Genome-Wide Screens Reveal that Resveratrol Induces Replicative Stress in Human Cells

Abstract

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.

A novel p53 regulator, C16ORF72/TAPR1, buffers against telomerase inhibition

“…The genome‐wide screen was carried out using the previously described extended knockout (EKO) pooled CRISPR‐Cas9 sgRNA library transduced into an inducible Cas9 NALM‐6 cell clone (Benslimane et al., 2020; Bertomeu et al., 2017) treated with either 0.1% DMSO (v/v) or 20 µM BIBR1532 for 20 days (Figure 1c‐d). Several gene deletions were identified that either buffered cells against (synthetic‐rescue) or sensitized (synthetic‐sick‐lethal, SSL) to treatment with BIBR1532.…”

“…Prior to this study, C16ORF72 / TAPR1 was identified as a hit in human genome‐wide CRISPR‐Cas9 chemogenetic screens for sensitizers to ATR kinase inhibition or hydroxyurea (Hustedt et al., 2019; Benslimane et al., 2020). C16ORF72 / TAPR1 is also a target of miR‐134, a microRNA associated with tumorigenesis and chemo‐resistance (Shuang et al., 2015), and is associated with rare de novo CNVs in autism spectrum disorder (ASD) and schizophrenia (Levinson et al., 2011; Sanders et al., 2011).…”

“…The CRISPR knockout screen to identify chemical‐genetic interactions with BIBR1532 was performed as previously described with the following changes (Benslimane et al., 2020). A NALM‐6 clone with inducible Cas9 expression previously transduced with the EKO library was treated with Doxycycline (2 µg/mL) for 8 days to induce Cas9 expression and knockout generation followed by treatment (28 million cells per treatment, corresponding to 100 cells/sgRNA) with 20 µM BIBR1532 or 0.1% (v/v) DMSO for 20 days.…”

“…A NALM‐6 clone with inducible Cas9 expression previously transduced with the EKO library was treated with Doxycycline (2 µg/mL) for 8 days to induce Cas9 expression and knockout generation followed by treatment (28 million cells per treatment, corresponding to 100 cells/sgRNA) with 20 µM BIBR1532 or 0.1% (v/v) DMSO for 20 days. For the genome‐wide CRISPR screen in TAPR1 ‐deficient cells, wild‐type NALM‐6 cells or two independent clones of TAPR1‐disrupted NALM‐6 cells were transduced with the TKOv3 sgRNA library as previously described (Benslimane et al., 2020; Hart et al., 2017). Briefly, 120 million cells were transduced at a MOI of 0.5, corresponding to a coverage of 800 cells/sgRNA.…”

Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication

“…In PrimPol depleted cells, HU treatment causes a decrease in fork progression, as measured by DNA fibre analysis, which can be rescued by expressing wild-type PrimPol, but not a primase-deficient version of the enzyme ( 132 ). A recent CRISPR screen implicated PrimPol in the response to resveratrol and its chemical analogue pterostilbene ( 142 ). Like HU treatment, Resveratrol also induces comparable dNTP depletion and fork speed decrease, highlighting the drug's ability to cause replication stress.…”