The Function And Molecular Mechanism Of HAT1and Siglec-G In Negatively Regulating Innate Immune Responses

Part I. B type acetyltransferase HAT1negatively regulates TLR-triggered inflammatory innate response and the underlying mechanism.Histone acetylation-deacetylation regulates many biological processes, and specific acetylation marks, either singly or in combination, produce distinct outcomes. For example, the acetylation pattern on newly synthesized histones H4K5Ac, H4K8Ac and H4K12Ac is important for their assembly into nucleosomes by histone chaperones; deacetylation of H4K16and H3K56is important for spreading of heterochromatin components, whereas acetylation of these sites serves as a marker of transcript activation. HATs are classified into two groups, HAT A and HAT B, depending on the mechanism of catalysis and on cellular localization. Type A HATs are found in the nucleus, where they transfer the acetyl group from Acetyl-CoA to an ε-NH2group of histone N-tails after the assembly into nucleosomes, including GNAT family, MYST family, P300/CBP. Conversely, Type B HATs act mostly in the cytoplasm and transfer the acetyl group from Acetyl-CoA to an ε-NH2group of free histones prior to their deposition on the DNA, maybe only incuding HAT1(Histone acetyltransferases-1, HAT1). Histone deacetylase can be roughly divided into two major components, HDAC1to HDAC11, and SIRT1to SIRT7. However, acetylation-deacetylation function in inflammatory innate immunity remained to be fully investigated.By RNA interference screening technique, we found that HAT1can negatively regulate TLR4-triggered interleukin6production in primary peritoneal macrophages. It seems not concordance with our conventional ideas "acetylation almost activates transcription". So we have to make sure of the above screening results. Q-PCR and ELISA results demonstrated that there was more increased mRNA and protein expression of IL-6and TNFa in HAT1-silenced peritoneal macrophages in response to stimuli, including challenge with TLR ligands PolyI:C, LPS, CpG-ODN, or infected with RNA virus SeV and DNA virus HSV-1, while the expression of type I interferons was almost unaffected. Immunoblotting experiments suggested that classic MAPK signaling and NF-κB signal remained unchanged in those HAT1-silenced macrophages. How does HAT1selectively target proinflammatory cytokines to regulate inflammatory innate response? Using chromatin immunoprecipitation technique (Chip), we discoveried that the H3K4Me3and H3Ac level at IL-6promoter was significantly increased once HAT1was silenced. Thus, we supposed that, B-type and A-type acetyltransferase type may compete for each other, including modifying the same or adjacent sites of histone, or HAT1may selectively target certain immunosuppressive molecule promoter region, or HAT1may acetylate non-histone substrates for controlling inflammatory transcription activity. The underlying mechanism of HAT1-mediated negative regulation of TLR-trioggered inflammatory innate immunity needs further study, for shedding light on prevention, diagnosis and therapy of autoimmune diseases. Part Ⅱ. Siglec-G negatively regulates RNA virus-mediated IFN β production by promoting c-Cbl-mediated RIG-I degradationRIG-I is the most important sensor for recognizing cytoplasmic viral RNA, which composed of two N-terminal caspase recruitment domains (CARDs), a central DEAD box helicase/ATPase domain, and a C-terminal regulatory domain (CTD). RIG-I recognizes relatively short dsRNA (up to1kb), and the presence of a5’triphosphate end greatly enhances its type Ⅰ IFN-inducing activity. In the resting state, CTD of RIG-I covers over its CARDs and the whole molecular comformation is closed. When virus RNAs invade into the cytoplasm, CTD is opened and recognizes dsRNA with Helicase domain, reseasing its own CARDs and associating with CARDs of MAVS, eventually more and more MAVS in mitochondrion outermembrane oligomerize to form a prion-like structure, and convert signal to activate IFNβ transcription. To invstigate the precise regulation of RIG-I-MASV-IFN pathway, especially RIG-I itself modification, we used two methods, Genearray analysis of gene expression profile in mouse primary peritoneal macrophages upon VSV infection, and LC-MS/MS analysis of RIG-I-Flag-Immunoprecipitation from HEK293T cells, then combined in vitro and in vivo experiments to confirm that sialic acid binding immunoglobulin-like lectin molecule Siglec-G can negatively regulate IFNβ production in response to RNA virus infection.In this subject, we went further to investigate the molecular mechanism by which Siglec-G controls the IFN β production. Followed by ImmunoBlot, Siglec-G knockout peritoneal macrophages generated more RIG-I protein after VSV infection, but the adapter MAVS relatively remained unchanged. By using the protein synthesis inhibitor cycloheximide to treatment the macrophages before VSV infection, we found that the half-life of RIG-I is extended in Siglec-G-deficient macrophages, indicating that Siglec-G could promote RIG-I degradation by an unkown mechanism. Using Siglec-G-Flag-Immunoprecipitation in RAW264.7and following LC-MS/MS, along with the proved idea "Siglec-G through its ITIM motif to recruit more SHP2molecule under VSV infection" and "c-Cbl can associate with SHP2upon SDF-1stimulation", we found c-Cbl might be a E3ligase for RIG-I. Overexpression experiemnts in HEK293T cells suggested that RIG-I could be greatly degradated by c-Cbl. In SHP2-knockout macrophages,VSV-induced IFN β production could be negatively regulated by c-Cbl siRNA but not Siglec-G siRNA, which stuggested Siglec-G was the upstream, then SHP2, and latter c-Cbl. Co-immunoprecipitation and Immunofluorescence colocalization experiments confirmed that Siglec-G-SHP2-c-Cbl-RIG-I tetramer exists objectively in mouse peritoneal macrophages, RAW264.7and HEK293cells, whereas the CTD domain of RIG-I is essential for its interaction with c-Cbl. In order to identify which amino acid sites of RIG-I was targeted by c-Cbl, we constructed a series of RIG-I point mutations. The K813R mutation caused near-complete loss of K48ubiquitination and degradation of RIG-I. Transfection of RIG-I-K813R induced IFN-β transcription more markedly in HEK293T cells, RAW264.7and RIG-I-deficient macrophages. In summary, RNA viruses can up regulate Siglec-G to recruit adaptor molecule SHP2and E3ligase c-Cbl, targeting RIG-I lysine813for degradation to inhibit anti-viral innate immunity.