阿拉伯芥組蛋白去乙醯酶HDA15結合性蛋白質體以及基因調控網路之研究

The Protein Interactome and Gene Regulatory Network of the Histone Deacetylase HDA15 in Arabidopsis

陳佳陽(Chia-Yang Chen)

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(2016年)

https://doi.org/10.6342/NTU201602661

摘要

組蛋白的乙醯化和去乙醯化是重要的轉譯後修飾作用，這個修飾是由組蛋白乙醯轉移酶和組蛋白去乙醯酶所催化的。組蛋白去乙醯酶能夠移除組蛋白離胺酸(Lysine)上的乙醯基，阿拉伯芥的HDA15是屬於RPD3/HDA1類型第二類的組蛋白去乙醯酶。在我們的研究中發現到從大腸桿菌或是阿拉伯芥中所純化得到的HDA15都具有去乙醯酶活性，並且若將HDA15的活性位點進行突變則會失去乙醯酶活性。此外，HDA15除了HDA15的組蛋白去乙醯酶結構域(HDAC domain)， N-端的鋅指結構域(Zinc finger domain)以及在C-端的出核訊號(Nuclear export signal)對於HDA15的活性以及功能都很重要。透過液相層析串聯式質譜 (LC-MS/MS)分析我們發現在第448個以及第452個絲胺酸有磷酸化的現象，並且這些磷酸化對於HDA15的活性以及功能都很重要。我們發現在模擬磷酸化(phosphomimetics)後會導致HDA15失去活性，並且會影響HDA15在細胞內的位置，HDA15從原本在核仁(Nucleolus)轉移到核質(Nucleoplasm)。
我們也利用液相層析串聯式質譜儀(LC-MS/MS)研究HDA15的結合性蛋白質體(Interactome)。發現HDA15可以和很多轉錄因子(Transcription factors)，染色質相關蛋白(Chromatin-associated proteins)以及各種核糖體蛋白(Ribosomal proteins)結合，並且也發現HDA15參與在Sin3-HDAC複合體以及MOS4相關的複合體中。在HDA15結合蛋白中，我們發現MOS4和MYB3也和HDA15一樣會參與在白化苗時期中的葉綠素生合成以及光合作用，並且MOS4還會正向調控在遠紅光下的下胚軸延長。這些結果說明MOS4可能會和HDA15共同調控光型態發生(Photomorphogenesis)中的基因表現。
我們也發現HDA15去乙醯的位置主要是在組蛋白H4上的第5個離胺酸(Lysine)(H4K5Ac)，並且透過染色質免疫沉澱定序(ChIP-seq)發現主要是影響基因本體(Gene body)上的3’末端(3’ end)。進一步分析發現HDA15可以結合在TCP轉錄因子的DNA結合區域(DNA binding motif)。此外HDA15也可以結合在W-box，E-box和G-box結合區域。值得注意的是，在HDA15結合的基因當中，參與在屬於”生物性刺激反應(response to biotic stimulus)”類別的基因占相當多的數量，這代表HDA15可以結合在具有W-box，E-box或G-box結合區域的生物性逆境反應相關的基因上。

關鍵字

阿拉伯芥 ； 組蛋白去乙醯? ； HDA15 ； 磷酸化 ； 液相層析串聯式質譜儀 ； Sin3-HDAC複合體 ； MOS4 ； 光型態發生 ； 染色質免疫沉澱定序

並列摘要

Histone acetylation and deacetylation catalyzed by histone acetyltransferases and histone deacetylases (HDACs) are important post-translational modifications. HDACs catalyze the removal of acetyl groups from lysine residues of histones and HDA15 is a RPD3/HDA1 type class II HDAC. It was found that the recombinant HDA15 protein purified from E.coli and in Arabidopsis displayed histone deacetylase activity and mutations in the active sites of HDA15 disrupted its enzymatic activity. In addition to the histone deacetylase domain, the N-terminal zinc finger domain and C-terminal nuclear export signal are also important for the activity and the function of HDA15. Moreover, two phosphorylation sites Ser 448 and Ser 452 important for the function and activity of HDA15 were identified by LC-MS/MS analysis. Phosphomimetics at Ser 448 and Ser 452 of HDA15 resulted in a loss of HDAC activity and altered localization of HDA15 from the nucleolus to the nucleoplasm.
The interactome landscape of HDA15 was identified through LC-MS/MS analysis. HDA15 could interact with multiple transcription factors, chromatin-associated proteins and diverse ribosomal proteins. Moreover, HDA15 was found to be associated with the Sin3-HDAC complex and the MOS4-associated complex. Two HDA15 interacting proteins, MOS4 and MYB3, were found to be involved in chlorophyll biosynthesis and photosynthesis in etiolated seedlings. MOS4 positively regulated hypocotyl elongation under FR conditions, indicating that MOS4 might associate with HDA15 to regulate gene expression in photomorphogenesis.
Chromatin immunoprecipitation-sequencing (ChIP-seq) revealed that HDA15 was required to deacetylate the histone H4K5 acetylation in the 3’ end of the gene body. Furthermore, HDA15 could target to the binding motif of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) transcription factors. HDA15 also bound to the W-box, E-box and G-box binding motif. In particular, the biological process “response to biotic stimulus” was enriched in the HDA15-bound genes, suggesting that HDA15 could target to biotic stress related genes containing the W-box, E-box and/or G-box binding motifs.

並列關鍵字

Arabidopsis ； histone deacetylases ； HDA15 ； phosphorylation ； LC-MS/MS ； Sin3-HDAC complex ； MOS4 ； photomorphogenesis ； ChIP-seq

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