ASH1L甲基转移酶在牛卵丘细胞中的表达与功能研究

doi:10.11843/j.issn.0366-6964.2020.08.010

Abstract

Abstract: The study aimed to investigate the expression and function of absent, small, or homeotic 1-like (ASH1L) methyltransferase in healthy bovine cumulus cells. Location of ASH1L methyltransferase and the methylation patterns of histone H3 Lysine 36 (H3K36) were detected using immunofluorescence staining in healthy bovine cumulus cells. The quantitative PCR (qPCR) and Western blotting were used to screen the effective siRNA targeting Ash1L gene in siRNA-1, siRNA-2, siRNA-3 and control group. The expression level of apoptosis-related genes and polycomb repressive complex 2(PRC2) genes were estimated by qPCR after the Ash1L gene was interfered with siRNA in treatment and control groups. The results showed that ASH1L methyltransferase was located in the nucleus of bovine cumulus cells and distributed in a dotted pattern. The effective siRNA (siRNA-2) targeting the bovine Ash1L gene was successfully screened and its interfering efficiency reached 60%-70%. The monomethylation, dimethylation and trimethylation levels of H3K36 in cumulus cells transfected with siRNA-2 were significantly lower than those in the control group (P<0.05). Additionally, interfering Ash1L gene could significantly up-regulated the expressions of apoptosis-related genes Bax, Bcl-2 and caspase-3. However, the expression levels of apoptosis-related genes Bax and caspase-3 were higher than that of anti-apoptosis-related gene Bcl-2 (1.311 and 1.179 vs 1.146). At the same time, the mRNA expressions of PRC2 protein subunit EZH2 and Suz12 were significantly increased after interfering Ash1L(P<0.05). The expression and function of ASH1L methyltransferase in bovine cumulus cells were studied, ASH1L showed dot distribution in bovine cumulus cells, and the inhibition of Ash1L gene expression resulted in a significant decrease in H3K36me1/2/3 levels and an increase in the expressions of apoptosis-related genes and PRC2 protein related subunits EZH2 and Suz12 genes, which will provide technical and theoretical basis for further studying the effects of ASH1L on embryos development in livestock animals.

Key words: ASH1L methyltransferase, bovine cumulus cells, H3K36 methylation, apoptosis

CLC Number:

S823.3

CUI Lixin, TIAN Yaqing, HAO Haisheng, ZOU Huiying, ZHAO Shanjiang, PANG Yunwei, ZHAO Xueming, ZHU Huabin, DU Weihua. Expression and Function of ASH1L Methyltransferase in Bovine Cumulus Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8): 1866-1877.

References 42

[1]	MATSUDA F,INOUE N,MANABE N,et al.Folli-cular growth and atresia in mammalian ovaries:regulation by survival and death of granulosa cells[J].J Reprod Dev,2012,58(1):44-50.
[2]	杨有福.Wnt/β-catenin协同褪黑素、PI3K信号通路对猪卵巢颗粒细胞发育的影响[D].杨凌:西北农林科技大学,2018.YANG Y F.Effect of Wnt/β-catenin in cooperation with melatonin, PI3K signaling on development of porcine granulosa cells[D]. Yangling:Northwest A&F University,2018.(in Chinese)
[3]	TIAN C,LIU L L,YE X Y,et al.Functional oocytes derived from granulosa cells[J].Cell Rep,2019,29(13):4256-4267.e9.
[4]	COSGROVE M S,WOLBERGER C.How does the histone code work?[J].Biochem Cell Biol,2005,83(4):468-476.
[5]	WILES E T,SELKER E U.H3K27 methylation:a promiscuous repressive chromatin mark[J].Curr Opin Genet Dev,2017,43:31-37.
[6]	SHEARN A,HERSPERGER E,HERSPERGER G.Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations[J].Roux's Arch Dev Biol,1987,196(4):231-242.
[7]	MIYAZAKI H,HIGASHIMOTO K,YADA Y,et al.Ash1l methylates Lys36 of histone H3 independently of transcriptional elongation to counteract Polycomb silencing[J].PLoS Genet,2013,9(11):e1003897.
[8]	YUAN W,XU M,HUANG C,et al.H3K36 methylation antagonizes PRC2-mediated H3K27 methylation[J].J Biol Chem,2011, 286(10):7983-7989.
[9]	BICOCCA V T,ORMSBY T,ADHVARYU K K,et al.ASH1-catalyzed H3K36 methylation drives gene repression and marks H3K27me2/3-competent chromatin[J].eLife,2018,7:e41497.
[10]	KLOCKO A D,UESAKA M,ORMSBY T,et al.Nucleosome positioning by an evolutionarily conserved chromatin remodeler prevents aberrant DNA methylation in Neurospora[J].Genetics,2019,211(2):563-578.
[11]	JAMIESON K,WILES E T,MCNAUGHT K J,et al.Loss of HP1 causes depletion of H3K27me3 from facultative heterochromatin and gain of H3K27me2 at constitutive heterochromatin[J].Genome Res,2016,26(1):97-107.
[12]	CASTIGLIONI I,CACCIA R,GARCIA-MANTEIGA J M,et al.The Trithorax protein Ash1L promotes myoblast fusion by activating Cdon expression[J].Nat Commun,2018,9(1):5026.
[13]	NAKAMURA T,BLECHMAN J,TADA S,et al.huASH1 protein,a putative transcription factor encoded by a human homologue of the Drosophila ash1 gene,localizes to both nuclei and cell-cell tight junctions[J].Proc Natl Acad Sci U S A,2000,97(13):7284-7289.
[14]	YIN B,HUI T Q,YU F Y,et al.ASH1L suppresses matrix metalloproteinase through mitogen-activated protein kinase signaling pathway in pulpitis[J].J Endod,2017,43(2):306-314.e2.
[15]	SUN B Y,TU J B,LIANG Q,et al.Expression of mammalian ASH1 and ASH4 in Drosophila reveals opposing functional roles in neurogenesis[J].Gene,2019,688:132-139.
[16]	PERUGORRIA M J,WILSON C L,ZEYBEL M,et al.Histone methyltransferase ASH1 orchestrates fibrogenic gene transcription during myofibroblast transdifferentiation[J].Hepatology,2012,56(3):1129-1139.
[17]	HUANG J H,MENG Y K,LIU Y,et al.MicroRNA-320a regulates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting HOXA10[J].Cell Physiol Biochem,2016,38(1):40-48.
[18]	ZHANG W,CHEN E,CHEN M,et al.IGFBP7 regulates the osteogenic differentiation of bone marrow-derived mesenchymal stem cells via Wnt/β-catenin signaling pathway[J].FASEB J,2018,32(4):2280-2291.
[19]	XIA M,LIU J,LIU S X,et al.Ash1l and lnc-Smad3 coordinate Smad3 locus accessibility to modulate iTreg polarization and T cell autoimmunity[J].Nat Commun,2017,8:15818.
[20]	LI G,YE Z S,SHI C,et al.The histone methyltransferase Ash1l is required for epidermal homeostasis in mice[J].Sci Rep,2017,7:45401.
[21]	BRINKMEIER M L,GEISTER K A,JONES M,et al.The histone methyltransferase gene absent,small,or homeotic discs-1 like is required for normal Hox gene expression and fertility in mice[J].Biol Reprod,2015,93(5):121.
[22]	JONES M,CHASE J,BRINKMEIER M,et al.Ash1l controls quiescence and self-renewal potential in hematopoietic stem cells[J].J Clin Invest,2015,125(5):2007-2020.
[23]	BADR R,HASHEMI M,JAVADI G,et al.Assessment of global ischemic/reperfusion and Tacrolimus administration on CA1 region of hippocampus:gene expression profiles of BAX and BCL2 genes[J].Bratisl Med J,2016,117(6):358-362.
[24]	SCHIAVONI V S,DA SILVA J P,LIZARTE NETO F S,et al.Morphological and immunohistochemical analysis of proteins CASPASE 3 and XIAP in rats subjected to cerebral ischemia and chronic alcoholism[J].Acta Cir Bras,2018,33(8):652-663.
[25]	WANG X L,LI C M,WANG Y R,et al.UFL1 alle-viates LPS-induced apoptosis by regulating the NF-κB signaling pathway in bovine ovarian granulosa cells[J].Biomolecules,2020,10(2):260.
[26]	YIN B,YU F Y,WANG C L,et al.Epigenetic control of mesenchymal stem cell fate decision via histone methyltransferase Ash1l[J].Stem Cells,2019,37(1):115-127.
[27]	TANAKA Y,KATAGIRI Z,KAWAHASHI K,et al.Trithorax-group protein ASH1 methylates histone H3 lysine 36[J].Gene,2007, 397(1-2):161-168.
[28]	ZHU L,LI Q,WONG S H K,et al.ASH1L links histone H3 lysine 36 dimethylation to MLL leukemia[J].Cancer Discov,2016, 6(7):770-783.
[29]	WAGNER E J,CARPENTER P B.Understanding the language of Lys36 methylation at histone H3[J].Nat Rev Mol Cell Biol,2012, 13(2):115-126.
[30]	DORIGHI K M,TAMKUN J W.The trithorax group proteins Kismet and ASH1 promote H3K36 dimethylation to counteract Polycomb group repression in Drosophila[J].Development,2013,140(20):4182-4192.
[31]	LU C,JAIN S U,HOELPER D,et al.Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape[J].Science,2016,352(6287):844-849.
[32]	DORAFSHAN E,KAHN T G,GLOTOV A,et al.Ash1 counteracts Polycomb repression independent of histone H3 lysine 36 methylation[J].EMBO Rep,2019,20(4):e46762.
[33]	KLYMENKO T,MÜLLER J.The histone methyltransferases Trithorax and Ash1 prevent transcriptional silencing by Polycomb group proteins[J].EMBO Rep,2004,5(4):373-377.
[34]	DORAFSHAN E,KAHN T G,GLOTOV A,et al.Genetic dissection reveals the role of Ash1 domains in counteracting Polycomb repression[J].G3(Bethesda),2019,9(11):3801-3812.
[35]	HOLOCH D,MARGUERON R.Mechanisms regulating PRC2 recruitment and enzymatic activity[J].Trends Biochem Sci, 2017, 42(7):531-542.
[36]	STREUBEL G,WATSON A,JAMMULA S G,et al.The H3K36me2 methyltransferase Nsd1 demarcates PRC2-mediated H3K27me2 and H3K27me3 domains in embryonic stem cells[J].Mol Cell,2018,70(2):371-379.e5.
[37]	WANG L J,JOSHI P,MILLER E L,et al.A role for monomethylation of histone H3-K27 in gene activity in Drosophila[J]. Genetics,2018,208(3):1023-1036.
[38]	CAO R,WANG L J,WANG H B,et al.Role of histone H3 lysine 27 methylation in polycomb-group silencing[J]. Science, 2002,298(5595):1039-1043.
[39]	FEI Q,YANG X Q,JIANG H,et al.SETDB1 modulates PRC2 activity at developmental genes indepen-dently of H3K9 trimethylation in mouse ES cells[J].Genome Res,2015,25(9):1325-1335.
[40]	SCHMITGES F W,PRUSTY A B,FATY M,et al.Histone methylation by PRC2 is inhibited by active chromatin marks[J].Mol Cell,2011,42(3):330-341.
[41]	HUANG C,YANG F,ZHANG Z Q,et al.Mrg15 stimulates Ash1 H3K36 methyltransferase activity and facilitates Ash1 Trithorax group protein function in Drosophila[J].Nat Commun,2017,8:1649.
[42]	SCHMÄHLING S,MEILER A,LEE Y,et al.Regulation and function of H3K36 di-methylation by the trithorax-group protein complex AMC[J].Development,2018,145(7):dev163808.

Expression and Function of ASH1L Methyltransferase in Bovine Cumulus Cells

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 42

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	CHEN Songbiao, LIU Feifei, SHANG Ke, YU Zuhua, HE Lei, WEI Ying, CHEN Jian, ZHANG Chunjie, CHENG Xiangchao, DING Ke. Molecular Mechanism of the “Battle” between Virus Infection and Host Antiviral Immunity-Apoptosis, Necroptosis and Pyroptosis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 59-70.
[2]	DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.
[3]	SONG Meijun, HAO Kexing, HAI Siyu, CHEN Yan, WANG Jing, HU Guangdong. Effects of SRIF-14 on Proliferation and Apoptosis of Endometrial Epithelial Cells in Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3325-3334.
[4]	XU Xi, YANG Baigao, ZHANG Hang, FENG Xiaoyi, HAO Haisheng, DU Weihua, ZHU Huabin, ZHANG Peipei, ZHAO Xueming. Effects of NMN on Lipid Droplet Content and Cryopreservation Effect of Bovine Oocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3348-3357.
[5]	WANG Wanjie, CHEN Nanzhu, ZOU Huiying, ZHOU Xinyi, HAO Haisheng, PANG Yunwei, ZHU Huabin, ZHAO Xueming, YU Dawei, DU Weihua. Effects of Histone Methyltransferase ASH1L Overexpression on Proliferation and Apoptosis of Bovine Cumulus Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3358-3368.
[6]	SHEN Tong, WANG Mengjie, WU Hua, LI Jinming, WANG Shuo, WU Xiaoqing, CHEN Xinlei, XING Qianwen, LIU Bo. Effect of Lycium ruthenicum Murray Anthocyanin on Hypoxia-induced Apoptosis in H9c2 Rat Cardiomyocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3490-3499.
[7]	AN Qi, YU Jialin, WU Xiaoling, DENG Guangcun. Regulation of BCG-induced Apoptosis in Murine Macrophages by Glutamine [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3054-3063.
[8]	ZHAO Yangfei, YU Yanghuan, WANG Jinming, ZHANG Jianhai, SUN Zilong, NIU Ruiyan, WANG Jundong. Effects of IL-17A Knockout on Fluoride-Induced Hepatic Inflammation and Hepatocyte Apoptosis [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3108-3117.
[9]	ZHAO Donghao, YUAN Meng, MA Kaiteng, DUAN Zhuo, ZHU Yixin, TANG Fang, HAN Keguang, HUO Nairui. Chelating Role of Sheep Bone Collagen Peptide to Cadmium and Its Protection Role against Liver Injuries Induced by Cadmium in Chickens [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2641-2652.
[10]	ZHU Junru, WEI Wei, PEI Dangshuai, ZHANG Fenque, DUAN Yu, GUO Yongfeng, JIANG Yue, XIA Shuli, HAN Jing, HOU Jinxing, AN Xiaopeng. Effects of PDCD4 on the Apoptosis of Dairy Goat Mammary Epithelial Cells and the Synthesis of β-casein and TG [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1429-1440.
[11]	DAN Yixin, YANG Lu, XIANG Hua, ZHANG Huanrong, REN Yupeng, YANG Falong, HE Honghong, ZHU Jiangjiang. Effects of BIRC5 on the Cycle and Apoptosis of Goat Testis Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1511-1524.
[12]	YANG Heng, LI Licai, FU Lin, XU Huihao, ZHANG Dezhi, LI Qianyong. Effect of Prostaglandin D₂ on Endocrine Function and Expression of Apoptosis-related Genes in Goat Luteal Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1652-1663.
[13]	REN Xiaoli, FAN Yuying, HUANGFU Heping, LIU Yun, SHI Dongmei. Effect of GSK126 on Epithelial-mesenchymal Transition of Canine Mammary Tumor Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1721-1729.
[14]	CHEN Yongping, KOU Yuhong, JIAO Wenjing, HOU Xiaoyu, FAN Honggang. Effect of Coenzyme Q10 on LPS-induced Acute Lung Injury in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1730-1741.
[15]	HU Yamei, SONG Xiangrong, HUANG Liang, ZHANG Lutong, GAO Lei, PANG Weijun, YANG Gongshe, CHU Guiyan. FGF21 Enhances Mitochondrial Function and Inhibits Apoptosis of Porcine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1034-1045.