Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study puts forth the model that under IFN-B stimulation, liquid-phase WTAP coordinates with the transcription factor STAT1 to recruit MTC to the promoter region of interferon-stimulated genes (ISGs), mediating the installation of m6A on newly synthesized ISG mRNAs. This model is supported by strong evidence that the phosphorylation state of WTAP, regulated by PPP4, is regulated by IFN-B stimulation, and that this results in interactions between WTAP, the m6A methyltransferase complex, and STAT1, a transcription factor that mediates activation of ISGs. This was demonstrated via a combination of microscopy, immunoprecipitations, m6A sequencing, and ChIP. These experiments converge on a set of experiments that nicely demonstrate that IFN-B stimulation increases the interaction between WTAP, METTL3, and STAT1, that this interaction is lost with the knockdown of WTAP (even in the presence of IFN-B), and that this IFN-B stimulation also induces METTL3-ISG interactions.
Strengths:
The evidence for the IFN-B stimulated interaction between METTL3 and STAT1, mediated by WTAP, is quite strong. Removal of WTAP in this system seems to be sufficient to reduce these interactions and the concomitant m6A methylation of ISGs. The conclusion that the phosphorylation state of WTAP is important in this process is also quite well supported.
Weaknesses:
The evidence that the above mechanism is fundamentally driven by different phase-separated pools of WTAP (regulated by its phosphorylation state) is weaker. These experiments rely relatively heavily on the treatment of cells with 1,6-hexanediol, which has been shown to have some off-target effects on phosphatases and kinases (PMID 33814344).
Given that the model invoked in this study depends on the phosphorylation (or lack thereof) of WTAP, this is a particularly relevant concern.
Related to this point, it is also interesting (and potentially concerning for the proposed model) that the initial region of WTAP that was predicted to be disordered is in fact not the region that the authors demonstrate is important for the different phase-separated states. Taking all the data together, it is also not clear to me that one has to invoke phase separation in the proposed mechanism.
We are grateful for the Reviewer’s positive comment and constructive feedback. In this article, we claim a novel and important mechanism that de-phosphorylation-driven solid to liquid phase transition of WTAP mediates its co-transcriptional m6A modification. We first observed that WTAP underwent phase transition during virus infection and IFN-β stimulation, and confirmed the phase transition driven force of WTAP through multiple experiments. Besides 1,6‐hexanediol (1,6-hex) treatment, we also introduced S/T to D/A mutations to mimic the phosphorylation and de-phosphorylation WTAP in vitro and in cells, identified 5ST-D mutant as SLPS mutant, and 5ST-A mutant as LLPS mutant. We then performed 1,6-hex experiment to confirm the importance of phase separation for WTAP function, and revealed that 5ST-D SLPS mutant and 5ST-A LLPS mutant had different influence on WTAP-promoter region interaction and co-transcriptional m6A modification. Following the reviewer’s suggestion, we need to further clarify the phosphorylation of WTAP phase separation. We plan to repeat the experiments by introducing potent PP4 inhibitor, fostriecin, and performed further experiments to explore the effect of WTAP IDR domain, which is reported to play a critical role for its phase separation.
1,6-hex was initially considered as the inhibitor of hydrophobic interaction which involved in various kinds of protein-protein interaction, indicating that off-target effects of 1,6-hex was inevitable. It is reported that 1,6-hex impaired RNA pol II CTD specific phosphatase and kinase activity at 5% concentration3. However, 1,6-hex is still widely used in the LLPS-associated functional studies despite its off-target effect. Related to this article, 10% 1,6-hex was reported to dissolve WTAP phase separation droplets2. Beside WTAP, 1,6-hex (5%-10% w/v) was also used to explore the phase separation characteristic and function on phosphorylated protein or even kinase, including p‐tau441, TAZ, HSF1 and so on4-6. 10% 1,6-hex inhibited the crucial role of phosphorylation-driven HSF1 LLPS in chromatin binding and transcriptional process presented by RNA-seq dataset6, indicating the function on kinase or phosphatase of 1,6-hex might not a global effect. To avoid the 1,6-hex-mediated kinase/phosphatase impairment in this project, we introduced the WTAP SLPS mutation and LLPS mutation besides 1,6-hex treatment to explore the m6A modification function of WTAP phase transition. We plan to repeat the experiments by lower the 1,6-hex concentration, check the WTAP phosphorylation status after 1,6-hex treatment, and discuss them in the discussion part.
A considerable number of proteins undergo phase separation via interactions between intrinsically disordered regions (IDRs). IDR contains more charged and polar amino acids to present multiple weakly interacting elements, while lacking hydrophobic amino acids to show flexible conformations7. In our article, we used PLAAC websites (http://plaac.wi.mit.edu/) to predict IDR domain of WTAP, and a fragment (234-249 amino acids) was predicted as prion-like domain. However, deletion of this fragment failed to abolish the phase separation properties of WTAP, which might be the main confusion to reviewers. To explain this issue, we checked the WTAP structure (within part of MTC complex) from protein data bank (https://www.rcsb.org/structure/7VF2) and found that prediction of IDR has been renewed due to the update of different algorithm. IDR of WTAP has expanded to 245-396 amino acids, containing the whole CTD region. According to our results, lack of CTD inhibited WTAP liquid-liquid phase separation both in vitro and in cells, while the phosphorylation status on CTD had dramatic impact on WTAP phase transition, which was consistent with the LLPS-regulating function of IDR. Therefore, we will revise our description on WTAP IDR, and performed further experiment to test its function.
Taken together, given the highly association between WTAP phosphorylation with phase separation status and its function during IFN-β stimulation, it is necessary to involve WTAP phase separation in our mechanism. We will perform further experiments to propose more convincing evidence and perfect our project.
Reviewer #2 (Public review):
In this study, Cai and colleagues investigate how one component of the m6A methyltransferase complex, the WTAP protein, responds to IFNb stimulation. They find that viral infection or IFNb stimulation induces the transition of WTAP from aggregates to liquid droplets through dephosphorylation by PPP4. This process affects the m6A modification levels of ISG mRNAs and modulates their stability. In addition, the WTAP droplets interact with the transcription factor STAT1 to recruit the methyltransferase complex to ISG promoters and enhance m6A modification during transcription. The investigation dives into a previously unexplored area of how viral infection or IFNb stimulation affects m6A modification on ISGs. The observation that WTAP undergoes a phase transition is significant in our understanding of the mechanisms underlying m6A's function in immunity. However, there are still key gaps that should be addressed to fully accept the model presented.
Major points:
(1) More detailed analyses on the effects of WTAP sgRNA on the m6A modification of ISGs:
a. A comprehensive summary of the ISGs, including the percentage of ISGs that are m6A-modified. merip-isg percentage
b. The distribution of m6A modification across the ISGs. topology
c. A comparison of the m6A modification distribution in ISGs with non-ISGs. topology
In addition, since the authors propose a novel mechanism where the interaction between phosphorylated STAT1 and WTAP directs the MTC to the promoter regions of ISGs to facilitate co-transcriptional m6A modification, it is critical to analyze whether the m6A modification distribution holds true in the data.
We appreciate the reviewer‘s summary of our manuscript and the constructive assessment. We plan to perform the related analysis accordingly to present the m6A modification in ISGs in our model.
(2) Since a key part of the model includes the cytosol-localized STAT1 protein undergoing phosphorylation to translocate to the nucleus to mediate gene expression, the authors should focus on the interaction between phosphorylated STAT1 and WTAP in Figure 4, rather than the unphosphorylated STAT1. Only phosphorylated STAT1 localizes to the nucleus, so the presence of pSTAT1 in the immunoprecipitate is critical for establishing a functional link between STAT1 activation and its interaction with WTAP.
We plan to repeat the immunoprecipitation experiments to clarify the function of pSTAT1 in WTAP interaction and m6A modification as the reviewer suggested.
(3) The authors should include pSTAT1 ChIP-seq and WTAP ChIP-seq on IFNb-treated samples in Figure 5 to allow for a comprehensive and unbiased genomic analysis for comparing the overlaps of peaks from both ChIP-seq datasets. These results should further support their hypothesis that WTAP interacts with pSTAT1 to enhance m6A modifications on ISGs.
We first performed the MeRIP-seq and RNA-seq and explored the critical role of WTAP in ISGs m6A modification and expression. By immunoprecipitation and immunofluorescence experiments, we found phase transition of WTAP enhanced its interaction to pSTAT1. These results indicate that WTAP mediated ISGs m6A modification and expression by enhanced its interaction with pSTAT1 during virus infection and IFN-β stimulation. However, we were still not sure how WTAP-mediated m6A modification related to pSTAT1-mediated transcription. By analyzing METTL3 ChIP-seq data or caPAR-CLIP-seq data, several researches have revealed the recruitment of m6A methylation complex (MTC) to transcription start sites (TSS) of coding genes and R-loop structure by interacting with transcriptional factors STAT5B or DNA helicase DDX21, indicating the engagement of MTC mediated m6A modification on nascent transcripts at the very beginning of transcription 8-10. Thus, we proposed that phase transition of WTAP could be recruited to the ISGs promoter region by pSTAT1, and verified this hypothesis by pSTAT1/WTAP-ChIP-qPCR. We believe ChIP-seq experiment is a good idea to explore the mechanism in depth, but the results in this article for now are enough to explain our mechanism. We will continuously focus on the whole genome chromatin distribution of WTAP and explore more functional effect of transcriptional factor-dependent WTAP-promoter region interaction in t.
Minor points:
(1) Since IFNb is primarily known for modulating biological processes through gene transcription, it would be informative if the authors discussed the mechanism of how IFNb would induce the interaction between WTAP and PPP4.
(2) The authors should include mCherry alone controls in Figure 1D to demonstrate that mCherry does not contribute to the phase separation of WTAP. Does mCherry have or lack a PLD?
(3) The authors should clarify the immunoprecipitation assays in the methods. For example, the labeling in Figure 2A suggests that antibodies against WTAP and pan-p were used for two immunoprecipitations. Is that accurate?
(4) The authors should include overall m6A modification levels quantified of GFPsgRNA and WTAPsgRNA cells, either by mass spectrometry (preferably) or dot blot.
We thank reviewer for raising these useful suggestions. We will perform related experiments and revised the manuscript carefully the as reviewer suggested.
Reviewer #3 (Public review):
Summary:
This study presents a valuable finding on the mechanism used by WTAP to modulate the IFN-β stimulation. It describes the phase transition of WTAP driven by IFN-β-induced dephosphorylation. The evidence supporting the claims of the authors is solid, although major analysis and controls would strengthen the impact of the findings. Additionally, more attention to the figure design and to the text would help the reader to understand the major findings.
Strength:
The key finding is the revelation that WTAP undergoes phase separation during virus infection or IFN-β treatment. The authors conducted a series of precise experiments to uncover the mechanism behind WTAP phase separation and identified the regulatory role of 5 phosphorylation sites. They also succeeded in pinpointing the phosphatase involved.
Weaknesses:
However, as the authors acknowledge, it is already widely known in the field that IFN and viral infection regulate m6A mRNAs and ISGs. Therefore, a more detailed discussion could help the reader interpret the obtained findings in light of previous research.
It is well-known that protein concentration drives phase separation events. Similarly, previous studies and some of the figures presented by the authors show an increase in WTAP expression upon IFN treatment. The authors do not discuss the contribution of WTAP expression levels to the phase separation event observed upon IFN treatment. Similarly, METTL3 and METTL14, as well as other proteins of the MTC are upregulated upon IFN treatment. How does the MTC protein concentration contribute to the observed phase separation event?
How is PP4 related to the IFN signaling cascade?
In general, it is very confusing to talk about WTAP KO as WTAPgRNA.
We are grateful for the positive comments and the unbiased advice by reviewer. To interpret the findings in previous research, we will revise the manuscript carefully and preform more detailed discussion on ISGs m6A modification during virus infection or IFN stimulation. As previous reported, WTAP protein level will be induced by long time IFN-β stimulation or LPS stimulation, while LPS-induced WTAP expression promoted its phase separation ability2,11. Although there was no significant upregulation of WTAP expression level in our short time treatment, we hypothesized that WTAP phase separation will be promoted due to higher protein concentration after long time IFN stimulation, enhancing m6A modification deposition on ISGs mRNA, revealing a feedback loop between WTAP phase separation and m6A modification during specific stimulation. To discuss the effect of MTC protein concentration in our proposed event, we will perform immunoblotting experiments of MTC proteins and check the phase separation effect in different WTAP concentration.
Protein phosphatase 4 (PP4) is a multi-subunit Ser/Thr phosphatase complex that participate in diverse cellular pathways including DDR, cell cycle progression, and apoptosis12. Protein phosphatase 4 catalytic subunit 4C (PPP4C) is one of the components of PP4 complex. Previous research showed that knockout of PPP4C enhanced IFN-β downstream signaling and gene expression, which was consistent with our findings that knockdown of PPP4C impaired WTAP-mediated m6A modification, enhanced the ISGs expression. Since there was no significant enhancement in PPP4C expression level during IFN-β stimulation in our results, we will consider to explore the post-translation modification that may influence the protein-protein interaction, such as ubiquitination.
In this project, all the WTAP-deficient THP-1 cells were bulk cells treated with WTAPsgRNA, but not monoclonal knockout cells. We confirmed that WTAP expression was efficiently knockdown in WTAPsgRNA THP-1 cells, and the m6A modification level has been impaired, avoiding the compensatory effect on m6A modification by other possible proteins. Thus, we prefer to call it WTAPsgRNA THP-1 cells rather than WTAP KO THP-1 cells.
References
(1) Raja, R., Wu, C., Bassoy, E.Y., Rubino, T.E., Jr., Utagawa, E.C., Magtibay, P.M., Butler, K.A., and Curtis, M. (2022). PP4 inhibition sensitizes ovarian cancer to NK cell-mediated cytotoxicity via STAT1 activation and inflammatory signaling. J Immunother Cancer 10. 10.1136/jitc-2022-005026.
(2) Ge, Y., Chen, R., Ling, T., Liu, B., Huang, J., Cheng, Y., Lin, Y., Chen, H., Xie, X., Xia, G., et al. (2024). Elevated WTAP promotes hyperinflammation by increasing m6A modification in inflammatory disease models. J Clin Invest 134. 10.1172/JCI177932.
(3) Duster, R., Kaltheuner, I.H., Schmitz, M., and Geyer, M. (2021). 1,6-Hexanediol, commonly used to dissolve liquid-liquid phase separated condensates, directly impairs kinase and phosphatase activities. J Biol Chem 296, 100260. 10.1016/j.jbc.2021.100260.
(4) Wegmann, S., Eftekharzadeh, B., Tepper, K., Zoltowska, K.M., Bennett, R.E., Dujardin, S., Laskowski, P.R., MacKenzie, D., Kamath, T., Commins, C., et al. (2018). Tau protein liquid-liquid phase separation can initiate tau aggregation. The EMBO journal 37. 10.15252/embj.201798049.
(5) Lu, Y., Wu, T., Gutman, O., Lu, H., Zhou, Q., Henis, Y.I., and Luo, K. (2020). Phase separation of TAZ compartmentalizes the transcription machinery to promote gene expression. Nat Cell Biol 22, 453-464. 10.1038/s41556-020-0485-0.
(6) Zhang, H., Shao, S., Zeng, Y., Wang, X., Qin, Y., Ren, Q., Xiang, S., Wang, Y., Xiao, J., and Sun, Y. (2022). Reversible phase separation of HSF1 is required for an acute transcriptional response during heat shock. Nat Cell Biol 24, 340-352. 10.1038/s41556-022-00846-7.
(7) Hou, S., Hu, J., Yu, Z., Li, D., Liu, C., and Zhang, Y. (2024). Machine learning predictor PSPire screens for phase-separating proteins lacking intrinsically disordered regions. Nat Commun 15, 2147. 10.1038/s41467-024-46445-y.
(8) Hao, J.D., Liu, Q.L., Liu, M.X., Yang, X., Wang, L.M., Su, S.Y., Xiao, W., Zhang, M.Q., Zhang, Y.C., Zhang, L., et al. (2024). DDX21 mediates co-transcriptional RNA m(6)A modification to promote transcription termination and genome stability. Mol Cell 84, 1711-1726 e1711. 10.1016/j.molcel.2024.03.006.
(9) Barbieri, I., Tzelepis, K., Pandolfini, L., Shi, J., Millan-Zambrano, G., Robson, S.C., Aspris, D., Migliori, V., Bannister, A.J., Han, N., et al. (2017). Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control. Nature 552, 126-131. 10.1038/nature24678.
(10) Bhattarai, P.Y., Kim, G., Lim, S.C., and Choi, H.S. (2024). METTL3-STAT5B interaction facilitates the co-transcriptional m(6)A modification of mRNA to promote breast tumorigenesis. Cancer Lett 603, 217215. 10.1016/j.canlet.2024.217215.
(11) Ge, Y., Ling, T., Wang, Y., Jia, X., Xie, X., Chen, R., Chen, S., Yuan, S., and Xu, A. (2021). Degradation of WTAP blocks antiviral responses by reducing the m(6) A levels of IRF3 and IFNAR1 mRNA. EMBO Rep 22, e52101. 10.15252/embr.202052101.
(12) Dong, M.Z., Ouyang, Y.C., Gao, S.C., Ma, X.S., Hou, Y., Schatten, H., Wang, Z.B., and Sun, Q.Y. (2022). PPP4C facilitates homologous recombination DNA repair by dephosphorylating PLK1 during early embryo development. Development 149. 10.1242/dev.200351.
