謝家慶Jia-Ching Shieh陳禮彬Li-Bin Chen生物醫學科學學系2026-03-189999-12-3120202020-07-24https://handle.ncl.edu.tw/11296/h366jmhttps://scholars.csmu.edu.tw/handle/123456789/5960白色念珠菌 (Candida albicans)是一種缺乏完整有性生殖週期的自然界二倍體生物。白色念珠菌能引發表層感染，或導致高致死率的系統性念珠菌症 (Systemic candidiasis)。型態可塑性 (Morphological plasticity)是白色念珠菌致病力的關鍵特徵，為白色念珠菌的研究重點。先前我們揭露進化保守的有絲分裂細胞週期調節因子，Skp1-Cullin-1 / Cdc53-F-box (SCF) Cdc4泛素連接酶，可抑制白色念珠菌酵母菌到菌絲的轉換，也鑑定出白色念珠菌Jhd2 (CaJhd2)為CaCdc4的相關蛋白，並發現CaJHD2編碼組蛋白H3第四離胺酸去甲基酶 (Histone H3 lysine 4 demethylase)可抑制白色念珠菌細胞聚集及生物膜形成，與白色念珠菌編碼E3泛素連接酶 (E3 ubiquitin ligase)編碼基因CaNOT4影響相反。在釀酒酵母中，Not4可透過對Jhd2的多泛素化-蛋白酶體降解，調控組蛋白H3第四離胺酸 (H3K4)三甲基狀態及特定的外表型。然而CaNOT4與CaJHD2之間功能性交互作用，導致H3K4甲基化改變影響致病相關基因表現及外表型，在白色念珠菌中仍待釐清。為釐清上述，我採用「NOT‒/‒ JHD2Tet-Off/‒」白色念珠菌品系，透過在CaNOT4同型合子無效突變株 (NOT4‒/‒)中建構CaJHD2的四環黴素誘導表達關閉系統 (Tet-Off system)。此品系在有或無去氧羥四環素(Doxycycline, Dox)時，能模擬CaNOT4及CaJHD2共同缺失或CaNOT4缺失而CaJHD2過量表現的情況。藉分析H3K4甲基化狀態及外表型，可了解CaNOT4和CaJHD2之間功能性交互作用的結果。 我發現CaNOT4缺失而CaJHD2過量表現時，可造成H3K4的二、三甲基修飾減少而一甲基修飾提升，顯示CaNot4可藉負調控CaJhd2來操縱H3K4的甲基化，三個H3K4甲基化狀態之間存在與CaJhd2競爭的情況。外表型試驗中，CaJHD2和CaNOT4對鈣離子依賴性絮凝力無影響，但CaJHD2能參與對纖維黏連蛋白 (Fibronectin, FN)的黏附。CaJHD2缺失可促進細胞對FN的黏附，CaNOT4剔除株及NOT‒/‒ JHD2Tet-Off/‒則無論有無Dox皆無差異，猜測白色念珠菌對FN之黏附有CaNot4-CaJhd2外的其他路徑參與。並且我發現CaJHD2缺失能增加細胞表面疏水性 (Cell surface hydrophobicity, CSH)，但CaNOT4缺失結果相反。當CaNOT4缺失同時過度表現CaJHD2會進一步降低CSH；CaJHD2表現受抑制則會恢復CSH至野生型水準。顯示CaJHD2抑制，而CaNOT4則促進CSH。此外，我發現CaJHD2缺失可促進芽管 (Germ tube)形成，CaNOT4缺失結果相反，表明CaJHD2和CaNOT4分別作為芽管形成之負向及正向調控者。 綜上所述，以NOT‒/‒ JHD2Tet-Off/‒品系搭配Dox控制CaJHD2表現，揭露CaNot4對CaJhd2之抑制性而與白色念珠菌致病力有關。Candida albicans, a natural diploid with an incomplete sexual cycle, can trigger either superficial infection or systemic candidiasis with high mortality. The morphological plasticity, a critical virulent attribute to C. albicans pathogenesis, is the focal point in C. albicans research. We have uncovered that the evolutionarily conserved cell cycle regulator, Skp1-Cullin-1/Cdc53-F-box (SCF)Cdc4 ubiquitin ligase, suppresses the yeast-to-hypha transition in C. albicans. Moreover, we have identified C. albicans Jhd2 (CaJhd2) as a C. albicans Cdc4-associated protein and found that C. albicans JHD2 (CaJHD2) encodes a histone H3 lysine 4 (H3K4) demethylase and suppresses biofilm formation, which is contrary to the C. albicans E3 ubiquitin ligase encoded NOT4. Importantly, the polyubiquitin-proteasome dependent degradation of Jhd2 by Not4 regulating the tri-methylation status of histone H3K4 for specific phenotypes in Saccharomyces cerevisiae is known. However, the functional interaction between CaNOT4 and CaJHD2, leading to changes in H3K4 methylation associated with expression of genes and the phenotypic consequences, particularly C. albicans virulence, remains elusive. To clarify those mentioned above, I took advantage of a C. albicans strain called “NOT4-/- JHD2Tet-Off/-”, by constructing tetracycline-repressible (Tet-Off system) CaJHD2 expression system into a CaNOT4 homozygous null mutant (NOT4-/-). The strain mimics the loss of both CaNOT4 and CaJHD2 genes with doxycycline (Dox), yet displays loss of CaNOT4 but overexpression of CaJHD2 without Dox. The outcomes of the functional interaction between CaJHD2 and CaNOT4 were assayed for the status of H3K4 methylation and the phenotypic consequences. I showed that C. albicans cells lacking CaNOT4 but overexpressing CaJHD2 reduced methylation on H3K4me2 and H3K4me3 but increased methylation on H3K4me1, which indicate that CaNot4 modulates H3K4 methylation through negatively regulating CaJhd2, but presence of competition among three H3K4 methylation states with CaJhd2. While no role in calcium-dependent flocculation of CaJHD2 and CaNOT4 was observed, CaJHD2 could bind with fibronectin. C. albicans cells lacking CaJHD2 increased in adhesion with fibronectin, but the results of those cells lacking CaNOT4 and cells of NOT4-/- JHD2Tet-Off/- strain with or without Dox were intangible. These results suggest that the binding with fibronectin may involve in other pathways beyond CaNot4-CaJhd2. Moreover, I found that while C. albicans cells lacking CaJHD2 increased the cell surface hydrophobicity (CSH), those cells lacking CaNOT4 showed the opposite. Cells lacking CaNOT4 but overexpressing CaJHD2 decreased CSH more significantly, whereas those cells with the repressed CaJHD2 expression restored the level to wild-type cells. These results indicate that CaJHD2 represses CSH, but CaNOT4 acts oppositely. Furthermore, I observed that C. albicans cells lacking CaJHD2 enhanced the ability in germ tube formation, but those lacking CaNOT4 exhibited the opposite, suggesting that CaJHD2 and CaNOT4 are negative and positive regulators of germ tube formation, respectively. Overall, the results indicate that the use of NOT4-/- JHD2Tet-Off/- strain coupled with Dox usage to control of CaJHD2 expression revealed the inhibitory nature of CaNot4 on CaJhd2 contributing C. albicans virulence.白色念珠菌組蛋白第四離胺酸甲基化JHD2NOT4致病力四環黴素誘導基因表達關閉系統Candida albicansHistone H3K4 methylationVirulenceTet-off systemthesis