指導教授：李建國臺灣大學：免疫學研究所謝郁萱Hsieh, Yu-HsuanYu-HsuanHsieh2014-11-262018-07-092014-11-262018-07-092014http://ntur.lib.ntu.edu.tw//handle/246246/262030第一型干擾素(type I interferon)可以活化訊號傳導與轉錄子 STAT1, STAT2 和 STAT3。 在第一型干擾素引起的抗病毒反應中，STAT1 和 STAT2 扮演著正調控的角色，而STAT3則是會負調控抗病毒反應。有趣的是，STAT3的N端區域就足以抑制第一型干擾素所引起的下游抗病毒基因之表現，但是詳細的機制還不清楚。目前已經知道，在第一型干擾素刺激之下，STAT3 N端區域不會影響STAT1的磷酸化和入核作用，然而，STAT3 N端區域可以與STAT1交互作用，而且STAT3 N端區域可以被乙醯化。首先，我們研究STAT3 N端區域乙醯化所造成的影響。將STAT3位於49及87的離胺酸 (Lysine) 同時突變為精胺酸 (Arginine) 後會阻礙STAT3在第一型干擾素下游基因表現的抑制效果，顯示STAT3N端區域的乙醯化可能涉及干擾素調控機制。因此，我們使用藥物抑制劑以及核糖核酸靜默技術去抑制或減少組織蛋白去乙醯酶 (Histone deacetylases)，進一步研究乙醯化在STAT3負調控能力所扮演的角色。在野生型及STAT3基因剔除的小鼠胚胎纖維母細胞株 (mouse embryonic fibroblast) 中，曲古菌素A (Trichostatin A) 對第一型干擾素下游基因表現的抑制程度不同，而使用煙鹼 (Nicotinamide) 則沒有這個效果，表示第一型和第二型組織蛋白去乙醯酶可能參與第一型干擾素反應的調控，但第三型組織蛋白去乙醯酶不參與其中。在Stat3f/f 及 Ad-Cre Stat3-/- MEFs中，雙重敲低HDAC1和HDAC2 會抑制第一型干擾素下游基因表現，但是在Ad-Cre Stat3-/- MEFs的效果較為明顯。我們推論STAT3可能會抑制HDAC和STAT1的交互作用，所以當缺少STAT3時就會增強HDAC對STAT1的影響。的確，在STAT3基因敲低的人類胚胎腎臟細胞株293T中，會增加STAT1和HDAC1的交互作用。總結來說，這些結果暗示STAT3可能會藉由和HDAC包含HDAC1及HDAC2的乙醯化機制來負調控第一型干擾素的反應。Type I interferons (IFN-I) activate STAT1, STAT2 and STAT3. While STAT1 and STAT2 play positive roles in IFN-I-mediated antiviral responses, STAT3 negatively regulates the responses. Interestingly, N-terminal domain (NTD) of STAT3 is sufficient to suppress IFN-I-induced downstream gene expression. Nevertheless, the detailed mechanisms remain unclear. STAT3 NTD did not affect IFN-I-induced phosphorylation and nuclear translocation of STAT1. Instead, STAT3 affects the binding of ISGF3 to the promoters of ISGs. However, STAT3 NTD interacts with STAT1 and STAT3 can be acetylated at its N-terminal domain. First of all, we investigated the effect of STAT3 acetylation in the NTD. STAT3K49R/K87R mutant blocked the suppressive effect of STAT3 on IFN-I-induced gene expression, suggesting that acetylation at STAT3 NTD may be involved in the negative regulation of IFN response. We further investigated the role of acetylation in detailed mechanism using pharmacological inhibitors and RNA silencing for histone deacetylase (HDACs). The susceptibility to Trichostatin A (TSA)-mediated suppression of IFN-dependent ISG induction was different in WT and STAT3KO MEFs. However, the treatment of nicotinamide did not have any effect, suggesting that STAT3 may be involved in the regulation of IFN-I response by class I and II but not class III HDACs. Double knockdown of HDAC1 and HDAC2 in either Stat3f/f or Ad-Cre Stat3-/- MEFs suppressed ISG expression in response to IFN-I. However, the suppressive effect was much prominent in STAT3-deficient MEFs than in control MEFs. We hypothesized that STAT3 may inhibit the interaction between HDAC and STAT1, therefore, increasing the impact of HDAC on STAT1 in the absence of STAT3. Indeed, increased interaction between STAT1 and HDAC1 was observed in 293T cells that had been treated with shSTAT3. In sum, these results suggest that STAT3 may negatively regulate IFN-I response through acetylation-dependent mechanism involves HDACs, including HDAC1 and maybe HDAC2.Contents 中文摘要 i Abstract ii Contents iv List of figures viii Chapter I introdution 1 1.1 IFN-I 2 1.2 IFN-I signaling pathway 2 1.3 Antiviral effect of type I IFN 3 1.4 The structure of STAT proteins 3 1.5 The STATs in IFN-I signaling 4 1.5.1 STAT1 4 1.5.2 STAT3 5 1.6 Regulation of STAT signaling by acetylation 6 1.6.1 Acetylation of STAT1 6 1.6.2 Acetylation of STAT3 8 1.7 HDAC and HDAC inhibitors 9 1.8 Rationale and aims 10 Chapter II Materials and methods 12 2.1 Cell lines 13 2.2 Plasmids, antibodies, reagents 13 2.3 Calcium phosphate precipitation transfection 15 2.4 Quantitative RT-PCR 16 2.5 Western blotting 17 2.6 Immunoprecipitation 17 2.7 Cytosolic and nuclear extracts 18 2.8 RNA interference 18 2.9 Buffers 19 Chapter III Results 20 3.1 Acetylation at K49 and K87 of STAT3 play an important role for inhibition of IFN-I induced gene induction 21 3.2 STAT3N does not affect the nuclear translocation of STAT1 in response to IFN-I 22 3.3 STAT1 interacts with STAT3N in response to IFN-I 22 3.4 STAT1 is acetylated in response to IFN-I 23 3.5 STAT3 may regulate IFN-I-mediated STAT1-dependent ISG expression by class I or class II HDAC activity 24 3.6 HDAC1 and HDAC2 may be involved in suppressive effect of STAT3 on ISG expression 24 3.7 STAT3 reduces the interaction between STAT1 and HDAC1 in response to IFN-I 26 Chapter IV Discussion 27 4.1 The acetylation of K49 and K87 at STAT3N is critical for STAT3 negative effect 28 4.2 STAT3 may regulate IFN-I-mediated STAT1-dependent ISG expression through HDAC1 and HDAC2 29 4.3 The acetylation of STAT1 increased by STAT3 in response to IFN-I needs to be confirmed 30 4.4 The involvement of CBP/p300 in suppressive effect of STAT3 on ISG expression remains to be determined 31 4.5 Opposing role of STAT1 and STAT3 31 4.6 Concluding remarks 32 Figures 33 References 49  1314970 bytesapplication/pdf論文使用權限：同意有償授權(權利金給回饋學校)第一型干擾素訊息傳導與轉錄子三抗病毒反應乙醯化組織蛋白去乙醯酶thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262030/1/ntu-103-R01449001-1.pdf