搜索

Chromatin Fractionation Assay in Fission Yeast
裂殖酵母的染色质分离实验   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The protein recruitment onto chromatin is a critical process for DNA metabolism, including DNA replication, DNA repair and DNA recombination. Especially DNA modification enzymes and checkpoint proteins are loaded onto DNA damage sites in a context-dependent manner. In our recent study (Kunoh and Habu, 2014), the chromatin association of Pcf1, a large subunit of Chromatin Assembly Factor-1 (CAF-1), was monitored after exposure of cells to hydroxyurea which slowed down the DNA replication. Results of the chromatin fractionation assay provided evidence that Pcf1 was recruited to chromatin upon DNA replication stress. A similar procedure enabled to reveal the chromatin association of Orp1, Mcm proteins, and Swi6 (Sadaie et al., 2008; Ogawa et al., 1999). This assay allows us to fractionate chromatin-bound and -unbound proteins from living cells. The following immunoblot of the respective fractions provides the information concerning the chromatin binding status of our target proteins.

Keywords: Chromatin(染色质), Fractionation(fractionation), Yeast(酵母), Schizosaccharomyces pombe(粟酒裂殖酵母), Pcf1(包括车灯)

Materials and Reagents

  1. Yeast strain (Schizosaccharomyces pombe)
  2. Flask (IWAKI PUMPS, catalog number: 4980FK500 )
  3. Conical tube (BD Biosciences, Falcon®, catalog number: 2070 )
  4. 1.5 ml microcentrifuge tube (Eppendorf, catalog number: 022364111 )
  5. Lysing enzymes from Trichoderma harzianum (Sigma-Aldrih, catalog number: L1412 )
  6. Zymolyase 100T (Seikagaku Corporation, catalog number: 120493-1 )
  7. Complete Mini (Roche Diagnostics, catalog number: 11836153001 )
  8. Anti-GFP antibody (Roche Diagnostics, catalog number: 11814460001 )
  9. Anti-histone H3 antibody (Millipore, Upstate Biotechnology, catalog number: 05-499 )
  10. Anti-alpha-tubulin antibody (generously provided by Dr. A. Baines)
    Note: Commercially available antibodies against alpha-tubulin (such as Abcam, catalog number: ab6161 ) can be used.
  11. Goat HRP conjugated-anti-mouse antibody (Life Technologies, Biosource, catalog number: A10551 )
  12. YES liquid medium (see Recipes)
  13. STOP buffer (see Recipes)
  14. PEMS buffer (see Recipes)
  15. 2x HBS buffer (see Recipes)
  16. Lysis buffer (see Recipes)
  17. 2x Laemmli protein sample buffer (see Recipes)

Equipment

  1. Air- (Tykyo Rikakikai, Eyela, model: FMC-1000 ) or water bath- (Taitec, model: MM-10 ) incubator shaker
  2. Centrifuges equipped with 50 ml tubes (Tomy Digital Biology, model: AX-501 ) and 1.5 ml microtubes (Tomy Digital Biology, model: MX-107 )
  3. Heat block (TAITEC, model: DTU-1BN) or water bath (Taitec, model: EXN-B )
  4. Light microscope (Nikon Corporation, model: Eclipse E200 )
  5. Electrophoresis apparatus (Bio-Rad Laboratories, catalog numbers: 165-8002JA and 164-5052 )
  6. Transfer unit (Bio-Rad Laboratories, catalog numbers: 170-3930JA and 170-3935JA )

Procedure

  1. Culture fission yeast cells in 200 ml of YES medium in a 500 ml flask at 26 °C.
    Note: Stop culturing during the mid-log phase (OD595 = ~1.0).
  2. Prepare approximately 2.5 x 108 cells by dilution of inoculum to 25 ml at OD595 = 0.5.
  3. Harvest the cells by centrifugation at 400 x g for 5 min at 4 °C.
  4. Resuspend the cell pellet in 1 ml of ice-cold STOP buffer and transfer the cell suspension to a 1.5 ml microtube.
  5. Centrifuge the cell suspension at 400 x g for 5 min at 4 °C and pour out the supernatant (STOP buffer) by aspiration.
  6. Place the microtube containing the cell pellet on ice for 5 min.
  7. Resuspend the cell pellet in 1 ml of PEMS buffer dissolving 1 mg/ml lysing enzymes and 1 mg/ml Zymolyase 100T.
  8. Incubate the cell suspension for 20 min at 37 °C for making spheroplasts which readily burst for enabling to fractionate chromatin-bound and -unbound proteins at the later step.
    Note: To test whether these lysing enzymes work adequately, take a small volume of the cell suspension, add the equal volume of 10% SDS, and then monitor yeast lysis progression by light microscopy. If lysing is successful, almost all cells (spheroplasts) are expected to burst in the presence of SDS.
  9. Centrifuge the spheroplast suspension at 400 x g for 5 min at 4 °C. Pour out the supernatant (PEMS buffer) carefully by aspiration.
  10. Wash the spheroplast pellet twice by resuspending in 1 ml of 1.2 M sorbitol to avoid bursting of spheroplasts, followed by centrifugation at 400 x g for 5 min at 4 °C.
  11. Lyse the spheroplasts by resuspending in 0.5 ml of ice-cold lysis buffer, followed by incubation on ice for 5 min.
  12. Take 1/10 aliquot of the resulting lysate and save it as the whole cell extract (optional).
    Note: To check the protein expression of our interest, we recommend an immunoblot of the whole cell extract with the other samples.
  13. Centrifuge the rest of the lysate at 22,000 x g for 15 min at 4 °C.
  14. Save the supernatant as the chromatin-unbound fraction.
  15. Wash the pellets twice by resuspending in 1 ml of lysis buffer, followed by centrifugation at 22,000 x g for 5 min at 4 °C.
  16. Resuspend the pellet in 0.45 ml of lysis buffer and save it as the chromatin-bound fraction.
  17. Add an equal volume of 2x Laemmli protein sample buffer to all of the saved fractions.
  18. Apply an equal volume of these fractions to the 8% SDS-PAGE gel and immunoblot using antibodies against histone H3 (1/5,000 dilution), alpha-tubulin (1/5,000 dilution) and specific protein(s).
    Note: We detected the chromatin association of Pcf1-GFP protein by using the anti-GFP antibody (1/1,000 dilution). The secondary antibody was diluted at 1/10,000.

Representative data



Figure 1. The cdc10-M17 mutant cells were grown in YES medium at 26 °C, synchronized to G1 phase by incubation for 4 h at 36 °C, and returned to 26 °C. Released cells into S phase were harvested at indicated time(s) and subjected to chromatin fractionation assay to monitor the chromatin association of Pcf1-GFP.

Notes

  1. The proper performance of the assay can be easily confirmed by immunoblots using anti-histone H3 and anti-alpha-tubulin antibodies, since histone H3 and alpha-tubulin are fractionated to the chromatin-bound and -unbound fractions, respectively. In some cases as shown in Figure 1, alpha-tubulin was fractionated into the chromatin-bound fraction. This contamination was probably due to low efficiency of cell lysis or insufficient washout of the chromatin-unbound proteins. Therefore, we recommend the reader(s) to check the former possibility by monitoring yeast lysis progression in the presence of SDS and adjusting cell number, concentration of lysing enzymes, and/or reaction time, if necessary. For the latter possibility, additional wash of the pellets should be done to remove the chromatin-unbound proteins completely.

Recipes

  1. YES liquid medium
    5 g/L Bacto yeast extract
    30 g/L glucose
    225 mg/L adenine, histidine, leucine, uracil and lysine hydrochloride
  2. STOP buffer
    150 mM NaCl
    50 mM NaF
    10 mM EDTA
    1 mM NaN3
    pH 8.0
  3. PEMS buffer
    100 mM PIPES
    50 mM EDTA
    10 mM MgSO4
    1.2 M sorbitol
    pH 6.9
  4. 2x HBS buffer
    50 mM MOPS
    120 mM beta-glycerophosphate
    30 mM MgCl2
    30 mM EGTA
    30 mM p-nitrophenylphosphate
    0.2 mM Na3VO4
    pH 7.2
  5. Lysis buffer
    2x HBS buffer 5 ml
    10 % Triton X-100 0.5 ml
    2 M sorbitol 2 ml
    100 mM dithiothreitol (DTT) 0.1 ml
    0.1 mM phenylmethanesulfonyl fluoride (PMSF) 0.1 ml
    Complete mini 1 tablet
    Fill up to 10 ml with distilled water
  6. 2x Laemmli protein sample buffer
    0.1 M Tris-HCl
    4 % sodium dodecyl sulfate (SDS)
    20 % glycerol
    0.2 % bromophenolblue (BPB)
    20 % 2-mercaptoethanol (2-ME)
    pH 6.8

Acknowledgments

This protocol was described in our previous report (Kunoh and Habu, 2014). We express our sincere appreciation to Dr. Mahito Sadaie (Graduate School of Biostudies, Kyoto University) for his technical advice on this protocol. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to T.H. and JST-CREST to Professor Jun Takada, Okayama University.

References

  1. Kunoh, T. and Habu, T. (2014). Pcf1, a large subunit of CAF-1, required for maintenance of checkpoint kinase Cds1 activity. Springerplus 3: 30.
  2. Ogawa, Y., Takahashi, T. and Masukata, H. (1999). Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins. Mol Cell Biol 19(10): 7228-7236.
  3. Sadaie, M., Kawaguchi, R., Ohtani, Y., Arisaka, F., Tanaka, K., Shirahige, K. and Nakayama, J.. (2008). Balance between distinct HP1 family proteins controls heterochromatin assembly in fission yeast. Mol Cell Biol  28(23): 6973-6988.

简介

染色质上的蛋白质募集是DNA代谢的关键过程,包括DNA复制,DNA修复和DNA重组。特别是DNA修饰酶和检查点蛋白以上下文依赖性方式装载到DNA损伤位点。在我们最近的研究中(Kunoh和Habu,2014),在将细胞暴露于羟基脲(其减慢DNA复制)后,监测Pcf1的染色质缔合,其为染色质装配因子-1(CAF-1)的大亚基。染色质分馏测定的结果提供了Pcf1在DNA复制应激时募集到染色质的证据。类似的程序能够揭示Orp1,Mcm蛋白和Swi6的染色质缔合(Sadaie等人,2008; Ogawa等人,1999)。该测定允许我们从活细胞分离染色质结合和非结合蛋白。以下各部分的免疫印迹提供了关于我们的靶蛋白的染色质结合状态的信息。

关键字:染色质, fractionation, 酵母, 粟酒裂殖酵母, 包括车灯

材料和试剂

  1. 酵母菌株(粟酒裂殖酵母)
  2. 烧瓶(IWAKI PUMPS,目录号:4980FK500)
  3. 锥形管(BD Biosciences,Falcon ,目录号:2070)
  4. 1.5ml微量离心管(Eppendorf,目录号:022364111)
  5. 来自哈茨木霉(Sigma-Aldrih,目录号:L1412)的裂解酶
  6. Zymolyase 100T(Seikagaku Corporation,目录号:120493-1)
  7. Complete Mini(Roche Diagnostics,目录号:11836153001)
  8. 抗GFP抗体(Roche Diagnostics,目录号:11814460001)
  9. 抗组蛋白H3抗体(Millipore,Upstate Biotechnology,目录号:05-499)
  10. 抗-α-微管蛋白抗体(由Dr.Baines慷ously提供)
    注意:可以使用市售的抗α-微管蛋白的抗体(如Abcam,目录号:ab6161)。
  11. 山羊HRP缀合的抗小鼠抗体(Life Technologies,Biosource,目录号:A10551)
  12. YES液体培养基(见配方)
  13. STOP缓冲区(参见配方)
  14. PEMS缓冲区(参见配方)
  15. 2x HBS缓冲区(见配方)
  16. 裂解缓冲液(见配方)
  17. 2x Laemmli蛋白质样品缓冲液(参见配方)

设备

  1. 空气 - (Tykyo Rikakikai,Eyela,型号:FMC-1000)或水浴 - (Taitec,型号:MM-10)
  2. 装备有50ml管(Tomy Digital Biology,型号:AX-501)和1.5ml微管(Tomy Digital Biology,型号:MX-107)的离心机
  3. 加热块(TAITEC,型号:DTU-1BN)或水浴(Taitec,型号:EXN-B)
  4. 光学显微镜(Nikon Corporation,型号:Eclipse E200)
  5. 电泳装置(Bio-Rad Laboratories,目录号:165-8002JA和164-5052)
  6. 转移单位(Bio-Rad Laboratories,目录号:170-3930JA和170-3935JA)

程序

  1. 文化裂殖酵母细胞在200ml的YES培养基中在500ml烧瓶中在26℃。
    注意:在对数中期(OD 595 =〜1.0)
  2. 通过在OD 595 = 0.5时将接种物稀释至25ml制备约2.5×10 8个细胞。
  3. 通过在4℃下以400×g离心5分钟收获细胞。
  4. 重悬细胞沉淀在1毫升冰冷的STOP缓冲液,并将细胞悬液转移到1.5毫升微管。
  5. 在4℃下将细胞悬浮液以400×g离心5分钟,并通过抽吸倒出上清液(STOP缓冲液)。
  6. 将含有细胞沉淀的微管置于冰上5分钟
  7. 重悬细胞沉淀在1毫升PEMS缓冲溶解1毫克/毫升裂解酶和1毫克/毫升Zymolyase 100T。
  8. 在37℃下孵育细胞悬液20分钟,以制备容易爆裂的原生质球,以便在后面的步骤分解染色质结合和结合蛋白。
    注意:为了测试这些裂解酶是否起作用,取少量细胞悬液,加入等体积的10%SDS,然后通过光学显微镜监测酵母裂解进展。如果裂解成功,几乎所有的细胞(原生质球)在SDS的存在下爆发。
  9. 在4℃下将原生质球悬浮液以400×g离心5分钟。通过抽吸小心地倒出上清液(PEMS缓冲液)
  10. 通过重悬于1ml的1.2M山梨醇中来洗涤原生质球沉淀两次以避免原生质球破裂,然后在4℃下以400×g离心5分钟。
  11. 通过重悬于0.5ml冰冷的裂解缓冲液中,然后在冰上孵育5分钟来裂解原生质球。
  12. 取1/10等分的所得溶胞产物,并保存为全细胞提取物(可选) 注意:为了检查我们感兴趣的蛋白质表达,我们建议全细胞提取物与其他样品的免疫印迹。
  13. 在4℃下以22,000×g离心其余的裂解物15分钟。
  14. 保存上清液作为染色质未绑定部分。
  15. 通过重悬于1ml裂解缓冲液中,随后在4℃下以22,000×g离心5分钟来洗涤沉淀两次。
  16. 将沉淀重悬于0.45 ml裂解缓冲液中,并保存为染色质结合级分
  17. 向所有保存的馏分中加入等体积的2x Laemmli蛋白样品缓冲液。
  18. 将等体积的这些级分应用于8%SDS-PAGE凝胶和使用抗组蛋白H3(1/5,000稀释),α-微管蛋白(1/5,000稀释)和特异性蛋白的抗体的免疫印迹。 注意:我们通过使用抗GFP抗体(1/1,000稀释)检测到Pcf1-GFP蛋白的染色质缔合。第二抗体以1/10,000稀释。

代表数据



图1. cdc10-M17突变体细胞在26℃下在YES培养基中生长,通过在36℃下孵育4小时与G1期同步,并返回到26℃。 在指定时间收集释放的细胞至S期,并进行染色质分级分析以监测Pcf1-GFP的染色质缔合。

笔记

  1. 通过使用抗组蛋白H3和抗-α-微管蛋白抗体的免疫印迹可以容易地证实测定的适当性能,因为组蛋白H3和α-微管蛋白分别分级分离为染色质结合部分和非结合部分。在一些情况下,如图1所示,将α-微管蛋白分馏成染色质结合级分。这种污染可能是由于细胞裂解的效率低或染色质未结合的蛋白质的洗出不足。因此,我们建议读者通过在SDS存在下监测酵母裂解进展并调节细胞数,裂解酶的浓度和/或反应时间(如果必要)来检查前一种可能性。对于后一种可能性,应该进行额外的颗粒洗涤以完全去除染色质未结合的蛋白。

食谱

  1. 是液体介质
    5g/L Bacto酵母提取物
    30g/L葡萄糖 225 mg/L腺嘌呤,组氨酸,亮氨酸,尿嘧啶和赖氨酸盐酸盐
  2. STOP缓冲区
    150mM NaCl 50mM NaF 10 mM EDTA
    1mM NaN 3
    pH 8.0
  3. PEMS缓冲区
    100 mM PIPES
    50mM EDTA
    10mM MgSO 4 1.2M山梨醇 pH 6.9
  4. 2x HBS缓冲区
    50 mM MOPS
    120 mMβ-甘油磷酸盐 30mM MgCl 2/v/v 30mM EGTA
    30mM对硝基苯基磷酸盐 0.2mM Na 3+ VO 4子/ pH 7.2
  5. 裂解缓冲液
    2x HBS缓冲液5ml ml / 10%Triton X-100 0.5ml
    2 M山梨醇2ml
    100mM二硫苏糖醇(DTT)0.1ml
    0.1mM苯甲磺酰氟(PMSF)0.1ml
    完成mini 1平板电脑
    用蒸馏水
    填充至10ml
  6. 2x Laemmli蛋白样品缓冲液
    0.1M Tris-HCl
    4%十二烷基硫酸钠(SDS)
    20%甘油 0.2%溴酚蓝(BPB)
    20%2-巯基乙醇(2-ME) pH 6.8

致谢

这个协议在我们前一份报告中描述(Kunoh和Habu,2014)。 我们衷心感谢京都大学生物科学研究生院教授Mahito Sadaie对本协议的技术建议。 这项工作得到了日本教育,文化,体育,科学和技术部给T.H.的资助。 和冈山大学教授Jun Takada教授的JST-CREST。

参考文献

  1. Kunoh,T。和Habu,T。(2014)。 Pcf1,CAF-1的大亚基,需要维持检查点激酶Cds1活性。 a> Springerplus 3:30.
  2. Ogawa,Y.,Takahashi,T.and Masukata,H。(1999)。 裂殖酵母Orp1和Mcm6蛋白与染色体复制起点的关联 Mol Cell Biol 19(10):7228-7236
  3. Sadaie,M.,Kawaguchi,R.,Ohtani,Y.,Arisaka,F.,Tanaka,K.,Shirahige,K.and Nakayama,J。(2008)。 不同HP1家族蛋白之间的平衡控制裂变酵母中的异染色质装配。 Mol Cell Biol  28(23):6973-6988
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kunoh, T. and Habu, T. (2014). Chromatin Fractionation Assay in Fission Yeast. Bio-protocol 4(14): e1185. DOI: 10.21769/BioProtoc.1185.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。