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Histone Methyltransferase Assay in vitro
组蛋白甲基转移酶活性体外检测试验   

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Abstract

Histone methylation is an important epigenetic modification that plays important roles in plant development and growth. Histone methytransferases are the enzymes to establish histone methylation and here we describe a simple and effective protocol for detecting methyltransferase activity and specificity in vitro.

Keywords: Plant(计划), Histone H3 lysine 36(组蛋白H3赖氨酸36), Methyltransferase assay(甲基转移酶检测)

Materials and Reagents

  1. 14C labeled-methyl-adenosyl-L-methionine (SAMS) (Sigma-Aldrich)
  2. Purified recombinant histones (from E. coli)
  3. Histone H3 (Roche Diagnostics, catalog number: 1034758 )
  4. Core histones (Sigma-Aldrich, catalog number: H9250 )
  5. Mononuclosomes and Oligonucleosomes (see procedure)
  6. Coomassie brilliant blue R-250
  7. MiliQ water
  8. SDS-PAGE
  9. KCl
  10. MgCl2
  11. Sucrose
  12. ZnCl2
  13. β-mercaptoethanol
  14. MAB buffer (see Recipes)

Equipment

  1. Water bath

Procedure

  1. Substrate
    Purified recombinant Histones (Ding et al., 2007), histone H3, core histones, mononuclosome and oligonucleosome (from Hela cells or recombinant ones from E.coli).
    Note: The mononucleosomes and oligonucleosomes prepared from Hela cells were gifts from Yi Zhang (UNC Chapel Hill, USA). The recombinant oligonucleosomes from E. coli were gifts from Bing Zhu (NIBS, Beijing, China).

  2. Reaction
    A mixture containing 1-5 μl enzyme (such as SDG725), 5 μl 4x MAB buffer, 250 nCi 14C labeled SAM and 1 μg substrates to 20 μl volume, is kept at 37 °C for 1-2 h. The reaction is stopped by adding 5 μl 5x SDS-PAGE loading buffer and boiling at 100 °C for 5 min, and 5 μl samples will be analyzed by 15% SDS-PAGE gel electrophoresis and visualized by Coomassie brilliant blue R-250. The SDS-PAGE gel is dried and exposed to X-ray films or scanned by Typhoon (GE) (15% refers to the concentration of Acr/Bis. Different histones can be separated more clearly by 15% SDS-PAGE).

Recipes

  1. MAB buffer
    1x MAB buffer
    50 mM Tris-Cl (pH 8.5)
    20 mM KCl
    10 mM MgCl2
    250 mM Sucrose
    100 μM ZnCl2
    10 mM β-mercaptoethanol

Acknowledgments

The protocol was adapted from previously published papers (Rea et al., 2000; Ding et al., 2007).

References

  1. Ding, B., Zhu, Y., Gao J., Yu, Y., Cao, K. M., Shen W. H., Dong, A. (2007) Molecular characterization of three rice SET-domain proteins. Plant Sci 172:1072-1078.
  2. Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B. D., Sun, Z. W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C. P., Allis, C. D. and Jenuwein, T. (2000). Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406(6796): 593-599.
  3. Sui, P., Jin, J., Ye, S., Mu, C., Gao, J., Feng, H., Shen, W. H., Yu, Y. and Dong, A. (2012). H3K36 methylation is critical for brassinosteroid-regulated plant growth and development in rice. Plant J 70(2): 340-347.

简介

组蛋白甲基化是重要的表观遗传修饰,在植物发育和生长中起重要作用。 组蛋白甲基转移酶是建立组蛋白甲基化的酶,在此我们描述了用于检测体外甲基转移酶活性和特异性的简单而有效的方案。

关键字:计划, 组蛋白H3赖氨酸36, 甲基转移酶检测

材料和试剂

  1. 14 -S标记的甲基腺苷-L-甲硫氨酸(SAMS)(Sigma-Aldrich)
  2. 纯化的重组组蛋白(来自大肠杆菌)
  3. 组蛋白H3(Roche Diagnostics,目录号:1034758)
  4. 核心组蛋白(Sigma-Aldrich,目录号:H9250)
  5. 单核细胞和寡核苷酸(参见程序)
  6. 考马斯亮蓝R-250
  7. MiliQ水
  8. SDS-PAGE
  9. KCl
  10. MgCl 2
  11. 蔗糖
  12. ZnCl 2
  13. β-巯基乙醇
  14. MAB缓冲区(请参阅配方)

设备

  1. 水浴

程序

  1. 基板
    纯化的重组组蛋白(Ding等人,2007),组蛋白H3,核心组蛋白,单核细胞和寡核小体(来自Hela细胞或来自大肠杆菌的重组细胞)
    注意:由Hela细胞制备的单核小体和寡核小体是来自Yi Zhang(UNC Chapel Hill,USA)的礼物。来自大肠杆菌的重组寡核小体是来自Bing Zhu(NIBS,北京,中国)的礼物。

  2. 反应
    将含有1-5μl酶(例如SDG725),5μl4x MAB缓冲液,250nCi 14 C标记的SAM和1μg底物至20μl体积的混合物在37℃下保持1小时-2小时。通过加入5μl5×SDS-PAGE上样缓冲液并在100℃煮沸5分钟终止反应,5μl样品将通过15%SDS-PAGE凝胶电泳分析并通过考马斯亮蓝R-250显色。将SDS-PAGE凝胶干燥并暴露于X射线胶片或通过Typhoon(GE)扫描(15%是指Acr/Bis的浓度。通过15%SDS-PAGE可以更清楚地分离不同的组蛋白)。 />

食谱

  1. MAB缓冲区
    1x MAB缓冲区
    50mM Tris-Cl(pH8.5) 20 mM KCl
    10mM MgCl 2/
    250 mM蔗糖 100μMZnCl 2/
    10mMβ-巯基乙醇

致谢

该方案改编自先前发表的论文(Rea等人,2000; Ding等人,2007)。

参考文献

  1. Ding,B.,Zhu,Y.,Gao J.,Yu,Y.,Cao,KM,Shen WH,Dong,A.(2007)植物科学172: 1072-1078。
  2. Rea,S.,Eisenhaber,F.,O'Carroll,D.,Strahl,BD,Sun,ZW,Schmid,M.,Opravil,S.,Mechtler,K.,Ponting,CP,Allis,CD和Jenuwein, T.(2000)。 通过位点特异性组蛋白H3甲基转移酶调节染色质结构。 406(6796):593-599。
  3. Sui,P.,Jin,J.,Ye,S.,Mu,C.,Gao,J.,Feng,H.,Shen,W. H.,Yu,Y.and Dong, H3K36甲基化对于油菜素类固醇调节的植物在水稻中的生长和发育至关重要。 Plant J 70(2):340-347
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sui, P., Yu, Y. and Dong, A. (2012). Histone Methyltransferase Assay in vitro. Bio-protocol 2(24): e311. DOI: 10.21769/BioProtoc.311.
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