阿拉伯芥組蛋白去乙醯化酶HDA6對開花時間及葉片發育調控之研究

Function of HDA6 in controlling flowering time and leaf development in Arabidopsis thaliana

游竣惟(Chun-Wei Yu)

指導教授 ： 吳克強

國立臺灣大學/生命科學院/植物科學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.10859

摘要

在真核生物中，組蛋白的乙醯化及去乙醯化對基因的活化或抑制扮演著非常重要的角色。HDA6是屬於RPD3類型的組蛋白去乙醯化酵素，我們的研究發現HDA6基因的突變株(axe1-5)會產生延遲開花的現象。經由遺傳學分析顯示，晚開花的現象是因為HDA6直接影響開花的負調控因子(FLC)所造成的。雙螢光互補(BiFC)、In vitro pull down和免疫共沉澱(Co-IP)實驗分析中顯示，HDA6可以直接與組蛋白去甲基化酵素(FLD)相互作用並調控阿拉伯芥開花。更進一步，我們證明了FLD是利用N端區域的SWIRM domain與HDA6的C端區域相互作用。在axe1-5及fld-6突變株中，染色質免疫沉澱證實較高的組蛋白乙醯化(H3K9K14Ac)及組蛋白甲基化(H3K4Me3)發生在FLC, MAF4及MAF5基因。這些結果顯示，組蛋白去乙醯化酵素與組蛋白去甲基化酵素可以經由HDA6與FLD的物理性相互作用產生crosstalk。染色質免疫沉澱實驗也證實了HDA6蛋白可以直接調控幾個下游的目標基因，包括FLC及MAF4基因。DNA 晶片(microarray)分析顯示，除了開花調控相關基因外，參與逆境和基因靜默調控的相關基因也受到HDA6所影響，證明了HDA6具有多樣化的功能。此外，我們也發現許多轉座子在HDA6基因的突變株中被大量表現，同時具有較高的組蛋白乙醯化(H3K9K14Ac)程度。這些結果顯示HDA6可以藉由影響轉座子的組蛋白去乙醯化程度，進而調控轉座子的基因表現。
在hda6突變株axe1-5和sil1中，葉子的邊緣會產生捲曲和鋸齒的性狀。藉由基因表現分析axe1- 5和sil1突變體，發現KNATM基因表現量明顯的增加。此外，我們還發現KNATM基因的組蛋白H3K9K14高度乙醯化，顯示HDA6可能通過調節組蛋白去乙醯化進而影響KNATM的表達。相對於單突變體，as1-1/axe1-5和as2-1/axe1-5雙突變體顯示更嚴重的葉片捲曲和葉柄變短的表型。雙突變體中，leaflob的頻率和leaflet like的結構也明顯增加了，這表示HDA6可能與AS1和AS2共同調節阿拉伯芥的葉片發育。通過使用雙螢光互補(BiFC)、In vitro pull-down和免疫共沉澱(Co-IP)實驗分析，我們證明了HDA6可以與 AS1和AS2相互作用。這些數據表明，HDA6是AS1- AS2蛋白質複合體的一部分，進而調節KNATM的基因表達。

關鍵字

組蛋白去乙醯化酶HDA6 ； FLD ； 開花 ； 基因晶片 ； AS1 ； AS2 ； KNOX ； KNATM ； 葉片發育

並列摘要

Histone acetylation and deacetylation play an important role in epigenetic controls of gene expression. HDA6 is a RPD3-type histone deacetylase and the hda6 mutant axe1-5 displayed a late flowering phenotype. axe1-5/flc-3 double mutants flowered earlier than axe1-5 plants, indicating that the late-flowering phenotype of axe1-5 was FLC dependent. Bimolecular fluorescence complementation, in vitro pull down and co-immunoprecipitation assays revealed the protein-protein interaction between HDA6 and the histone demethylase FLD. It was found that the SWIRM domain in the N-terminal region of FLD and the C-terminal region of HDA6 are responsible for the interaction between these two proteins. Increased levels of histone H3 acetylation and H3K4 trimethylation at FLC, MAF4 and MAF5 were found in both axe1-5 and fld-6 plants, suggesting functional interplay between histone deacetylase and demethylase in flowering control. These results support a scenario in which histone deacetylation and demethylation crosstalk mediated by physical association between HDA6 and FLD. Chromatin immunoprecipitation analysis indicated that HDA6 bound to the chromatin of several potential target genes including FLC and MAF4. Genome-wide gene expression analysis revealed that in addition to genes related to flowering, genes involved in gene silencing and stress response were also affected in hda6 mutants, revealing multiple functions of HDA6. Furthermore, a subset of transposons was up-regulated and displayed increased histone hyperacetylation, suggesting that HDA6 can also regulate transposons through deacetylating histone.
The hda6 mutants, axe1-5 and sil1, also displayed curling and serrated leaves. The expression of one of the KNOX family genes, KNATM, was up-regulated in axe1-5 and sil1 mutants. In addition, hyperacetylation of histone H3K9K14 at KNATM was found in both axe1-5 and sil1 mutants, suggesting the HDA6 may regulate KNATM expression through histone deacetylation. Compared with the single mutants, the as1-1/axe1-5 and as2-1/axe1-5 double mutants displayed more severe curling leaf and short petiole phenotypes. In addition, the frequencies of leaf lobes and leaflet-like structures were also increased in as1-1/axe1-5 and as2-1/axe1-5 double mutants, suggesting that HDA6 may function together with AS1 and AS2 to regulated the leaf development in Arabidopsis. By using the in vitro pull-down, BiFC and Co-IP assays, we demonstrated that HDA6 can interact with AS1 and AS2. These data indicate that HDA6 is part of the AS1-AS2 repression complex to regulate the expression of KNATM.

並列關鍵字

histone deacetylase ； HDA6 ； FLD ； flowering ； microarray ； AS1 ； AS2 ； KNOX ； KNATM ； leaf development

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