FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

  1. Dawei Zhou
  2. Zhenyu Wu
  3. Jun-Gyu Park
  4. Guillaume N Fiches
  5. Tai-Wei Li
  6. Qin Ma
  7. Huachao Huang
  8. Ayan Biswas
  9. Luis Martinez-Sobrido
  10. Netty G Santoso
  11. Jian Zhu

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Abstract

FACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated in both epithelial and natural killer (NK) cells. The histone acetyltransferase TIP60 contributes to the acetylation of SUPT16H middle domain (MD) at lysine 674 (K674). Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H in both epithelial and NK cells. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (HDAC1, EZH2), and further regulates their activation status and/or promoter association as well as affects the relevant histone marks (H3ac, H3K9me3 and H3K27me3). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, depletion or inhibition of SUPT16H is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that depletion or inhibition of SUPT16H also causes the remarkable activation of IFN signaling in NK cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in coordinating with BRD4 and regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of the FACT inhibitor CBL0137 to treat viral infections.

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  1. Version published to 10.1093/nar/gkac645
  2. ScreenIT

    SciScore for 10.1101/2021.04.21.440833: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-acetyl lysine antibody was purchased from ImmuneChem.
    Anti-acetyl
    suggested: (ImmuneChem Cat# ICP0380, RRID:AB_2801477)
    Anti-SUPT16H, anti-SSRP1, anti-TIP60, anti-IFI16, anti-MX1, anti-ISG15, anti-GAPDH and normal mouse IgG antibodies were purchased from Santa Cruz Biotechnology.
    Anti-SUPT16H,
    suggested: None
    Anti-SUPT16H
    suggested: None
    anti-SSRP1
    suggested: None
    anti-TIP60
    suggested: None
    anti-IFI16
    suggested: None
    anti-MX1
    suggested: None
    anti-ISG15
    suggested: None
    anti-GAPDH
    suggested: None
    mouse IgG
    suggested: None
    Anti-BRD4 antibody was purchased from Bethyl Laboratories.
    Anti-BRD4
    suggested: None
    Anti-FLAG, anti-V5, and normal rabbit IgG antibodies were purchased from Invitrogen.
    Anti-FLAG
    suggested: None
    anti-V5
    suggested: None
    rabbit IgG
    suggested: None
    Anti-K48Ub, anti-histone H3, anti-mouse HRP-linked and anti-rabbit HRP-linked antibodies were purchased from Cell Signaling Technology.
    Anti-K48Ub, anti-histone
    suggested: None
    Anti-K48Ub, anti-histone H3
    suggested: None
    anti-histone
    suggested: None
    anti-mouse
    suggested: None
    anti-rabbit HRP-linked antibodies were purchased from Cell Signaling Technology.
    suggested: None
    Anti-HDAC1 antibody was purchased from Novus Biologicals.
    Anti-HDAC1
    suggested: None
    Anti-H3K9me3, anti-H3K27me3, anti-H3ac (pan-acetyl), and anti-EZH2 antibodies were purchased from Active Motif.
    Anti-H3K9me3
    suggested: None
    anti-H3K27me3
    suggested: None
    anti-H3ac
    suggested: (Millipore Cat# 06-599, RRID:AB_2115283)
    anti-EZH2
    suggested: None
    PE/Cyanine7 anti-human CD107a (LAMP-1), PE anti-human IFNγ and anti-IL-6 antibodies were purchased from BioLegend.
    PE/Cyanine7 anti-human CD107a
    suggested: (BioLegend Cat# 328618, RRID:AB_11147955)
    anti-human CD107a
    suggested: None
    anti-human IFNγ
    suggested: None
    anti-IL-6
    suggested: None
    Anti-flavivirus group antigen antibody that probes ZIKV E protein and anti-SARS-CoV-1/2 NP 1C7C7 antibody was purchased from Sigma-Aldrich.
    Anti-flavivirus group antigen
    suggested: None
    anti-SARS-CoV-1/2 NP
    suggested: None
    Anti-influenza A virus nucleoprotein (NP) antibody was obtained from BEI Resources.
    Anti-influenza A virus nucleoprotein (NP
    suggested: None
    Anti-IL-4, anti-IL-8, and FITC Mouse anti-rat IgG1 antibodies was purchased from BD Biosciences.
    Anti-IL-4,
    suggested: None
    Anti-IL-4
    suggested: None
    anti-IL-8
    suggested: None
    anti-rat IgG1
    suggested: None
    To determine acetylation, ubiquitination, and other protein binders of the targeted proteins, cell lysates were incubated with the specific antibodies recognizing the targeted proteins or control IgG, followed by the incubation with protein A/G magnetic beads.
    control IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and plasmids: HEK293T, HeLa, Vero E6, and TZM-bl cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich).
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    TZM-bl
    suggested: NIH-ARP Cat# 8129-442, RRID:CVCL_B478)
    Jurkat cells were maintained in RPMI 1640 medium (Gibco).
    Jurkat
    suggested: None
    HeLa or A549 cells were seeded at the density of 8,000 cells/well on 96-well culture plates.
    HeLa
    suggested: None
    A549
    suggested: None
    In brief, K562 target cells were pre-stained with the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen) according to the manufacturer’s protocols.
    K562
    suggested: NCI-DTP Cat# K-562, RRID:CVCL_0004)
    1 × 106 NK-92 cells were co-cultured with the same number of K562 cells (1 : 1 of effector : target [E : T] ratio) for 4 h.
    NK-92
    suggested: None
    In brief, Vero E6 cells were seeded on 96-well plates with 1 × 104 cells/well at 24 h prior to viral infection.
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    Four domains of SUPT16H, NTD, DD, MD and CTD, were cloned in pQCXIP (Clontech) with a N-terminal FLAG tag.
    pQCXIP
    suggested: RRID:Addgene_15714)
    Site-specific mutation of K674R was introduced in pQCXIP-FLAG-MD by using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer’s instructions.
    pQCXIP-FLAG-MD
    suggested: None
    TIP60 shRNA (5’-TCG AAT TGT TTG GGC ACT GAT-3’) and firefly luciferase (FLuc) shRNA (5’-CAC AAA CGC TCT CAT CGA CAA G-3’) were cloned in a pAPM lentiviral vector as previously described (Huang et al., 2019).
    pAPM
    suggested: None
    cis-reporting vectors of IFN activity, pISRE-Luc and pGAS-Luc, were purchased (Agilent Technologies).
    pISRE-Luc
    suggested: None
    HDAC1 was cloned in pcDNA-DEST40 with a C-terminal V5 tag.
    pcDNA-DEST40
    suggested: None
    pLX317-EZH2-V5 expression vector was acquired from Sigma-Aldrich.
    pLX317-EZH2-V5
    suggested: None
    At 72 h post-transfection, cells were further transfected with pISRE-Luc or pGAS-Luc vector along with pRL-TK by using the TurboFect™ Transfection Reagent (Thermo Scientific) for 24 h.
    pGAS-Luc
    suggested: None
    pRL-TK
    suggested: RRID:Addgene_11313)
    Software and Algorithms
    SentencesResources
    The intensity of protein bands was quantified by using the ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Percentages of virus-infected cells was quantified by using the Gen5 Image+ software (BioTek).
    Gen5 Image+
    suggested: None
    Differential expression of genes was analyzed by DESeq2 (Love et al., 2014).
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    R packages, pheatmap and clusterProfiler, were used for heatmap construction and pathway analysis, respectively.
    clusterProfiler
    suggested: (clusterProfiler, RRID:SCR_016884)
    MFI was calculated by using the FlowJo V10 software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Results were presented as either means ± standard deviation (SD) or means ± standard error of the mean (SEM), and graphed by using the GraphPad Prism 9.0 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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.

    About SciScore

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  3. Version published to 10.1101/2021.04.21.440833v1 on bioRxiv