DNA damage response at telomeres boosts the transcription of SARS‐CoV‐2 receptor ACE2 during aging

  1. Sara Sepe
  2. Francesca Rossiello
  3. Valeria Cancila
  4. Fabio Iannelli
  5. Valentina Matti
  6. Giada Cicio
  7. Matteo Cabrini
  8. Eugenia Marinelli
  9. Busola R Alabi
  10. Alessia di Lillo
  11. Arianna Di Napoli
  12. Jerry W Shay
  13. Claudio Tripodo
  14. Fabrizio d’Adda di Fagagna

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Abstract

No abstract available

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  1. Version published to 10.15252/embr.202153658
  2. ScreenIT

    SciScore for 10.1101/2021.06.09.447484: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All mice were bred and maintained under pathogen-free condition at the Scripps Research Institute and were handled according to Institutional Animal Care and Use Committee guidelines.
    Consent: Patients provided written informed consent to the use of their tissue samples for research purposes (Ethical Approval SA250)
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power AnalysisAfter deparaffinization, antigen retrieval in pH8 buffer was brought to a boil at 100% power, followed by 20% power for 15 minutes using microwave technology (MWT).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For multiple-marker immunostainings, sections were incubated with ACE2 and TTF-1 primary antibodies and the binding of the primary antibodies to their respective antigenic substrates was revealed by made-specific secondary antibodies conjugated with Alexa-488 (Life Technologies, 1:250) and Alexa-568 (Life Technologies, 1:300) fluorochromes.
    TTF-1
    suggested: None
    Antibodies: The following primary antibodies were adopted for IHC and IF on mouse and human tissues: rabbit polyclonal ACE2 (1:500 pH8, ab15348, Abcam), rabbit polyclonal Prosurfactant Protein C (1:200 pH9, AB3786, Merck Millipore)
    ACE2
    suggested: (LSBio (LifeSpan Cat# LS-C347-500, RRID:AB_1271966)
    Experimental Models: Cell Lines
    SentencesResources
    RNA extraction from cultured cells: For MEFs, HeLa, and BJ cells total RNA was extracted using Maxwell® RSC Instrument, with Maxwell® RSC simplyRNA Tissue Kit (AS134, Promega), following manufacturer’s instructions.
    HeLa
    suggested: None
    Primer sequences (5−3′ orientation): Luciferase assay: HeLa shTRF2 cells were transfected with ACE2(−1119)-luc plasmid, a gift from Gerhart Ryffel (
    HeLa shTRF2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Rosa26-CreERT2 Trf2F/F MEFs (Okamoto et al, 2013), a kind gift from Eros Lazzerini Denchi (National Cancer Institute, NIH, Bethesda, USA), were grown in DMEM supplemented with 10% fetal bovine serum and 1% glutamine; for CreER activation, cells were treated with 600nM 4 hydroxytamoxifen (4OHT, H7904, Sigma-Aldrich).
    Rosa26-CreERT2 Trf2F/F MEFs
    suggested: None
    For the analysis of young and old mice, C57BL/6 J mice were purchased from the Charles River Laboratories.
    C57BL/6 J
    suggested: None
    Terc mice (B6.Cg-Terctm1Rdp/J Stock No: 004132 | mTR−/−)
    B6.Cg-Terctm1Rdp/J
    suggested: RRID:IMSR_JAX:004132)
    Terc+/− mice were intercrossed to generate first-generation (G1) homozygous Terc−/− knockout mice.
    Terc+/−
    suggested: None
    Second-generation (G2) Terc−/− mice were generated by successive breeding of G1 Terc−/− and then G3 Terc−/− mice by crosses between G2 Terc−/− mice.
    Terc−/−
    suggested: None
    Finally, wild type C57BL/6J and G3 Tert−/− mice were used for the experiments.
    Tert−/−
    suggested: None
    Rosa26-CreERT mice (Jackson Laboratory) and TRF2 conditional knockout mice (Celli & de Lange, 2005) and mice carrying a p53 conditional allele (Jackson Laboratory) were crossed to generate Trf2/p53/Rosa26 mice.
    Rosa26-CreERT
    suggested: None
    Trf2/p53/Rosa26
    suggested: None
    Mice were maintained in 129/c57Bl6 genetic background.
    129/c57Bl6
    suggested: None
    Recombinant DNA
    SentencesResources
    Primer sequences (5−3′ orientation): Luciferase assay: HeLa shTRF2 cells were transfected with ACE2(−1119)-luc plasmid, a gift from Gerhart Ryffel (
    −1119)-luc
    suggested: None
    Addgene plasmid # 31110; http://n2t.net/addgene:31110; RRID:Addgene_31110), 5 days after doxycycline treatment to induce shTRF2 expression or 1 day after the ionizing radiations treatment.
    detected: RRID:Addgene_31110)
    Software and Algorithms
    SentencesResources
    IHC staining was revealed using Novolink Polymer Detection Systems (Leica Novocastra) or IgG (H#L) specific secondary antibodies (Life Technologies, 1:500) and DAB (3,3’-Diaminobenzidine
    Novolink Polymer Detection Systems
    suggested: None
    Microphotographs were collected using a Zeiss Axiocam 503 Color digital camera with the Zen 2.0 Software (Zeiss).
    Zen
    suggested: None
    NanoDrop spectrophotometer was used to detect RNA quantity and purity.
    NanoDrop
    suggested: None
    TFBS motifs for the human Ace2 gene (refseq ID: NM_021804) were searched around −450 +50 of the TSS, using the Jaspar 2018_NR matrix as a descriptor.
    Jaspar
    suggested: (JASPAR, RRID:SCR_003030)
    The EnrichR tool (Kuleshov et al, 2016) was used to perform a gene set enrichment analysis of the resulting Top100 significantly over-represented transcription factors.
    EnrichR
    suggested: (Enrichr, RRID:SCR_001575)
    P value was calculated by the indicated statistical tests, using Prism software.
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    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.

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

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.06.09.447484v1 on bioRxiv