Research ArticleGENETICS

Pooled CRISPR screening identifies m6A as a positive regulator of macrophage activation

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Science Advances  28 Apr 2021:
Vol. 7, no. 18, eabd4742
DOI: 10.1126/sciadv.abd4742
  • Fig. 1 CRISPR screening identifies METTL3 as a regulator of TNF-α production in macrophages.

    (A) Scheme of pooled CRISPR-Cas9 screening of RBPs playing critical roles in macrophage activation. Briefly, Cas9-expressing Raw 264.7 cells were infected with lentivirus library containing sgRNAs targeting RBP genes in the mouse genome. After selection with puromycin for 7 days, the cells were stimulated with LPS and sorted by flow cytometry on the basis of the expression levels of TNF-α. (B) Venn diagrams showing the overlap between the top 100 ranked candidate genes enriched in TNF-α–Low and TNF-α–Hi populations in two replicate screens. (C) Volcano plot showing sgRNA-targeted genes enriched in the TNF-α–Hi (blue) and TNF-α–Low (red) populations. Known positive regulators (purple), negative regulators (green), and m6A modulators (black) of TNF-α production in macrophages are highlighted. (D) Protein level of METTL3 and the overall RNA m6A methylation levels in WT and Mettl3-KO Raw 264.7 cells were measured by Western blotting and m6A dot blot assay. (E) Expression of TNF-α in METTL3-depleted and control Raw 264.7 cells after LPS stimulation measured by flow cytometry. MFI, median fluorescence intensity; NT, not treated. (F) GO enrichment analysis of down-regulated transcripts in Mettl3-KO Raw 264.7 cells compared to WT control cells. (G) Heatmap illustrating the expression of transcripts downstream of the TLR4 signaling pathway in Mettl3-deficient and WT Raw 264.7 cells. (H) Expression of TNF-α in bone marrow–derived macrophages (BMDMs) from Mettl3flox/flox;Lyzm-Cre and Mettl3flox/flox control mice upon LPS stimulation measured by flow cytometry. Data are shown from two experiments (B and C), as a representative result of three independent experiments (D), or as means ± SEM (E and H).*P < 0.05 and ****P < 0.0001 (unpaired two-tailed Student’s t test).

  • Fig. 2 Mettl3flox/flox;Lyzm-Cre mice are more susceptible to S. typhimurium infection.

    (A) Body weight of Mettl3flox/flox;Lyzm-Cre (n = 14) and their Mettl3flox/flox littermates (n = 10) measured 2 and 3 days after S. typhimurium infection. (B to F) Bacteria load of the feces (B and C), cecum (D), spleen (E), and liver (F) of infected Mettl3flox/flox;Lyzm-Cre (n = 14) and Mettl3flox/flox littermates (n = 10) measured by counting colony-forming units (CFU) in cultures of serially diluted homogenates of organs on MacConkey agar plates. Data are shown as representative results of three independent experiments (A to F) or as means ± SD of indicated determinants (B to F). *P < 0.05 and **P < 0.01 (unpaired two-tailed Student’s t test).

  • Fig. 3 Mettl3flox/flox;Lyzm-Cre mice exhibit faster tumor growth and a lower level of macrophage-derived TNF-α than WT littermates.

    MC38 cells were subcutaneously injected into the flanks of Mettl3flox/flox;Lyzm-Cre mice and their Mettl3flox/flox littermates. The tumor was excised, and tumor-infiltrating cells were characterized by flow cytometry and real-time qPCR. (A) Representative images of tumors excised from Mettl3flox/flox;Lyzm-Cre mice (n = 10) and Mettl3flox/flox littermates (n = 10) 21 days after cell injection. Photo credit: Jiyu Tong, Shanghai Jiao Tong University School of Medicine. (B) Tumor growth in Mettl3flox/flox;Lyzm-Cre mice (n = 10) and Mettl3flox/flox littermates (n = 10). (C and D) Relative expression of Mettl3 (C) and Tnf-α (D) in TAMs from Mettl3flox/flox;Lyzm-Cre mice (n = 4) and Mettl3flox/flox littermates (n = 4) measured by real-time qPCR. (E and F) Flow cytometry profile and MFI of CD86-positive (E) and CD206-positive (F) TAMs from Mettl3flox/flox;Lyzm-Cre mice (n = 4) and Mettl3flox/flox littermates (n = 4). (G and H) Fractions of intratumoral PD-1–positive CD4+ T cells (G) and PD-1–positive CD8+ T cells (H) measured by flow cytometry (n = 3). (I and J) BMDMs from Mettl3flox/flox;Lyzm-Cre mice and Mettl3flox/flox littermates were stimulated with medium conditioned by MC38 tumor cells in vitro. The expression of TNF-α was measured by flow cytometry and shown as a histogram (I) and MFI (J). Arg-1 expression in TAMs from Mettl3flox/flox;Lyzm-Cre mice and Mettl3flox/flox littermates measured by real-time qPCR after TCM or IL-4 treatment. Data are shown as representative results of three independent experiments (A, E, F, and I), as means ± SD (B, G, and H), or as means ± SEM (C to F, J, and K). *P < 0.05 and ***P < 0.001 (unpaired two-tailed Student’s t test).

  • Fig. 4 METTL3 deficiency impairs the TLR4 signaling pathway by modulating IRAKM expression.

    (A) Activation of the TLR4 signaling pathway in Mettl3-KO and Mettl3-WT BMDMs upon LPS stimulation was analyzed by Western blotting of p65, JNK, p38, ERK, and IRAKM. (B) Relative expression of Tirap, Myd88, Traf6, Irak, Irak4, Trif, and Tram in Mettl3flox/flox;Lyzm-Cre mice and Mettl3flox/flox littermates measured by real-time qPCR. (C) Relative expression of Irakm in control (NT) and LPS-treated Mettl3flox/flox;Lyzm-Cre and Mettl3flox/flox littermates measured by real-time qPCR. (D) Knockdown efficiency of shRNA targeting Irakm in BMDMs measured by real-time qPCR (n = 3). (E) TNF-α synthesis in WT BMDMs transfected with control shRNA (sh-CTL) and Mettl3-deficient BMDMs transfected with sh-CTL or IRAKM shRNA (sh-IRAKM) measured by flow cytometry (n = 3). Data are shown as representative results of three independent experiments (A) or as means ± SEM (B to E). **P < 0.01 and ***P < 0.001 (unpaired two-tailed Student’s t test).

  • Fig. 5 IRAKM expression is down-regulated by m6A modification.

    (A) Specific m6A peaks enriched in the 3′UTR of Irakm mRNAs in Raw 264.7 macrophages profiled using MeRIP-seq. CDS, coding sequences. (B) m6A peaks enriched in the 3′UTR of Irakm mRNAs in WT cells were lost in Mettl3-KO Raw 264.7 cells. (C) WT or Mettl3-deficient BMDMs were treated with the transcription inhibitor actinomycin D, and the level of Irakm transcripts was measured over time. (D) Relative luciferase activity of pGL4-luc2 with WT-3′UTR (IRAKM-WT) or with IRAKM-3′UTR containing mutated m6A sites (IRAKM-MUT) transfected into HEK293T cells was measured. The firefly luciferase activity was normalized to Renilla luciferase activity. Data are shown as representative results of three independent experiments (A) or as means ± SEM (B to D). **P < 0.01 and ****P < 0.0001; NS, not significant (unpaired two-tailed Student’s t test).

Supplementary Materials

  • Supplementary Materials

    Pooled CRISPR screening identifies m6A as a positive regulator of macrophage activation

    Jiyu Tong, Xuefei Wang, Yongbo Liu, Xingxing Ren, Anmin Wang, Zonggui Chen, Jiameng Yao, Kaiqiong Mao, Tingting Liu, Fei-Long Meng, Wen Pan, Qiang Zou, Jun Liu, Yu Zhou, Qiang Xia, Richard A. Flavell, Shu Zhu, Hua-Bing Li

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