Novel gene-specific translation mechanism of dysregulated, chronic inflammation reveals promising, multifaceted COVID-19 therapeutics
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Abstract
Hyperinflammation and lymphopenia provoked by SARS-CoV-2-activated macrophages contribute to the high mortality of Coronavirus Disease 2019 (COVID-19) patients. Thus, defining host pathways aberrantly activated in patient macrophages is critical for developing effective therapeutics. We discovered that G9a, a histone methyltransferase that is overexpressed in COVID-19 patients with high viral load, activates translation of specific genes that induce hyperinflammation and impairment of T cell function or lymphopenia. This noncanonical, pro-translation activity of G9a contrasts with its canonical epigenetic function. In endotoxin-tolerant (ET) macrophages that mimic conditions which render patients with pre-existing chronic inflammatory diseases vulnerable to severe symptoms, our chemoproteomic approach with a biotinylated inhibitor of G9a identified multiple G9a-associated translation regulatory pathways that were upregulated by SARS-CoV-2 infection. Further, quantitative translatome analysis of ET macrophages treated progressively with the G9a inhibitor profiled G9a-translated proteins that unite the networks associated with viral replication and the SARS-CoV-2-induced host response in severe patients. Accordingly, inhibition of G9a-associated pathways produced multifaceted, systematic effects, namely, restoration of T cell function, mitigation of hyperinflammation, and suppression of viral replication. Importantly, as a host-directed mechanism, this G9a-targeted, combined therapeutics is refractory to emerging antiviral-resistant mutants of SARS-CoV-2, or any virus, that hijacks host responses.
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SciScore for 10.1101/2020.11.14.382416: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibodies against G9a (07-551) and H3K9me2 (07-441) were from Millipore; Antibodies against EIF4E(11149-1-AP), EIF3B(10319-1-AP), HNRNPA2B1(14813-1-AP),METTL3(15073-1-AP), YTHDF2(24744-1-AP),PD-L1 (66248-1-lg), CDC20(10252-1-AP), CDCA7(15249-1-AP), BIRC5(10508-1-AP), TPX2(11741-1-AP), TOP2A(24641-1-AP), NUSAP1(12024-1-AP), SPP1(22952-1-AP), SQSTM1(18420-1-AP), ACTIN(60008-1-lg) were from Proteintech. G9asuggested: (Millipore Cat# 07-551, RRID:AB_310709)H3K…SciScore for 10.1101/2020.11.14.382416: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibodies against G9a (07-551) and H3K9me2 (07-441) were from Millipore; Antibodies against EIF4E(11149-1-AP), EIF3B(10319-1-AP), HNRNPA2B1(14813-1-AP),METTL3(15073-1-AP), YTHDF2(24744-1-AP),PD-L1 (66248-1-lg), CDC20(10252-1-AP), CDCA7(15249-1-AP), BIRC5(10508-1-AP), TPX2(11741-1-AP), TOP2A(24641-1-AP), NUSAP1(12024-1-AP), SPP1(22952-1-AP), SQSTM1(18420-1-AP), ACTIN(60008-1-lg) were from Proteintech. G9asuggested: (Millipore Cat# 07-551, RRID:AB_310709)H3K9me2suggested: NoneEIF4E(11149-1-APsuggested: NoneEIF3B(10319-1-APsuggested: NoneHNRNPA2B1suggested: (Proteintech Cat# 14813-1-AP, RRID:AB_2279638)METTL3suggested: (Proteintech Cat# 15073-1-AP, RRID:AB_2142033)YTHDF2suggested: (Proteintech Cat# 24744-1-AP, RRID:AB_2687435)PD-L1 ( 66248-1-lg) , CDC20suggested: NoneCDCA7suggested: (Proteintech Cat# 15249-1-AP, RRID:AB_2878119)BIRC5suggested: NoneTPX2suggested: (Proteintech Cat# 11741-1-AP, RRID:AB_2208895)TOP2A(24641-1-APsuggested: NoneNUSAP1suggested: (Proteintech Cat# 12024-1-AP, RRID:AB_2157789)SPP1suggested: None22952-1-APsuggested: (Proteintech Cat# 22952-1-AP, RRID:AB_2783651)SQSTM1suggested: NoneAntibody against p-p65 (S536)(3033) was from Cell Signaling. p-p65suggested: (Millipore Cat# PP65-K, RRID:AB_1603731)S536suggested: NoneAntibody against Brg1 (H-88) (sc-10768x) was from Santa Cruz Brg1suggested: NoneAnti-HA (clone HA-7) and anti-flag M2 (clone M2) antibodies were from Sigma. m6A RNA methylation quantification kit was from Abcam(ab185912). Anti-HAsuggested: Noneanti-flagsuggested: NoneTen percent of 150 ng mRNA was used as Input mRNA, and the remainder was incubated with 3 ug anti-m6A polyclonal antibody (Synaptic Systems; 202003) which was preconjugated to Dynabeads Protein A (Thermo Fisher; 10001D) in 500uL IP buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Igepal CA-630) for 2 hours at 4°C. anti-m6Asuggested: (Synaptic Systems Cat# 202 003, RRID:AB_2279214)Experimental Models: Cell Lines Sentences Resources HEK 293 stable TLR4-MD2-CD14 cell line was also purchased from InvivoGen. HEK 293suggested: NoneRaw264.7 and THP1 cells were purchased from ATCC (Manassas,VA). THP1suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)For MeRIP-Seq, THP-1 cells were first incubated in the presence of 60 nM PMA overnight to differentiate into macrophages followed by 48 h resting in PMA-free medium. THP-1suggested: NoneTransfection and CRISPR Knockout: 293TLR4-MD2-CD14 cells were seeded in 6-well plates for 24 h before transfection, and the constructs were transfected or co-transfected into MCF7 cells using reagent jetPRIME (Polyplus). 293TLR4-MD2-CD14suggested: NoneMCF7suggested: NCI-DTP Cat# MCF7, RRID:CVCL_0031)In brief, a total of 10 μg of plasmid, including target plasmid, pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260) with a ratio of 10:5:9, was co-transfected into 293T cells with jetPRIME™. 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)MDA-MB-231 cells at 60-80% confluency were incubated with the virus containing media for 24-48 hours, and then subjected to 1.0 μg/mL puromycin selection. MDA-MB-231suggested: NoneMETTL3 KO, G9a KO, and control Raw 264.7 cells were seeded in 6-well plates with indicated treatments followed by 50□μg/mL Mitomycin C treatment for 30□min at 37□°C. Raw 264.7suggested: NoneSoftware and Algorithms Sentences Resources Protein quantitation was performed using TMT reporter intensity found in MaxQuant and a one-sample t-test statistics on three technical replicates was used with a p-value of 5% to report statistically significant protein abundance fold-changes. MaxQuantsuggested: (MaxQuant, RRID:SCR_014485)Analysis of Functional Category and Networks: The biological processes and molecular functions of the G9a-interacting proteins were categorized by IPA (http://www.ingenuity.com/), DAVID (http://david.abcc.ncifcrf.gov/), and STRING (http://string-db.org/). http://www.ingenuity.com/suggested: (Ingenuity Pathways Knowledge Base, RRID:SCR_008117)DAVIDsuggested: (DAVID, RRID:SCR_001881)STRINGsuggested: (STRING, RRID:SCR_005223)Ten percent of 150 ng mRNA was used as Input mRNA, and the remainder was incubated with 3 ug anti-m6A polyclonal antibody (Synaptic Systems; 202003) which was preconjugated to Dynabeads Protein A (Thermo Fisher; 10001D) in 500uL IP buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Igepal CA-630) for 2 hours at 4°C. Synaptic Systemssuggested: (Synaptic Systems, RRID:SCR_013612)Thermo Fishersuggested: (Thermo Fisher Scientific, RRID:SCR_008452)Then the Input and m6A-IP reads were mapped to human genome version hg38 by STAR v. STARsuggested: (STAR, RRID:SCR_015899)The turnover rates (kdeg/ksyn), curve maxima (Mo/Ho), offsets (γ) and goodness-of-fit statistics (SSE, RMSE, rsquared, adjrsquared) were obtained for each protein using a nonlinear least square (NLS) method in MATLAB (vR2017b). MATLABsuggested: (MATLAB, RRID:SCR_001622)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.
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