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標題: | STAT1的轉譯後修飾調節B細胞的活化及分化 Regulation of B-cell Activation and Differentiation by Post-translational Modification of STAT1 |
作者: | 李婕愉 Chieh-Yu Lee |
指導教授: | 李建國 Chien-Kuo Lee |
關鍵字: | B細胞,STAT1,磷酸化,胸腺非依賴性,胸腺依賴性,表觀遺傳調控, B cells,STAT1,phosphorylation,T-independent,T-dependent,epigenetic regulation, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | B細胞是參與適應性免疫反應的一個重要成員,它的活化及分化受到轉錄因子網絡的緊密調控。雖然STAT3和STAT5的角色在調控B細胞過程中的作用已經被研究許多,STAT1的功能仍然不清楚。在我們的研究中,我們對於STAT1的後轉譯修飾(Post-translational modification, PTM),包括磷酸化和組蛋白甲基化以及乙醯化進行探討。胸腺非依賴性(T-independent, TI)以及胸腺依賴性(T-dependent, TD)的刺激都能誘導STAT1絲胺酸 (STAT1-pS727) 和酪胺酸 (STAT1-pY701) 的磷酸化。功能上,STAT1-pS727能夠正向調節B細胞的活化,生發中心 (Germinal center, GC) B細胞的形成,漿细胞的分化和免疫球蛋白的產生。而在B細胞中過量表達STAT1能夠增加TI或TD誘導的漿细胞形成以及抗體的產生,且這些作用在Y701F或S727A突變時會被部分阻斷,表示這兩個STAT1磷酸化位點在控制B細胞分化中都有正向調控的作用。另一方面,TI和TD刺激還能使表觀遺傳學H3K4和H3K27有顯著變化。此外,STAT1-pS727在濾泡型B細胞(follicular B cell, FO B)和邊緣區B細胞(Marginal zone B cell, MZ B)中能夠在脂多醣(lipopolysaccharide, LPS)刺激下調控H3K4me1和H3K27me3的表現。通過在B細胞中進行CUT&Tag (Cleavage under targets and tagmentation)實驗,我們發現經由 LPS刺激後,STAT1-pS727能夠結合上一些組蛋白甲基轉移酶(lysine methyltransferase, KMTs)和去甲基化酶(lysine demethylase, KDMs)的啟動子 (promoter) 的位置,包括Kmt5b,Kmt2d,Kdm5a和Kdm4c。幾個已知會調節B細胞分化的轉錄因子也發現會被STAT-pS727所結合,包括Prdm1,Irf4,Irf8,Xbp1和Bcl6。此外,STAT1-pS727的突變能夠降低LPS刺激時這些基因的H3K4甲基化。而FO B在受到LPS刺激後的H3K27me3的表現也顯著減少。為了研究H3K27甲基化的作用,我們給予B細胞Valemetostat,一種EZH1/2雙重抑制劑,已知可以產生H3K27me3和STAT3甲基化。有趣的是,這種抑制劑在LPS刺激下降低了B細胞中STAT1-pS727的表達。我們的研究結果顯示STAT1-pS727能夠透過表觀遺傳學,包括可能調節表觀遺傳修飾因子(epigenetic modifier)的表達或功能來調節B細胞對於刺激的反應,同時也提出甲基化和 STAT1-pS727之間在調節B細胞反應中具有潛在的關聯性。 B cells are an important arm of humoral immunity, in which a network of transcription factors tightly regulates their activation and differentiation. While the role of STAT3 and STAT5 in this process is well-characterized, the functions of STAT1 remain largely unclear. Here, we investigated the effect of post-translational modifications (PTMs) of STAT1, including phosphorylation and histone methylation/acetylation. Not only T-independent (TI) but also T-dependent (TD) stimulation induced both STAT1 serine and tyrosine phosphorylation. STAT1 serine phosphorylation positively regulated B cell activation, GC B cell formation, plasma cell (PC) differentiation and immunoglobulin (Ig) production. Overexpression of WT STAT1 in primary B cells increased TI- or TD-induced PC formation and Ig production, which were partially blocked by either Y701F or S727A mutation, suggesting a positive role of both STAT1 phosphorylation sites in controlling B cell differentiation. Both TI and TD stimulation also induced significant epigenetic changes on H3K4 and H3K27. In addition, STAT1-pS727 positively regulated the level of H3K4me1 and H3K27me3 in FO B and MZ B cells in response to LPS stimulation. By performing CUT&Tag, an improved ChIP-seq in B cells, we found several histone methyltransferases (KMTs) and demethylases (KDMs), including Kmt5b, Kmt2d, Kdm5a, and Kdm4c, to be downstream of STAT1-pS727. Several known transcription factors (TFs) regulating B cell differentiation were also found to be STAT-pS727 targets, including Prdm1, Irf4, Irf8, Xbp1 and Bcl6. Moreover, STAT1-pS727 mutation decreased H3K4 methylation of these genes upon LPS-stimulation. In addition, total H3K27me3 was reduced in FO B following LPS stimulation. To assess the role of H3K27 methylation, we treated B cells with Valemetostat, a dual inhibitor of EZH1/2 and a methyltransferase known to mediate H3K27me3 and STAT3 methylation. Interestingly, the inhibitor reduced STAT1-pS727 in LPS-stimulated B cells. Together, our findings demonstrate a crucial role of STAT1-pS727 in regulating diverse B cell functions and highlight its impact on modulating the epigenetic landscapes, probably through regulating the expression or function of epigenetic modifiers. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90412 |
DOI: | 10.6342/NTU202304033 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 免疫學研究所 |
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