3).

Statistics

For all boxplots, the centre lines represent medians; box limits indicate the 25th and 75th percentiles; and whiskers extend 1.5 times the interquartile range (IQR) from the 25th and 75th percentiles. For qPCR, data were analysed using Prism (v7.00) and presented as mean ± s.d., as indicated in figure legends. At least two biological replicates were conducted for RNA-seq, ChIP-seq or Hi-C sequencing. Student’s t-test (two-tailed) was commonly used to compare means between two qPCR groups; P < 0.05 was considered significant (*P < 0.05; **P < 0.01; ***P < 0.001). Statistical analyses for sequencing data were performed with Python (v3.6, pandas v1.1.5, numpy v1.17.3, matplotlib v3.1.2, scipy v1.5.4) or R (v3.6.0) scripts.

Reporting summary

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