乙酸胁迫条件下酿酒酵母全基因组启动子区组蛋白H4 K16 乙酰化修饰分析

doi:10.15933/j.cnki.1004-3268.2022.01.009

摘要/Abstract

摘要： 为探究酵母乙酸耐受性的表观遗传调控新机制，采用染色质免疫共沉淀-芯片（Chromatin immunoprecipitation‐chip，ChIP-chip）技术，分析乙酸胁迫条件下酿酒酵母全基因组启动子区组蛋白H4 K16乙酰化修饰的变化。结果表明，酵母细胞经乙酸胁迫处理（60 mmol/L、30 min）后，与对照组（添加等体积无菌水）相比，73个酵母基因的启动子区域组蛋白H4 K16位点乙酰化修饰发生了改变，乙酰化修饰上升基因19个，主要分布在2、4、5、7、13、15、16号染色体上；乙酰化修饰下降基因54个，主要分布在1、2、4、5、6、7、8、10、11、12、13、14、15、16号染色体上。基因功能分析结果表明，按生物学过程分类乙酰化修饰改变基因主要富集在碱基切除修复、细胞骨架组装、细胞周期进程和含蛋白质的复合物组装；按细胞组分分类乙酰化修饰改变基因主要富集在液泡质子转运V-型ATP酶、DNA聚合酶复合体、质子转运双扇区ATP酶复合物/质子转运域、有丝分裂纺锤体和核染色体；按分子功能分类乙酰化修饰改变基因主要富集在单链DNA 3′—5′外脱氧核糖核酸酶活性、蛋白酶体结合、ATP酶活性/耦联离子跨膜运动。随机选取3个启动子区组蛋白H4 K16乙酰化修饰发生差异变化的基因（ATP12、VPS55、DLT1）进行染色质免疫沉淀-实时定量PCR检测，结果与ChIP-chip分析结果一致，验证了ChIP-chip试验的准确性。综上，酿酒酵母基因启动子区组蛋白H4 K16乙酰化修饰与乙酸耐受性密切相关。

关键词: 酿酒酵母, 乙酸胁迫, 组蛋白H4, 乙酰化, 染色质免疫共沉淀-芯片

Abstract: For the exploration of new epigenetic regulation mechanism of yeast tolerance to acetic acid，changes of the histone H4 K16 acetylation modification in genome‐wide promoters in Saccharomyces cerevisiae under acetic acid stress were analyzed using the chromatin immunoprecipitation‐chip technique（ChIP‐chip）. Results showed that the acetylation levels of histone H4 K16 of 73 yeast genes in the promoter region changed in contrast to those in the control group after acetic acid（60 mmol/L for 30 min）stress treatment. A total of 19 genes with increased histone H4 K16 acetylation levels were mainly distributed on chromosomes 2，4，5，7，13，15 and 16，and 54 genes with decreased histone H4 K16 acetylation levels were mainly distributed on chromosomes 1，2，4，5，6，7，8，10，11，12，13，14，15 and 16.Gene function analysis showed that the genes were mainly concentrated in base‐excision repair，cytoskeleton organization，cell cycle process，and protein‐containing complex assembly，classified by biological process；The genes were mainly concentrated in vacuolar proton‐transporting V‐type ATPase，DNA polymerase complex，proton‐transporting two‐sector ATPase complex/proton‐transporting domain，mitotic spindle，and nuclear chromosome，classified by cellular component；The genes were mainly concentrated in the single‐stranded DNA 3′—5′ exodeoxyribonuclease activity，proteasome binding，and ATPase activity/coupled to transmembrane movement of ions，classified by molecular function.Three genes（ATP12，VPS55，and DLT1） with histone H4 K16 acetylation alterated in their gene promoter regions were randomly selected for chromatin immunoprecipitation‐quantitative real‐time PCR detection，and the results were consistent with the ChIP‐chip analysis，verifying the accuracy of ChIP‐chip data.Overall，these results indicate that the acetylation of histone H4 K16 in the gene promoter region is closely related to acetic acid tolerance in Saccharomyces cerevisiae.

Key words: Saccharomyces cerevisiae, Acetic acid stress, Histone H4, Acetylation, Chromatin ：S141, TS261.11 文immunoprecipitation‐chip

中图分类号:

马田, 王玉婧, 程爽, 张小华, 刘向勇. 乙酸胁迫条件下酿酒酵母全基因组启动子区组蛋白H4 K16 乙酰化修饰分析[J]. 河南农业科学, 2022, 51(1): 71-78.

MA Tian, WANG Yujing, CHENG Shuang, ZHANG Xiaohua, LIU Xiangyong. Analysis of Histone H4 K16 Acetylation in Genome‐Wide Promoter Region in Saccharomyces cerevisiae under Acetic Acid Stress[J]. Journal of Henan Agricultural Sciences, 2022, 51(1): 71-78.

图/表 6

参考文献

［1］ROBAK K，BALCEREK M. Review of second generation bioethanol production from residual biomass［J］.Food Technol Biotechnol，2018，56（2）：174‐187.
［2］ABO B O，GAO M，WANG Y，et al.Lignocellulosic biomass for bioethanol：An overview on pretreatment，hydrolysis and fermentation processes［J］.Rev Environ Health，2019，34（1）：57‐68.
［3］KIM D.Physico‐chemical conversion of lignocellulose：Inhibitor effects and detoxification strategies：A mini
review［J］.Molecules，2018，23（2）：309.
［4］KUMAR V，YADAV S K，KUMAR J，et al.A critical review on current strategies and trends employed for removal of inhibitors and toxic materials generated during biomass pretreatment ［J］.Bioresour Technol，2020，299：122633.
［5］杨莉，谭丽萍，刘同军.木质纤维素预处理抑制物产生及脱除方法的研究进展［J］.生物工程学报，2021，37（1）：15‐29.
YANG L，TAN L P，LIU T J.Progress in detoxification of inhibitors generated during lignocellulose pretreatmen［J］.Chinese Journal of Biotechnology，2021，37（1）：15‐29.
［6］赵心清，张明明，徐桂红，等.酿酒酵母乙酸耐性分子机制的功能基因组进展［J］.生物工程学报，2014，30（3）：368‐380.
ZHAO X Q，ZHANG M M，XU G H，et al.Advances in functional genomics studies underlying acetic acid tolerance of Saccharomyces cerevisiae［J］.Chinese Journal of Biotechnology，2014，30（3）：368‐380.
［7］GENG P，ZHANG L，SHI G Y. Omics analysis of acetic acid tolerance in Saccharomyces cerevisiae［J］.World J Microbiol Biotechnol，2017，33（5）：94.
［8］PALMA M，GUERREIRO J F，SA‐CORREIA I.Adaptive response and tolerance to acetic acid in Saccharomyces cerevisiae and Zygosaccharomyces bailii：A physiological genomics perspective［J］.Front Microbiol，2018，9：274.
［9］TESSARZ P，KOUZARIDES T.Histone core modifications regulating nucleosome structure and dynamics［J］.Nat Rev
Mol Cell Biol，2014，15（11）：703‐708.
［10］JAISWAL D，TURNIANSKY R，GREEN E M.Choose your own adventure：The role of histone modifications in yeast cell fate［J］.J Mol Biol，2017，429（13）：1946‐1957.
［11］FENLEY A T，ANANDAKRISHNAN R，KIDANE Y H，et al.Modulation of nucleosomal DNA accessibility via charge‐altering post‐translational modifications in histone core［J］.Epigenetics Chromatin，2018，11（1）：11.
［12］CALLUM J，O’KANE C J，HYLAND E M.Yeast epigenetics：The inheritance of histone modification states［J］.Biosci Rep，2019，39（5）：BSR20182006.
［13］TOPAL S，VASSEUR P，RADMAN‐LIVAJA M，et al.Distinct transcriptional roles for histone H3‐K56 acetylation during the cell cycle in yeast［J］.Nat Commun，2019，10（1）：4372.
［14］VIEITEZ C，MARTINEZ‐CEBRIAN G，SOLE C，et al.A genetic analysis reveals novel histone residues required for transcriptional reprogramming upon stress［J］.Nucleic Acids Res，2020，48（7）：3455‐3475.
［15］DANG W，STEFFEN K K，PERRY R，et al.Histone H4 lysine 16 acetylation regulates cellular lifespan［J］.Nature，2009，459：802‐807.
［16］REITER C，HEISE F，CHUNG H R，et al.A link between Sas2‐mediated H4 K16 acetylation，chromatin assembly in S‐phase by CAF‐Ⅰ and Asf1，and nucleosome assembly by Spt6 during transcription［J］.FEMS Yeast Res，2015，15（7）：fov073.
［17］RAY A，KHAN P，NAG CHAUDHURI R.Regulated acetylation and deacetylation of H4 K16 is essential for efficient NER in Saccharomyces cerevisiae［J］.DNA Repair，2018，72：39‐55.
［18］YOUNG T J，CUI Y，IRUDAYARAJ J，et al.Modulation of gene silencing by Cdc7p via H4 K16 acetylation and
phosphorylation of chromatin assembly factor CAF‐1 in Saccharomyces cerevisiae［J］.Genetics，2019，211（4）：1219‐1237.
［19］BOLTENGAGEN M，SAMEL‐POMMERENCKE A，FECHTIG D，et al.Dynamics of SAS‐Ⅰ mediated H4 K16 acetylation during DNA replication in yeast［J］.PLoS One，2021，16（5）：e0251660.
［20］LIU X，ZHANG X，ZHANG Z.Point mutation of H3/H4 histones affects acetic acid tolerance in Saccharomyces cerevisiae［J］.J Biotechnol，2014，10（187）：116‐123.
［21］WANG J，VASAIKAR S，SHI Z，et al.WebGestalt 2017：A more comprehensive，powerful，flexible and interactive gene set enrichment analysis toolkit［J］.Nucleic Acids Res，2017，45（1）：130‐137.
［22］ERIC L.A laboratory practical illustrating the use of the ChIP‐qPCR method in a robust model：Estrogen receptor alpha immunoprecipitation using Mcf‐7 culture cells［J］.Biochem Mol Biol Educ，2017，45（2）：152‐160.
［23］OH E J，WEI N，KWAK S，et al.Overexpression of RCK1 improves acetic acid tolerance in Saccharomyces cerevisiae［J］.J Biotechnol，2019，292：1‐4.
［24］ZHANG M M，XIONG L，TANG Y J，et al.Enhanced acetic acid stress tolerance and ethanol production in Saccharomyces cerevisiae by modulating expression of the de novo purine biosynthesis genes［J］.Biotechnol Biofuels，2019，12：116.
［25］HU J J，DONG Y，WANG W，et al.Deletion of Atg22 gene contributes to reduce programmed cell death induced by acetic acid stress in Saccharomyces cerevisiae［J］.Biotechnol Biofuels，2019，12：298.
［26］KONARZEWSKA P，SHERR G L，AHMED S，et al.Vma3p protects cells from programmed cell death through the regulation of Hxk2p expression［J］.Biochem Biophys Res Commun，2017，493（1）：233‐239.
［27］MIHO K，KAZUO M，TSUTOMU F，et al.Yeast genes involved in response to lactic acid and acetic acid：Acidic conditions caused by the organic acids in Saccharomyces cerevisiae cultures induce expression of intracellular metal metabolism genes regulated by Aft1p［J］.FEMS Yeast Res，2006，6（6）：924‐936.

［28］FERNANDEZ‐NINO M，PULIDO S，STEFANOSKA D，et al.Identification of novel genes involved in acetic acid tolerance of Saccharomyces cerevisiae using pooled‐segregant RNA sequencing［J］.FEMS Yeast Res，2018，18（8）：foy100.

［29］ALMEIDA B，OHLMEIER S，ALMEIDA A J，et al.Yeast protein expression profile during acetic acid‐induced apoptosis indicates causal involvement of the TOR pathway［J］. Proteomics，2009，9（3）：720‐732.
［30］MIRA N P，PALMA M，GUERREIRO J F，et al.Genome‐wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid［J］.Microb Cell Fact，2010，9：79.
［31］LI Y C，GOU Z X，LIU Z S，et al.Synergistic effects of TAL1 over‐expression and PHO13 deletion on the weak acid inhibition of xylose fermentation by industrial Saccharomyces cerevisiae strain ［J］.Biotechnol Lett，2014，36（10）：2011‐2021.
［32］杜昭励，程艳飞，朱卉，等.絮凝基因FLO1及FLO1c高表达提高工业酿酒酵母乙酸耐受性及发酵性能［J］.生物工程学报，2015，31（2）：231‐241.
DU Z L，CHENG Y F，ZHU H，et al.Improvement of acetic acid tolerance and fermentation performance of industrial Saccharomyces cerevisiae by overexpression of flocculent gene FLO1 and FLO1c［J］.Chinese Journal of Biotechnology，2015，31（2）：231‐241.
［33］KITANOVIC A，BONOWSKI F，HEIGWER F，et al.Acetic acid treatment in S. cerevisiae creates significant energy deficiency and nutrient starvation that is dependent on the activity of the mitochondrial transcriptional complex Hap2‐3‐4‐5［J］.Front Oncol，2012，2：118.
［34］KURDISTANI S K，GRUNSTEIN M.Histone acetylation and deacetylation in yeast［J］.Nat Rev Mol Cell Biol，2003，4（4）：276‐284.
［35］VALL‐LLAURA N，MIR N，GARRIDO L，et al.Redox control of yeast Sir2 activity is involved in acetic acid resistance and longevity［J］.Redox Biol，2019，24：101229.
［36］CHENG C，ZHAO X，ZHANG M，et al.Absence of Rtt109p，a fungal‐specific histone acetyltransferase，results in improved acetic acid tolerance of Saccharomyces cerevisiae［J］.FEMS Yeast Res，2016，16（2）：fow010.
［37］ISHIDA Y，NGUYEN T T M，LZAWA S.The yeast ADH7 promoter enables gene expression under pronounced translation repression caused by the combined stress of vanillin， furfural， and 5‐hydroxymethylfurfural［J］.J Biotechnol，2017，252：65‐72.
［38］FLETCHER E，GAO K，MERCURIO K，et al.Yeast chemogenomic screen identifies distinct metabolic pathways required to tolerate exposure to phenolic fermentation inhibitors ferulic acid，4‐hydroxybenzoic acid and coniferyl aldehyde［J］.Metab Eng，2019，52：98‐109.
［39］PEREIRA R，MOHAMED E T，RADI M S，et al.Elucidating aromatic acid tolerance at low pH in Saccharomyces cerevisiae using adaptive laboratory evolution［J］.Proc Natl Acad Sci，2020，117（45）：27954-27961.

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