SARS-CoV-2 Restructures the Host Chromatin Architecture

  1. Ruoyu Wang
  2. Joo-Hyung Lee
  3. Feng Xiong
  4. Jieun Kim
  5. Lana Al Hasani
  6. Xiaoyi Yuan
  7. Pooja Shivshankar
  8. Joanna Krakowiak
  9. Chuangye Qi
  10. Yanyu Wang
  11. Holger K. Eltzschig
  12. Wenbo Li

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Abstract

SARS-CoV-2 has made >190-million infections worldwide, thus it is pivotal to understand the viral impacts on host cells. Many viruses can significantly alter host chromatin 1 , but such roles of SARS-CoV-2 are largely unknown. Here, we characterized the three-dimensional (3D) genome architecture and epigenome landscapes in human cells after SARS-CoV-2 infection, revealing remarkable restructuring of host chromatin architecture. High-resolution Hi-C 3.0 uncovered widespread A compartmental weakening and A-B mixing, together with a global reduction of intra-TAD chromatin contacts. The cohesin complex, a central organizer of the 3D genome, was significantly depleted from intra-TAD regions, supporting that SARS-CoV-2 disrupts cohesin loop extrusion. Calibrated ChIP-Seq verified chromatin restructuring by SARS-CoV-2 that is particularly manifested by a pervasive reduction of euchromatin modifications. Built on the rewired 3D genome/epigenome maps, a modified activity-by-contact model 2 highlights the transcriptional weakening of antiviral interferon response genes or virus sensors (e.g., DDX58 ) incurred by SARS-CoV-2. In contrast, pro-inflammatory genes (e.g. IL-6 ) high in severe infections were uniquely regulated by augmented H3K4me3 at their promoters. These findings illustrate how SARS-CoV-2 rewires host chromatin architecture to confer immunological gene deregulation, laying a foundation to characterize the long-term epigenomic impacts of this virus.

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    SciScore for 10.1101/2021.07.20.453146: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Then, the membranes were incubated in TBST with primary antibodies (GAPDH (Proteintech, 60004-1), RAD21 (Abcam, Ab992, Lot: GR214359-10), CTCF (Millipore, 07-729), SMC3 (Abcam, Ab9263, Lot:GR466-7), Total Histone H3 (Abcam, Ab1791, Lot:GR206754-1), H3K4me3 (Abcam, Ab8580, Lot: GR3264490-1), H3K9me3 (Abcam, Ab8898, Lot: GR164977-4), H3K27ac (Abcam, Ab4729, Lot: GR3357415-1), and H3K27me3 (Cell Signaling Technology, #9733S, Lot19)) at 4°C overnight.
    GAPDH
    suggested: (Proteintech Cat# 60004-1-Ig, RRID:AB_2107436)
    RAD21
    suggested: (Abcam Cat# ab992, RRID:AB_2176601)
    CTCF
    suggested: (Millipore Cat# 07-729, RRID:AB_441965)
    Lot:GR466-7), Total Histone H3
    suggested: None
    H3K27ac
    suggested: (Abcam Cat# ab4729, RRID:AB_2118291)
    H3K27me3
    suggested: None
    Cells were incubated with 1:500 anti-SARS-CoV-2 Spike glycoprotein antibody (Abcam, Catalog#ab272504) diluted in blocking buffer for overnight at 4 degree.
    anti-SARS-CoV-2 Spike glycoprotein
    suggested: (Abcam Cat# ab272504, RRID:AB_2847845)
    The next morning, the antibody-protein-chromatin complex was retrieved by adding 25μl pre-washed Protein G Dynabeads (Thermo Fisher Scientific, 10004D).
    antibody-protein-chromatin
    suggested: None
    The antibodies used for ChIP-Seq include RNA Polymerase II (RPB1 N terminus, Cell Signaling Technology, #14958S, Lot4), RAD21 (Abcam, Ab992, Lot: GR214359-10), SMC3 (Abcam, Ab9263, Lot:GR466-7), CTCF (Millipore, 07-729), H3K4me3 (Abcam, Ab8580, Lot: GR3264490-1), H3K9me3 (Abcam, Ab8898, Lot: GR164977-4), H3K27ac (Abcam, Ab4729, Lot: GR3357415-1), H3K27me3 (Cell Signaling Technology, #9733S, Lot19) and HA (Abcam, Ab9110, Lot:GR3231414-3).
    RPB1 N terminus, Cell Signaling Technology,
    suggested: None
    SMC3
    suggested: (Abcam Cat# ab9263, RRID:AB_307122)
    Lot:GR466-7), CTCF
    suggested: None
    H3K4me3
    suggested: None
    H3K9me3
    suggested: (Abcam Cat# ab8898, RRID:AB_306848)
    HA
    suggested: (Abcam Cat# ab9110, RRID:AB_307019)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: Human lung adenocarcinoma cells A549 expressing human ACE2 (A549-ACE2, #NR53821) was acquired from BEI Resources (Manassas, VA).
    A549
    suggested: None
    293T cells were from ATCC and were cultured in DMEM with 10% FBS.
    293T
    suggested: None
    Prior to infection, Vero-E6 cells were washed once with PBS and the viral inoculum was added to the flask in the presence of 3 ml of serum-free and antibiotics-free MEM medium supplemented with 1mM HEPES, and incubated for 1 hour at 37°C with 5% CO2.
    Vero-E6
    suggested: None
    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, and were cross-linked using 1% formaldehyde for 10mins at room temperature, quenched with 0.75M Tris-HCl pH 7.5 for 5mins.
    A549-ACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    Lenti-viral 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 insert it to the pLenti-EF1a-dCas9-VP64-2A-Blast backbone (Addgene 61425) to replace th dCas9-VP64.
    pB-CAGGS-dCas9-KRAB-MeCP2
    suggested: RRID:Addgene_110824)
    pLenti-EF1a-dCas9-VP64-2A-Blast
    suggested: None
    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, respectively, by lipofectamine 2000.
    psPAX2
    suggested: RRID:Addgene_12260)
    pMD2.G
    suggested: RRID:Addgene_12259)
    Software and Algorithms
    SentencesResources
    For RBP1 ChIP-Seq gene transcription quantification, hg19 RefSeq gene annotation coordinates were used.
    RefSeq
    suggested: (RefSeq, RRID:SCR_003496)
    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 v 2.7.027.
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Differential gene expression analyses were performed with EdgeR, and genes with |FC|>2, FDR<0.05 were considered as significantly differential expressed genes.
    EdgeR
    suggested: (edgeR, RRID:SCR_012802)
    The pair of reads were mapped to the human reference genome assembly hg19, and multi-mapped pairs, duplicated pairs, and other unvalid 3C pairs were filtered out following the standard procedure of HiC-Pro.
    HiC-Pro
    suggested: (HiC-Pro, RRID:SCR_017643)
    Compartmental domains are genomic regions with continuous positive or negative compartmental scores (E1), identified by applying HOMER tool (findHiCCompartments.pl) on E1 scores.
    HOMER
    suggested: (HOMER, RRID:SCR_010881)
    We considered loops with a DESeq2 FDR <0.1 and a log2FC >0 or <0 as virus-strengthened or weakened chromatin loops.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Statistics: qPCR data was analyzed by Prism and presented as mean±SD, which are indicated in figure legends.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Statistical analyses for sequencing data were performed with Python or R scripts.
    Python
    suggested: (IPython, RRID:SCR_001658)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

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  2. Version published to 10.1101/2021.07.20.453146v1 on bioRxiv