染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em>在体内</em
Summary
染色质免疫沉淀是一 个功能强大的技术的DNA结合蛋白拟南芥的体内位点的鉴定。此过程包括染色质交联和碎裂，免疫沉淀与针对感兴趣的蛋白的选择性抗体，和结合的DNA的定量PCR分析。我们描述了拟南芥植物优化的一个简单的ChIP实验。
Abstract
Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.
Introduction
近年来广泛的遗传，分子和基因组工具被开发在模型物种拟南芥。该技术促进了极大的进展，了解如何植物生长发育的调节。在使用拟南芥为模式研究的发展过程中，开花时间的基因控制已经被广泛分析。这些研究表明，植物开花响应于内源性线索非常精确的时间调制如激素和植物的年龄，并且还认为则同步开花时间随着季节1的自然循环环境信号诸如光周期和温度， 2。拟南芥突变体的分离和鉴定与改建开花的时候已经决定在公布的基因，调节开花时间响应内源性因素和环境因素的复杂网络。这些遗传电路集成在充当开花交换机的几个主基因水平和花芽分化的确切时间取决于促进开花，压制这项工作的花卉积分基因1,3上游活动的平衡。
确定了其在开花开始的控制作用的基因的功能特性，由最近使用基因组学方法的帮助下，已揭示转录调控中的开花时间的调制的中心作用。事实上，许多的开花编码转录的主控基因因子4。此外，一些染色质重塑蛋白复合物影响开花的主基因的表达。许多拟南芥突变体中分离得到的改变的开花时间的横空出世，携带突变基因编码的各种染色质修饰的。不同的染色质remodelers的引入华宏翻译后修饰Ë尾巴，重新定位相对于DNA核或组蛋白变体交换规范的组蛋白所必需的开花拟南芥5,6的适当调节。其中的一些染色质重塑活动催化沉积或除去的共价修饰，如乙酰化或甲基化的组蛋白的特定残基。这些组蛋白标记由该招募其他染色质重塑复合物，转录因子或转录机制的成分以调节开花的基因的转录活性专门效应特异性识别。
染色质免疫沉淀（ChIP）允许在体内的DNA结合位点的感兴趣的蛋白质（图1）的识别。这个过程利用了某些化学品的能力交联的蛋白质的DNA。得到的DNA蛋白质复合物可以通过使用特异性抗体来阿加然后免疫沉淀研究所转录因子，染色质结合蛋白，或特定变型和异源表位（通常被称为“标签”）附连到所选择的蛋白质。从这些免疫沉淀物进行纯化的DNA可以用作定量PCR（qPCR的）反应的模板，以评估所关注的特定序列的富集。以这种方式，转录因子的结合位点或在特定的基因组蛋白标记的分布可以建立7,8。此外，结合新一代测序（NGS），使大规模平行测序，芯片技术已经成为可能的转录因子结合位点，以及组蛋白修饰揭幕表观景观的全基因组的鉴定。此外，基因表达的同时分析可以监控转录调节如何结合或特定组蛋白标记的沉积相关的转录活性状况TY基因9。
利用拟南芥的ChIP协议允许评估的影响，多种转录调节因子有开花的主控基因的染色质组织，如何将这些结构变化影响基因表达5,6。以前的结果表明，拟南芥基因先期抽薹在短日照 （EBS）作为开花突变体中该基因节目开花和开花的FT的主基因的上调的加速度的阻遏。此外，丧失功能的突变的FT充分抑制EBS突变植物的早期开花表型，这表明FT需要EBS突变体的过早开花和EBS是必要的这个主开花10的基因的压制，11。EBS编码含有PHD-蛋白可以特异性结合组蛋白H3的二和三甲基在第Ë赖氨酸残基4（H3K4me2 / 3），这表明对EBS在FT 12的染色质介导的抑制的作用。使用该芯片的做法表明，拟南芥含有PHD蛋白EBS 10,11结合花卉积分FT基因的调控区，以抑制其表达12。通过使用芯片技术所获得的额外数据表明，该蛋白质是必需的，以保持在拟南芥发展初期低水平组蛋白乙酰化，活性转录的标志的，在这个主开花的基因的染色质。这些观察，连同遗传和基因表达数据，证明该拟南芥含有PHD-蛋白具有在开花时间的微调通过调制花香积分基因FT 12的表达的中心作用。这里提出的工作提供了优化的方法不仅对组蛋白的分析，但也可用于其他有用染色质相关蛋白质，和以更高的效率和降低的实验时间。此外，该报告说明了如何使用的ChIP协议提供了新的见解中染色质修饰改变和植物基因的转录状态之间的关系，以及如何将这些染色质介导的基因表达的控制影响的机制上开花拟南芥的发作。
Protocol
Representative Results
Discussion
此处所描述的沉淀协议是一个可再现的和强大的技术来分析蛋白质和特定的DNA序列之间的相互作用在体内在拟南芥植物。一个成功的鉴定结合位点的目的蛋白质需要适当的选择植物器官或发育阶段，其中相关的相互作用实际上是正在发生的。此外，重要的是，获得的植物材料的适当的固定和染色质的通过超声处理的最佳剪切。是对于所选择的蛋白质/标签的有效免疫沉淀的高度特异性抗体也…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge The Plant Cell for allowing the use of some data published in this journal to elaborate the representative results described here in Figure 4. This work was supported by the EU 7FP Marie Curie-Initial Training Network EpiTRAITS (Grant Agreement 316965), and by the Spanish Ministerio de Economìa y Competitividad (grants BIO2010-15589 and BIO2013-43098-R).
Materials
| MES | Sigma | M8250 | |
| MS (Murashige and Skoog Basal Salt Mixture) | Sigma | M5524 | |
| Formaldehyde 37% | Sigma | F8775-25 | Use under the fume hood |
| Protease inhibitor mix cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack | Roche | 5892791001 | |
| Bioruptor Standard sonication device | Diagenode | B01010002 (UCD200TO) | |
| Glycine | Sigma | 50046 | |
| QIAquick PCR Purification Kit | Qiagen | 28104 | |
| Dynabeads® magnetic beads coupled with protein A or protein G | Life Technologies | 10003D/10001D | Check manufacturer’s manual for antibody affinity |
| Miracloth | Merck Millipore | 475855 | |
| Triton™ X-100 Surfact-Amps™ Detergent Solution | Life Technologies | 85112 | |
| (mouse, rat, rabbit…)-IgG | Diagenode | C15400001, C15420001, C15410206 | |
| Magnetic rack – DynaMag™-2 | Life Technologies | 12321D | |
| H3K9/14ac polyclonal antibody – Premium | Diagenode | C15410200-10 | |
| Chelex® 100 Resin | Bio-Rad | 142-2832 | |
| Proteinase K | Life Technologies | 17916 | |
| Anti-Myc Tag Antibody, clone 4A6 | Millipore | 05-724 | |
| LightCycler® 480 SYBR Green I Master | Roche | 4707516001 |
Referências
- Andres, F., Coupland, G. The genetic basis of flowering responses to seasonal cues. Nat Rev Genet. 13 (9), 627-639 (2012).
- Capovilla, G., Schmid, M., Pose, D. Control of flowering by ambient temperature. J Exp Bot. 66 (1), 59-69 (2015).
- Jarillo, J. A., Piñeiro, M. Timing is everything in plant development. The central role of floral repressors. Plant Science. 181 (4), 364-378 (2011).
- Pajoro, A., et al. The (r)evolution of gene regulatory networks controlling Arabidopsis plant reproduction: a two-decade history. J Exp Bot. 65 (17), 4731-4745 (2014).
- He, Y. Chromatin regulation of flowering. Trends Plant Sci. 17 (9), 556-562 (2012).
- Jarillo, J. A., Pineiro, M., Cubas, P., Martinez-Zapater, J. M. Chromatin remodeling in plant development. International Journal of Developmental Biology. 53 (8-10), 1581-1596 (2009).
- Saleh, A., Alvarez-Venegas, R., Avramova, Z. An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc. 3 (6), 1018-1025 (2008).
- Haring, M., et al. Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods. 3, 11 (2007).
- Kaufmann, K., et al. Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP). Nat Protoc. 5 (3), 457-472 (2010).
- Gomez-Mena, C., et al. early bolting in short days: An Arabidopsis mutation that causes early flowering and partially suppresses the floral phenotype of leafy. Plant Cell. 13 (5), 1011-1024 (2001).
- Piñeiro, M., Gomez-Mena, C., Schaffer, R., Martinez-Zapater, J. M., Coupland, G. EARLY BOLTING IN SHORT DAYS is related to chromatin remodeling factors and regulates flowering in Arabidopsis by repressing FT. Plant Cell. 15 (7), 1552-1562 (2003).
- Lopez-Gonzalez, L., et al. Chromatin-dependent repression of the Arabidopsis floral integrator genes involves plant specific PHD-containing proteins. Plant Cell. 26 (10), 3922-3938 (2014).
- Sambrook, J., Fritsch, E. F., Maniatis, T. . Molecular Cloning: A Laboratory Manual. , (1989).
- Lorenz, T. C. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. J. Vis. Exp. , e3998 (2012).
- Wong, M. L., Medrano, J. F. Real-time PCR for mRNA quantitation. Biotechniques. 39 (1), 75-85 (2005).
- Dedon, P. C., Soults, J. A., Allis, C. D., Gorovsky, M. A. A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions. Anal Biochem. 197 (1), 83-90 (1991).
- Yu, C. W., et al. HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis. Plant Physiology. 156 (1), 173-184 (2011).
- Zhou, J., et al. Genome-wide profiling of histone H3 lysine 9 acetylation and dimethylation in Arabidopsis reveals correlation between multiple histone marks and gene expression. Plant Mol Biol. 72 (6), 585-595 (2010).
- Lafos, M., et al. Dynamic regulation of H3K27 trimethylation during Arabidopsis differentiation. PLoS Genet. 7 (4), e1002040 (2011).
- Barcala, M., Fenoll, C., Escobar, C. Laser microdissection of cells and isolation of high-quality RNA after cryosectioning. Methods Mol Biol. 883, 87-95 (2012).
- Carter, A. D., Bonyadi, R., Gifford, M. L. The use of fluorescence-activated cell sorting in studying plant development and environmental responses. Int J Dev Biol. 57 (6-8), 545-552 (2013).
- Deal, R. B., Henikoff, S. The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat Protoc. 6 (1), 56-68 (2011).
- Zeng, P. Y., Vakoc, C. R., Chen, Z. C., Blobel, G. A., Berger, S. L. In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation. Biotechniques. 41 (6), 694-698 (2006).
- Das, P. M., Ramachandran, K., vanWert, J., Singal, R. Chromatin immunoprecipitation assay. Biotechniques. 37 (6), 961-969 (2004).
- Kaufmann, K., et al. Target genes of the MADS transcription factor SEPALLATA3: integration of developmental and hormonal pathways in the Arabidopsis flower. PLoS Biol. 7 (4), e1000090 (2009).
