Research On The Role And Mechanism Of ZC3H10 Negatively Regulating The Downstream Signaling Pathway Of Type I Interferon

Interferons are member of cytokine superfamily.They play key roles in anti-viral infection.In addition,they also participate in mediating cell proliferation,apoptosis,inflammation and adaptive immunity.Interferons can be divided into three types:I,II and Ⅲ.Type Ⅰ interferons are important parts of innate immunity.Interferon-α and β are main components of type Ⅰ interferons.They can be expressed by various cells.After virus,bacteria and other pathogens infection,host cells recognize pathogen associated molecular patterns through pattern recognition receptors.Then type I interferons are induced.The produced type Ⅰ interferons are secreted out of the cell and function through autocrine and paracrine pathways.Type Ⅰ interferons activate Janus Kinase 1(JAK1)and Tyrosine Kinase 2(TYK2)by binding to interferon receptor.Then signal transducer and activator of transcription(STAT1)and STAT2 undergo phosphorylation and form an interferon-stimulated gene factor 3(ISGF3)complex with interferon regulatory factor 9(IRF9).ISGF3 can stimulate the expression of interferon stimulated genes(ISGs)by binding the interferon stimulated response element(ISRE)in the promoter region of ISGs.The antiviral effect of interferons is realized by activating the expression of the ISGs.The production and downstream function of interferons are accurately regulated.Negative regulatory molecules can control the reaction range and intensity of interferons.On one hand,negative regulation of interferon signaling can prevent tissue pathological damage caused by overreaction.On the other hand,it can inhibit the immune response of host cells,thus leading to the decline of immune function against infection.At present,there are few researches about negative regulation of interferon downstream signal pathway.To explore the negative regulation of type I interferons downstream signal pathway,we screened a cDNA library.We found a batch of molecules that can inhibit downstream response of type Ⅰ interferons and thus promote the process of viral infection.Through a secondary screening of candidate molecules,ZC3H10 which belongs to zinc finger protein was selected as the target gene to further study its mechanism of inhibiting type I interferon downstream signal pathway.Zinc finger protein family molecules have zinc finger domain,which play an important role in transcription,ubiquitin-mediated protein degradation,DNA damage,cell migration and other physiological processes.The C2H2 zinc finger protein directly combines with DNA through its zinc finger domain to play the role of transcription factor and regulate the transcription of target genes.The RING zinc finger protein contain many E3 ubiquitin ligases,which mediate ubiquitination of target proteins and regulate proteins degradation or activation.CCCH zinc finger protein is generally believed to have RNA binding ability and regulate RNA metabolism.There is little research about ZC3H10,and the only research proves that it can regulate splicing of microRNA and mediate tumor migration through RNA binding ability.In addition,scholars have recently proved that ZC3H10 can promote transcription of UCP1 by binding to its promoter region,thus regulating the thermogenic function of brown fat.However,the function of ZC3H10 in immune response has not been reported.Through overexpression of ZC3H10,we found that ISGs expression is inhibited when stimulated by interferon-β.Surprisingly,this inhibition still existed in the resting state without stimulation of type Ⅰ interferons.Under the stimulation of type Ⅰ interferons,the exogenous overexpression of ZC3H10 inhibited the expression of ISGs,which also lead to the reduction of the ability to resist against virus infection such as Newcastle disease virus,Sendai virus,enterovirus 71,Rhinovirus 16.ZC3H10 antagonized the inhibition of type Ⅰ interferon on virus replication.Then we constructed ZC3H10 knockout cell line by CRISPR/cas9 technology.The expression of ISGs was upregulated in ZC3H10 knockout cells,which was consistent with the result ofZC3H10 over expression.Transcriptome data of ZC3H10 knockout cells also proved the above conclusion.Then we restored the expression of ZC3H10 by transfecting ZC3H10 in knockout cells,and the expression of ISGs was also restored accordingly.To explore the inhibiting function,we constructed ZC3H10 mutations by deleting each domain of ZC3H10 protein.Through exogenous overexpression,we found that the zinc finger domain and nuclear sequence were crucial to its function.When the nuclear sequence of ZC3H10 was deleted,ZC3H10 molecule lost its nuclear localization ability,at the same time,its ability to inhibit the expression of ISGs was also lost.This proving that its function needs to be realized in the nucleus.We found that ZC3H10 did not inhibit phosphorylation of STAT1 and STAT2.Through immune coprecipitation we obtained proteins bound to ZC3H10 and analyzed by mass spectrometry.We found that it did not bind to STAT1，STAT2 and IRF9.GO analysis showed that proteins bound to ZC3H10 were mostly related to mRNA metabolism，methylation,chromatin remodeling,and regulation of chromatin tissue.Therefore,we presumed that ZC3H10 can inhibit ISGs expression by binding DNA or RNA.Through chromatin coprecipitation experiment.ZC3H10 binding DNA sequence was obtained,and motif analysis showed that ZC3H10 can bind to the characteristic sequence of 5’-TTTC-3’,which was part of ISRE sequence.In addition,when type Ⅰ interferon stimulated,the nuclear localization and expression level of ZC3H10 were not affected,but result in slight change in the binding characteristic sequence and tendentiousness towards 5’-ATTT-3’.To sum up,through high-throughput screening of cDNA library,we found that ZC3H10 could negatively regulate the downstream signal pathway of type Ⅰ interferons.Because of antagonism to type Ⅰ interferons,ZC3H10 promoted the infection process of virus.Further mechanism research indicated that ZC3H10 did not play roles by regulating the activation and nucleation of STAT1 and STAT2.This study not only further revealed the regulation mechanism of type Ⅰ interferon downstream signaling pathway,but also provided more theoretical basis for anti-infection immunity and new targets for the development of antiviral drugs.