Elevated WTAP promotes hyperinflammation by increasing m6A modification in inflammatory disease models
Yong Ge, Rong Chen, Tao Ling, Biaodi Liu, Jingrong Huang, Youxiang Cheng, Yi Lin, Hongxuan Chen, Xiongmei Xie, Guomeng Xia, Guanzheng Luo, Shaochun Yuan, Anlong Xu
Yong Ge, Rong Chen, Tao Ling, Biaodi Liu, Jingrong Huang, Youxiang Cheng, Yi Lin, Hongxuan Chen, Xiongmei Xie, Guomeng Xia, Guanzheng Luo, Shaochun Yuan, Anlong Xu
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Research Article Immunology Inflammation

Elevated WTAP promotes hyperinflammation by increasing m6A modification in inflammatory disease models

Abstract

Emerging evidence has linked the dysregulation of N6-methyladenosine (m6A) modification to inflammation and inflammatory diseases, but the underlying mechanism still needs investigation. Here, we found that high levels of m6A modification in a variety of hyperinflammatory states are p65-dependent because Wilms tumor 1–associated protein (WTAP), a key component of the “writer” complex, is transcriptionally regulated by p65, and its overexpression can lead to increased levels of m6A modification. Mechanistically, upregulated WTAP is more prone to phase separation to facilitate the aggregation of the writer complex to nuclear speckles and the deposition of m6A marks on transcriptionally active inflammatory transcripts, thereby accelerating the proinflammatory response. Further, a myeloid deficiency in WTAP attenuates the severity of LPS-induced sepsis and DSS-induced IBD. Thus, the proinflammatory effect of WTAP is a general risk-increasing mechanism, and interrupting the assembly of the m6A writer complex to reduce the global m6A levels by targeting the phase separation of WTAP may be a potential and promising therapeutic strategy for alleviating hyperinflammation.

Authors

Yong Ge, Rong Chen, Tao Ling, Biaodi Liu, Jingrong Huang, Youxiang Cheng, Yi Lin, Hongxuan Chen, Xiongmei Xie, Guomeng Xia, Guanzheng Luo, Shaochun Yuan, Anlong Xu

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Figure 5

WTAP promotes the protein expression of proinflammatory genes through m6A modification.

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WTAP promotes the protein expression of proinflammatory genes through m6...
(A and B) Immunoblots showing the expression of critical adapters in the IL-6/ STAT3 signaling pathway in WT and WTAPΔ1–77 THP-1 cells. (C) Flow cytometry showing IL6ST-FITC fluorescence in cell surface of peritoneal macrophages and BMDMs. (D) Immunoblots showing the expression of WTAP and IL6ST in the lung or colon tissues from Wtapfl/fl and LyzM-cre+ WtapΔ1–77 mice that were intraperitoneally injected with LPS for 12 hours. (E) Immunoblots showing the levels of phosphorylated STAT3, p65, and p38 in WT and IL6ST–/– THP-1 cells stimulated with LPS at the indicated time points. (F) ELISAs detecting IL-6 secretion in the supernatants of WT and IL6ST–/– THP-1 cells stimulated with indicated inflammatory stimuli. (G) Schematic representation showing the position of m6A motifs within the IL6ST transcripts. F1/R1 represents detection site 1 and F2/R2 represents detection site 2. (H) MeRIP-qPCR showing the abundance of IL6ST transcripts in WT and WTAPΔ1–77 THP-1 cells stimulated with LPS for 6 hours. (I) WT or mutant (Mut) m6A consensus sequences (A–T mutation) on the IL6ST-3′ UTR were fused with the Renilla luciferase reporter in the psiCHECK-2 vector. (J and K) Relative luciferase activities in 293T cells after transfection with indicated reporter vectors. (L) qRT–PCR showing the proportion of IL6ST mRNA in polysome fractions from WT and WTAPΔ1–77 THP-1 cells stimulated with LPS for 6 hours. (M) Schematic representation of the biotin-labeled probes of IL6ST transcripts. (N) RNA pull-down analyses showing the interaction between different IL6ST RNA probes and the YTHDF1/3 protein. (O) RIP-qPCR showing the interaction between IL6ST mRNA and YTHDF1/3 in WT and WTAPΔ1–77 THP-1 cells. (P) Immunoblot analyses showing the abundance of IL18Rα and IL15Rα in WT and WTAPΔ1–77 THP-1 cells stimulated with LPS at the indicated time points. (Q) Relative luciferase activities in 293T cells after transfection with indicated reporter vectors. Data are representative of 3 independent biological experiments in A–E, N, and P. Data are presented as the mean ± SD in F, H, J–L, O, and Q, with individual measurements overlaid as dots. Statistical analysis was performed using 1-way ANOVA multiple comparisons in F, H, J, O, and Q, or 2-tailed Student’s t test in K.