阿拉伯芥組蛋白去乙醯基酶HDA5在熱逆境中之功能性研究

Abstract

先前研究已經闡明部分與植物熱逆境反應相關的轉錄調控網絡，然而關於轉錄靜默和染色質重塑的機制仍然未知。組蛋白去乙醯基酶 (HDACs) 是與轉錄靜默緊密相連的關鍵染色質調控因子，對植物適應不良環境至關重要。阿拉伯芥微陣列分析顯示，熱逆境誘導HDA5的表達，但其功能與機制仍然不明。在本研究中透過兩種熱耐性測試：基礎耐熱性及後天耐熱性確認HDA5在熱逆境反應途徑中作為負調控因子。進一步透過蛋白交互作用實驗發現，在19個熱休克因子（HSFs）中，HDA5會與HSFA1d，HSFA8，HSFA9以及HSFB2b在細胞核中有相互作用。qPCR分析表明，在hda5-1突變株中，與野生型相比，多個HSFs的表達量增加，並且其下游基因HSP22與HSP101的表現量顯著增加。ChIP-qPCR分析發現，在hda5-1突變體中，HSP22啟動子的H3K9乙醯化的程度顯著提升，而在HSP101啟動子則未出現變化。此外，HSP22啟動子上的H3K9乙醯化與H3K4二甲基化的豐度之間存在拮抗關係，且HSP101啟動子的H3K4二甲基化的程度呈下降趨勢。在原生質體次細胞定位實驗中中發現HDA5在正常生長環境下出現在細胞核中，在熱逆境的環境下則是完全轉移至細胞核外，這表明HDA5可能間接調控熱相關基因 （heat-responsive genes）。Dual-luciferase reporter實驗顯示，HDA5與HSFB2b共同作用，顯著抑制了HSP22的轉錄活性。根據上述研究結果推測，HDA5在熱反應中可能透過非組蛋白為目標的蛋白質去乙醯化（deacetylation）與調控組蛋白修飾相關蛋白來實現對轉錄組的轉錄沉默，而非直接參與組蛋白修飾（histone modification）。

Research has highlighted the complex regulatory networks involved in plant heat shock responses (HSR), yet the mechanisms underlying chromatin remodeling and transcriptional silencing during HS adaptation remain elusive. Histone deacetylases (HDACs) are key chromatin regulators linked to transcriptional silencing, essential for plant resilience in adverse environments. Arabidopsis microarray data indicates that heat stress induces HDA5, but its precise role remains unclear. This study reveals that HDA5 functions as a negative regulator in the heat stress response and that thermotolerance tests confirm its regulatory role under both baseline and acquired heat stress conditions. Further analysis of protein-protein interactions data showed that among 19 heat shock factors (HSFs), HDA5 interacts with HSFA1d, HSFA8, HSFA9, and HSFB2b in the nucleus. qPCR analysis revealed that in the hda5-1 mutant, several HSFs were upregulated compared to the wild type (Col-0), with downstream genes HSP22 and HSP101 showing significant increases in expression. ChIP-qPCR analysis revealed an increase in H3K9 acetylation at the HSP22 loci in the hda5-1 mutant, while no changes were observed at the HSP101 loci. Additionally, an antagonistic relationship between H3K9 acetylation and H3K4 dimethylation was observed at the HSP22 loci, accompanied by a downtrend in H3K4 dimethylation at the HSP101 loci. Subcellular localization studies showed that HDA5 relocates from the nucleus to the cytoplasm under HS, suggesting that HDA5 may regulate heat-responsive genes indirectly. Dual-luciferase reporter assays revealed that HDA5, in conjunction with HSFB2b, significantly inhibits the transcriptional activity of HSP22. These findings propose a novel role for HDA5 in transcriptional silencing during HS, potentially through the deacetylation of non-histone proteins or modulation of histone modification-related proteins, rather than direct histone modification.