Tet2 Promotes Pathogen Infection-induced Myelopoiesis Through MRNA Oxidation

A variety of RNA modifications form the epitranscriptome for post-transcriptional regulation.5-methylcytosine(5-mC)is a kind of major RNA modification in kinds of RNAs,including messenger RNA(mRNA).However,the dynamic regulation and function of 5-mC in mammalian mRNAs have been largely unknown.Tet-eleven Translocation(Tet)proteins mediates active DNA demethylation through oxidizing 5-mC,and Tet proteins also can oxidize 5-mC in RNAs in vitro.However,the function of Tet proteins in regulating 5-mC turnover in RNAs and the specific-pattern and function of 5-mC in mRNAs in mammalian system,especially in lineage-specific primary cells needs to be identification.Innate immunity is the first line of host defense against pathogen infection.How to recognize and elimilate pathogens quickly and efficiently is the fundamental question in innate immunity.Emergency myelopoisis elicited by pathogen infection-initiated immune response and inflammation further amplifies innate immune response,contributing to efficient pathogen elimination.Myelopoiesis is a common response of host in acute and chronic infection,however,its epigenetic mechanism needs to be identification.Tet2 is essential for normal myelopoiesis.Tet2 acts as a critical tumor suppressor in myeloid malignancies mostly in an enzymatic activity-dependent manner and is also important in resolving inflammatory response via enzymatic activity-independent way.Whether Tet2 involves infection-induced myelopoiesis is unclear,and if so,the targets and the epigenetic mechanism of Tet2-mediated regulation of pathogen infection-induced myelopoiesis remains to be discovered.Here,we demonstrate that Tet2 promotes infection-induced myelopoiesis in an RNA oxidation-dependent manner through Adar1-mediated repression of Socs3 expression at post-transcription level.Tet2 promotes both abdominal sepsis-induce emergency myelopoiesis and parasite-induced chronic myelopoiesis through decreasing mRNA level of Socs3,a key negative regulator of JAK-STAT pathway which is critical for cytokine-induced myelopoiesis during inflammation.Tet2 represses Socs3 expression through Adar1 which binds and destabilizes Socs3 mRNA in an RNA editing-independent manner.For the underlying mechanism of Tet2 regulation at mRNA level,Tet2 represses the level of 5-mC in mRNA in an enzymatic activity-dependent manner.Tet2-deficiency leads to appearance of more methylated cytosines in Socs3 mRNA which might influence dsRNA formation at 3? UTR of Socs3 which is essential for Adar1 binding,probably through cytosine methylation-specific readers,such as RNA helicaes.Our study reveals a previously unknown regulatory role of Tet2 at post-transcription level,promoting Adar1 function,and also promoting myelopoiesis during infection at an immunological view,in mammalian system.Moreover,inhibitory function of cytosine methylation repressed by Tet2 in Adar1 binding in mRNAs reveals its new physiological role in mammalian system.Part ?.Tet2 promotes pathogen-induced myelopoiesis through decreasing mRNA level of Socs3In the process of pathogen infection,sensing of pathogen and inflammatory cytokines skews haematopoiesis towards myeloid development with the intent to clear the invading pathogen quickly and efficiently;however,the epigenetic mechanism for this pathogen infection-induced myelopoiesis is unclear.We investigated the role of Tet2,a myeloid tumour suppressor,in pathogen infection-induced myelopoiesis.First,we subjected Tet2-deficient(knockout)and the littermate control(wild-type)mice to cecal ligation and puncture,a model of abdominal polymicrobial sepsis with acute mobilization and expansion of myeloid cells.Compared with the control mice,Tet2-deficient mice were protected from sepsis with lower mortality rates and had lower clinical scores.The increased neutrophil and monocyte numbers in control mice were associated with higher serum levels of inflammatory cytokines compared with Tet2-deficient mice.In a mouse model infected with parasite Schistosoma japonicum,Tet2 deficiency inhibited differentiation and expansion of tissue mast cells derived from bone marrow progenitor cells during chronic parasite infection.These data showed that Tet2 played an important role in the process of pathogen infection-induced myeloposiesis.We subjected significant varied genes in RNA sequencing data from Tet2-deficient bone-marrow-derived mast cells(BMMCs)and control cells to pathway analysis,and expression of the genes in JAK-STAT and PI3K-AKT pathways,which are critical for inflammatory cytokine-induced myelopoiesis,were varyring.Among these genes,Socs3,a key suppressor of JAK-STAT signalling,was significantly increased in the Tet2-deficient group.Upregulation of Socs3 in both BMMCs and haematopoietic stem and progenitor cells stimulated by interleukin-3,a critical cytokine for both acute infection-induced myelopoiesis and parasite infection-induced mast cell expansion,was observed at both mRNA and protein levels in Tet2-deficient groups.We also found decreased expression of IL-3 signal-induced genes in Tet2-deficient BMMCs and the impaired phosphorylation of Akt and STAT5 in both IL-3-stimulated BMMCs and bone marrow cells from Tet2-deficient mice,further validating the defective JAK-STAT signalling in Tet2-deficient myeloid cells.Silencing of Socs3 in Tet2-deficient BMMCs increased IL-3 signalling,indicating that Tet2 promoted infection-induced myelopoiesis by repressing Socs3 expression for efficient cytokine signaling.Part.? Adar1 binds unmethylated Socs3 mRNA and inhibits Socs3 expression in a Tet2-dependent mannerTo reveal the molecular mechanism of Tet2-mediated Socs3 expression suppression,we first detected the DNA methylation levels of CpGs in two predicted CpG islands near the Socs3 promoter and found that all of these CpGs were hypomethylated in both wild-type and Tet2-deficient BMMCs;while loss of Tet2 still increased the mRNA level of Socs3 when de novo transcription was inhibited,indicating the potential role of Tet2 in regulating Socs3 at post-transcriptional level.This finding implied that Tet2 acted as an RNA binding protein and played a general role in post-transcriptional regulation.We performed three biological replicates of crosslinking and immunoprecipitation sequencing for obtaining potential Tet2-binding RNAs in BMMCs.Among thousands of peaks identified in the three replicates,more than 80% were located in genic regions,including Socs3 loci.In RNA-seq data,we found transcriptome-wide more A-to-G mutations in the wild-type group than in the Tet2-deficient group,including an A-to-G mutation in the 3? UTR of Socs3 mRNA.Silencing Adar1 increased mRNA and protein levels of Socs3 in wild-type BMMCs.Adar1 repressed Socs3 expression independent of enzymatic activity but dependent of dsRNA binding ability.Through immunoprecipitation-coupled liquid chromatography-tandem mass spectrometry(LC-MS)analysis,we found that Adar1 associated with two RNA binding proteins,which were involved in regulating mRNA processing and stability.This implies that Adar1 post-transcriptionally represses Socs3,probably through its binding pattern,which needs further investigation.To explore the regulatory role of Tet2 in Adar1-mediated inhibition of Socs3 expression,we found that silencing Adar1 in Tet2-deficient BMMCs barely increased the mRNA level of Socs3 further.Overexpression of Tet2 or its mutants indicate that Tet2 promotes Adar1-mediated repression of Socs3 mRNA in an enzymatic activity-dependent manner,but not DNA interaction-dependent manner.Patt ?.Tet2 decreases mRNA methylation level in an oxidation dependent manner for Adar1 targeting.Tet proteins catalyze the oxidation of methyl group of 5-mC to 5-hydroxymethylcytosine(5-hmC)in DNA.Recently,5-mC and 5-hmC have been found in RNA in Drosophila and human cell lines.We found that recombinant Tet2 could decrease 5-mC levels of transcribed RNAs in vitro in a substrate-dependent manner.Tet2 knockout in BMMCs increased 5-mC levels in mRNAs compared with the control cells.However,both dot blot and LC-MS analysis barely detected signals of 5-hmC,5-fC and 5-caC in mRNAs in the tested cells,indicating much lower levels of these oxidation products than 5-mC in vivo.To reveal 5-mC regulation by Tet2 transcriptome-wide,we performed bisulfite-sequencing of mRNAs from Tet2-deficient BMMCs and the control cells.Both technical and biological replicates had good overlapping rates of identified 5-mCs,especially in Tet2-deficient group.More methylcytosines were consistently identified,and with higher methylation levels in Tet2-deficient replicates than in controls.These results indicate that Tet2 could decrease overall levels of 5-mCs in mRNAs in an oxidation-dependent manner.DNA/RNA modifications can recruit specific readers to mediate epigenetic regulation.With the methylated 3? UTR of Socs3 in pull-down assay,we identified several ATP-dependent RNA helicases,which are involved in altering RNA secondary structure and unwinding of dsRNA.As dsRNA structure was essential for Adar1 binding,Adar1 indeed bound fewer methylated 3? UTRs of Socs3 than unmethylated ones.These results imply that 5-mC in mRNA inhibits Adar1 function,probably through inhibiting dsRNA formation,which is essential for Adar1 binding.Taken together,our current study first revealed that Tet2 decreased 5-mC levels in mRNAs in an oxidation-dependent manner,and revealed a new function of increased 5-mC in mRNA owing to Tet2 loss in inhibiting the function of Adar1,especially in Socs3 mRNAs.Furthermore,our study revealed that Tet2 loss impaired dsRNA structure,which is essential for Adar1 function.With the data on transcriptome-wide editing sites that appeared in the wild-type control group but not the Tet2-deficient group,our study provided a general relationship between Tet2 and Adar1 function.According to our data,amounts of 5-hmC were much lower than 5-mC in mRNA from the tested mammalian cells.Thus,additional enzymatic steps by an unknown protein may convert 5-hmC back to cytosine in mRNA.Our study linked Tet2 to pathogen infection-induced myelopoiesis in an immunological way and found that Tet2 strengthened cytokine signalling in myeloid differentiation by suppressing the repressor.This work also provided a new mechanism for epigenetic regulation in innate immunity.