探究H3組蛋白4號賴氨酸去甲基酶JHD2於氮素逆境誘導假菌絲發育過程所扮演的角色

The Role of Histone H3 Lysine 4 Demethylase JHD2 in Pseudohyphae Development During Nitrogen Starvation

杜柏儀(Bo-Yi Du)

指導教授 ： 林敬哲

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202100891

摘要

Saccharomyces cerevisiae藉由二型態之酵母菌型態到假菌絲型態的轉換來因應環境的變化。菌絲化的生長可在氮素的逆境下觀察到細胞的體長延伸、單極性出芽生殖以及子代細胞串連並且朝遠離群落的方向生長。組蛋白N端的轉譯後修飾在表觀遺傳層次的調控上參與了細胞的分化。JHD2作為酵母菌唯一的H3K4去甲基酶，在剔除JHD2後難以觀察正常營養供給條件下的生長缺陷，然而在氮素缺失的固態培養基上jhd2∆菌株產生大量的假菌絲，由此大膽假設失去JHD2促進了氮素逆境下假菌絲的生長。
為了進一步驗證此假說，首先，測量細胞長寬比後，包括jhd2∆在內其他jhd2突變株H427A、PHD∆、JmjN∆均在24小時有較高比例的細胞伸長，如此說明JHD2需要所有的結構域來運行完整的功能。為了更明確捕捉形態變化的時間點，從顯微鏡間時攝影技術可以觀察到jhd2∆在細胞分裂的第一子代就可分化出菌絲化的酵母菌，相反地，過度表現JHD2則在歷經幾個世代均無法產生穩定的假菌絲，然而在過度表現JHD2卻可以看到大量行軸向出芽生殖的雙套染色體酵母菌，從結果可以推測JHD2更是參與了細胞型的出芽位置選擇。
更進一步探討JHD2調控的分子機制，首先檢測了決定假菌絲生長與生物膜的重要基因FLO11的轉錄表現量，發現jhd2∆在正常營養條件培養與氮素缺乏條件培養均維持了較高的表現量；反觀過度表現JHD2的菌株無法有效誘導FLO11，再者發現無法誘導FLO11是因為其上游扮演抑制功能的順式干擾性非編碼核糖核酸ICR1的高表現所導致。最後透過核糖核酸定序分析測試野生型與jhd2∆菌株在正常或氮素缺乏條件下轉錄體的差異，發現在氮素逆境下野生型表現量上升的基因群組卻在正常條件下的jhd2∆也高程度的有較高的表現量，再利用階層式分群的方法分析也看到jhd2∆轉錄體表現圖譜與野生型在氮素逆境的圖譜相似性較高。總結，在失去JHD2功能的細胞狀態更趨近於處於氮素逆境下的細胞基因表現，而JHD2也參與非編碼核糖核酸ICR1與細胞分裂時出芽位置選擇的調控。

關鍵字

表觀遺傳學 ； 去甲基酶 ； H3 組蛋白 ； 酵母菌 ； 假菌絲 ； 氮素逆境 ； 核糖核酸定序分析 ； 轉譯後修飾

並列摘要

Saccharomyces cerevisiae goes through dimorphic transition, the switch from yeast to pseudohyphae, in response to environmental changes. During Nitrogen starvation (NS), cells elongate, bud in unipolar, and connect into a long-chained fashion evading out from central cluster, called filamentous growth. Reversible post-translational modifications of histone tails participate in epigenetic regulation of cell identity and differentiation. JHD2, encoding Jumonji-C domain containing histone demethylase, acts as a histone H3K4 demethylase in budding yeast. jhd2-deletion (jhd2∆) strains showed no obvious growth defects in physiological condition. However, jhd2∆ formed much more pseudohyphae on the nitrogen-depleted solid agar. Hence, we hypothesized that loss of JHD2 promotes filamentous growth in response to NS.
To elucidate the role of JHD2 in filamentous growth, the length-width ratio of the cells surrounding the clusters was measured. jhd2∆ and its mutants including catalytic-dead mutant, jhd2-H427A, PHD∆ (plant homeodomain deleted) and JmjN∆ (Jumonji N deleted), showed higher ratio than wild type (WT) in 24 hours. It suggests that loss of JHD2 promotes cell elongation, and full function of JHD2 activity requires each of the domain together. To capture the timing of transition, time-lapse imaging was performed. During filamentous growth, jhd2∆ was able to generate elongated cells in short period of time. On the contrary, overexpression of JHD2 failed to maintain stable filamentous growth through generations. Unexpectedly, axial budding pattern was largely appeared in JHD2 overexpressing diploid strain indicating that JHD2 may participate in cell-type specific bud-site selection.
To further understand the molecular mechanism of JHD2 regulating filamentous growth, FLO11, essential gene for pseudohyphae development and biofilm formation, expression level was tested. jhd2∆ remained significantly high expression of FLO11 during NS. In contrast, overexpression of JHD2 failed to induce FLO11 during NS. Moreover, measurement of cis-interfering ncRNA, ICR1, toggling the promoter region of FLO11 hence repressing adjacent gene transcription, showed higher expression in overexpression of JHD2. These discoveries suggest that JHD2 regulates FLO11 expression through ICR1. Finally, RNA sequencing was performed in WT and jhd2∆ strains with or without nitrogen starvation (NS). Gene set, up-regulated in WT under NS, was enriched in jhd2∆ but under nitrogen-rich condition. In addition, hierarchical analysis showed that gene expression pattern of jhd2∆ was grouped more closely to the WT under NS. In sum, loss of JHD2 mimics the gene signature of cells going through NS, and regulates dimorphic transition through ncRNA ICR1 and bud-site selection.

並列關鍵字

Epigenetics ； H3K4 demethylase ； Demethylase ； Saccharomyces ； Pseudohyphae ； Filamentous growth ； Dimorphic transition ； Nitrogen starvation ； RNA sequencing ； GSEA

參考文獻

Avramova, Z. (2015). "Transcriptional 'memory' of a stress: transient chromatin and memory (epigenetic) marks at stress-response genes." Plant J 83(1): 149-159.

Barral, S., Y. Morozumi, H. Tanaka, E. Montellier, J. Govin, M. de Dieuleveult, G. Charbonnier, Y. Coute, D. Puthier, T. Buchou, F. Boussouar, T. Urahama, F. Fenaille, S. Curtet, P. Hery, N. Fernandez-Nunez, H. Shiota, M. Gerard, S. Rousseaux, H. Kurumizaka and S. Khochbin (2017). "Histone Variant H2A.L.2 Guides Transition Protein-Dependent Protamine Assembly in Male Germ Cells." Mol Cell 66(1): 89-101 e108.

Barrales, R. R., P. Korber, J. Jimenez and J. I. Ibeas (2012). "Chromatin modulation at the FLO11 promoter of Saccharomyces cerevisiae by HDAC and Swi/Snf complexes." Genetics 191(3): 791-803.

Benayoun, B. A., E. A. Pollina, D. Ucar, S. Mahmoudi, K. Karra, E. D. Wong, K. Devarajan, A. C. Daugherty, A. B. Kundaje, E. Mancini, B. C. Hitz, R. Gupta, T. A. Rando, J. C. Baker, M. P. Snyder, J. M. Cherry and A. Brunet (2014). "H3K4me3 breadth is linked to cell identity and transcriptional consistency." Cell 158(3): 673-688.

Brown, A. J. and N. A. Gow (1999). "Regulatory networks controlling Candida albicans morphogenesis." Trends Microbiol 7(8): 333-338.