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Biologie

通过荧光共振能量转移研究组蛋白修饰酶与体内转录因子之间的相互作用

Published: October 14, 2022
doi:
10.3791/64656
, ,
1Department of Biochemistry and Cell Biology,State University of New York

Summary

荧光共振能量转移（FRET）是一种用于检测活细胞中蛋白质相互作用的成像技术。在这里，提出了一种FRET协议来研究组蛋白修饰酶与转录因子的关联，转录因子将它们募集到靶启动子中，用于植物组织中基因表达的表观遗传调控。

Abstract

基因表达的表观遗传调控通常受到组蛋白修饰酶（HME）的影响，这些酶分别产生异色或正色组蛋白标记，用于转录抑制或激活。HME通过转录因子（TF）募集到其靶染色质中。因此，检测和表征 HME 和 TF 之间的直接相互作用对于更好地理解它们的功能和特异性至关重要。如果在 活 组织中体内进行，这些研究将更具生物学相关性。在这里，描述了一种协议，用于使用荧光共振能量转移（FRET）可视化植物组蛋白去泛素酶和植物转录因子之间的植物叶片相互作用，该协议允许检测彼此相距<10nm内的蛋白质分子之间的复合物。介绍了FRET技术的两种变体：SE-FRET（敏化发射）和AB-FRET（受体漂白），其中能量以非辐射方式从供体传递到受体，或者在受体光漂白时由供体辐射发射。SE-FRET和AB-FRET方法都可以很容易地适应，以发现 植物中其他蛋白质之间的其他相互作用。

Introduction

植物组蛋白去泛素酶通过组蛋白的翻译后修饰在控制基因表达中起重要作用，特别是通过擦除其单泛素化标记1。到目前为止，OTLD1是拟南芥2，3中为数不多的在分子水平上表征的植物组蛋白去泛素酶之一。OTLD1从H2B组蛋白分子中去除单泛素基团，从而促进靶基因染色质4，5中H3组蛋白的正色乙酰化和甲基化修饰的去除或添加。此外，OTLD1与另一种染色质修饰酶组蛋白赖氨酸去甲基化酶KDM1C相互作用，影响靶基因6，7的转录抑制。

大多数组蛋白修饰酶缺乏DNA结合能力，因此不能直接识别其靶基因。一种可能性是它们与DNA结合转录因子蛋白合作，这些转录因子蛋白结合这些酶并将它们引导到染色质靶标。具体而言，在植物中，已知几种主要的组蛋白修饰酶（即组蛋白甲基转移酶8，9，组蛋白乙酰转移酶10，组蛋白去甲基化酶11和Polycomb抑制复合物12，13，14）被转录因子招募。与这一想法一致，最近提出了一种将OTLD1募集到目标启动子的可能机制，该机制基于OTLD1与转录因子LSH1015的特定蛋白质 – 蛋白质相互作用。

LSH10属于植物ALOG（拟南芥LSH1和Oryza G1）蛋白家族，其作为中枢发育调节因子16，17，18，19，20，21，22。AOG蛋白家族的成员含有DNA结合基序23并表现出转录调节22，核定位19和同源二聚化24的能力，这一事实进一步支持了这些蛋白质（包括LSH10）可能在转录的表观遗传调控过程中充当特异性转录因子的观点。用于表征体内LSH10-OTLD1相互作用的主要实验技术之一是荧光共振能量转移（FRET）15。

FRET是一种成像技术，用于直接检测活细胞内彼此相距<10 nm以内的蛋白质之间的近距离相互作用 25.FRET方法26有两种主要变体：敏化发射（SE-FRET）（图1A）和受体漂白（AB-FRET）（图1B）。在SE-FRET中，相互作用的蛋白质 – 其中一个用供体荧光染料（例如，绿色荧光蛋白，GFP）标记，另一个用受体荧光染料（例如，单体红色荧光蛋白，mRFP27，28）标记 – 非辐射地将激发态能量从供体转移到受体。由于在此转移过程中不发射光子，因此会产生荧光信号，其辐射发射光谱与受体的辐射发射光谱相似。在AB-FRET中，当受体被光漂白永久灭活时，根据供体的辐射发射升高来检测和量化蛋白质相互作用，因此无法接收从供体转移的非辐射能量（图1）。重要的是，FRET荧光的亚细胞位置表明细胞中相互作用蛋白的定位。

在活组织中部署FRET并在检测这种相互作用的同时确定相互作用蛋白质的亚细胞定位的能力，使FRET成为体内蛋白质 – 蛋白质相互作用研究和初步表征的首选技术。一种可比的体内荧光成像方法，双分子荧光互补（BiFC）29，30，31，32，是一种很好的替代方法，尽管与FRET不同，BiFC可能由于自发荧光BiFC报告33的自发组装而产生假阳性，并且其数据的定量不太精确。

本文分享了实施SE-FRET和AB-FRET技术的成功经验，并提出了一种部署协议，以研究OTLD1和LSH10在植物细胞中的相互作用。

Protocol

本氏烟草、根癌农杆菌菌株EHA105或GV3101用于本研究。 1. FRET矢量构建 为供体/受体FRET对选择荧光标签。使用来自pPZP-RCS2A-dest-EGFP-N115，28 的EGFP（参见 材料表）生成供体载体。 使用来自pPZP-RCS2A-DEST-mRFP-N1的mRFP（参见 材料表）生成受体载体。 使?…

Representative Results

图2 说明了SE-FRET实验的典型结果，其中细胞核同时记录在三个通道中（即供体GFP，受体mRFP和SE-FRET）。这些数据用于生成以伪色标编码的SE-FRET效率图像。在这个尺度上，从蓝色到红色的转变对应于FRET效率的提高，FRET效率是蛋白质 – 蛋白质接近度从0%到100%的衡量标准。在该代表性实验中，SE-FRET信号记录在细胞核中，其LSH10和OTLD1共表达后的强度与mRFP-GFP表达后观察到的强度相?…

Discussion

该FRET协议简单且易于复制;它还需要最少的供应投资，并利用许多现代实验室的标准设备。具体而言，五个主要技术特征区分了该程序的多功能性。首先，FRET构建体是使用位点特异性重组生成的，与传统的限制性内切酶克隆相比，这是一种易于使用的克隆方法，可产生准确的结果并节省时间。其次， 本氏猪笼草 植物生长简单，产生相对大量的组织，大多数实验室都可以买到。第三，农业?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

V.C.实验室的工作得到了NIH（R35GM144059和R01GM50224），NSF（MCB1913165和IOS1758046）和BARD（IS-5276-20）对VC的资助。

Materials

Acetosyringone (3′,5′-Dimethoxy-4′-hydroxyacetophenone) Sigma-Aldrich #D134406-1G
Bacto Agar BD Biosciences #214010
Bacto trypton BD Biosciences #211705
Bacto yeast extract BD Biosciences #212750 
Confocal laser scanning microscope (CLSM) Zeiss LSM900 Any CLSM with similar capabilities is suitable
EHA105 VWR 104013-310 We use the stock in the Citovsky bacterial lab stock collection
Gateway BP Clonase II  Invitrogen #11789100
Gateway LR Clonase II Invitrogen #11791020
GV3101 VWR 104013-296 We use the stock in the Citovsky bacterial lab stock collection
ImageJ https://imagej.nih.gov/ij/download.html
MES Sigma-Aldrich #69889-10G
MgCl2 Sigma-Aldrich #63068-250G
NaCl Sigma-Aldrich #S5886-500G
Nicotiana benthamiana seeds Herbalistics Pty RA4 or LAB We use the stock in the Citovsky seed lab stock collection
pDONR207 Invitrogen #12213013
pPZP-RCS2A-DEST-EGFP-N1  N/A Refs. 15, 28
pPZP-RCS2A-DEST-mRFP-C1 N/A Generated based on the pPZP-RCS2A-DEST-EGFP-C1 construct (see refs. 15, 28)
pPZP-RCS2A-DEST-mRFP-N1  N/A Generated based on the pPZP-RCS2A-DEST-EGFP-N1 construct
Rifampicin Sigma-Aldrich #R7382-5G
Spectinomycin Sigma-Aldrich #S4014-5G
Syringes without needles BD 309659
Zen software for CLSM imaging Zeiss ZEN 3.0 version The software should be compatible with the CLSM used
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References

  1. March, E., Farrona, S. Plant deubiquitinases and their role in the control of gene expression through modification of histones. Frontiers in Plant Science. 8, 2274 (2018).
  2. Isono, E., Nagel, M. K. Deubiquitylating enzymes and their emerging role in plant biology. Frontiers in Plant Science. 5, 56 (2014).
  3. Feng, J., Shen, W. H. Dynamic regulation and function of histone monoubiquitination in plants. Frontiers in Plant Science. 5, 83 (2014).
  4. Keren, I., Citovsky, V. Activation of gene expression by histone deubiquitinase OTLD1. Epigenetics. 12 (7), 584-590 (2017).
  5. Keren, I., Citovsky, V. The histone deubiquitinase OTLD1 targets euchromatin to regulate plant growth. Science Signaling. 9 (459), 125 (2016).
  6. Krichevsky, A., Lacroix, B., Zaltsman, A., Citovsky, V. Involvement of KDM1C histone demethylase-OTLD1 otubain-like histone deubiquitinase complexes in plant gene repression. Proceedings of the National Academy of Sciences. 108 (27), 11157-11162 (2011).
  7. Keren, I., Lapidot, M., Citovsky, V. Coordinate activation of a target gene by KDM1C histone demethylase and OTLD1 histone deubiquitinase in Arabidopsis. Epigenetics. 14 (6), 602-610 (2019).
  8. Kim, S. Y., Michaels, S. D. SUPPRESSOR OF FRI 4 encodes a nuclear-localized protein that is required for delayed flowering in winter-annual Arabidopsis. Development. 133 (23), 4699-4707 (2006).
  9. Kim, S., Choi, K., Park, C., Hwang, H. J., Lee, I. SUPPRESSOR OF FRIGIDA4, encoding a C2H2-type zinc finger protein, represses flowering by transcriptional activation of Arabidopsis FLOWERING LOCUS C. The Plant Cell. 18 (11), 2985-2998 (2006).
  10. Sridhar, V. V., Surendrarao, A., Gonzalez, D., Conlan, R. S., Liu, Z. Transcriptional repression of target genes by LEUNIG and SEUSS, two interacting regulatory proteins for Arabidopsis flower development. Proceedings of the National Academy of Sciences. 101 (31), 11494-11499 (2004).
  11. Ning, Y. Q., et al. Two novel NAC transcription factors regulate gene expression and flowering time by associating with the histone demethylase JMJ14. Nucleic Acids Research. 43 (3), 1469-1484 (2015).
  12. Hecker, A., et al. The Arabidopsis GAGA-binding factor BASIC PENTACYSTEINE6 recruits the POLYCOMB-REPRESSIVE COMPLEX1 component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA motifs. Plant Physiology. 168 (3), 1013-1024 (2015).
  13. Xiao, J., et al. Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis. Nature Genetics. 49 (10), 1546-1552 (2017).
  14. Yuan, W., et al. A cis cold memory element and a trans epigenome reader mediate Polycomb silencing of FLC by vernalization in Arabidopsis. Nature Genetics. 48 (12), 1527-1534 (2016).
  15. Phan, M. S. V., Keren, I., Tran, P. T., Lapidot, M., Citovsky, V. Arabidopsis LSH10 transcription factor interacts with the co-repressor histone deubiquitinase OTLD1 to recruit it to the target promoters. bioRxiv. , (2022).
  16. Zhao, L., et al. Overexpression of LSH1, a member of an uncharacterised gene family, causes enhanced light regulation of seedling development. The Plant Journal. 37 (5), 694-706 (2004).
  17. Lei, Y., Su, S., He, L., Hu, X., Luo, D. A member of the ALOG gene family has a novel role in regulating nodulation in Lotus japonicus. Journal of Integrative Plant Biology. 61 (4), 463-477 (2019).
  18. MacAlister, C. A., et al. Synchronization of the flowering transition by the tomato TERMINATING FLOWER gene. Nature Genetics. 44 (12), 1393-1398 (2012).
  19. Takeda, S., et al. CUP-SHAPED COTYLEDON1 transcription factor activates the expression of LSH4 and LSH3, two members of the ALOG gene family, in shoot organ boundary cells. The Plant Journal. 66 (6), 1066-1077 (2011).
  20. Yan, D., et al. BEAK-SHAPED GRAIN 1/TRIANGULAR HULL 1, a DUF640 gene, is associated with grain shape, size and weight in rice. Science China Life Sciences. 56 (3), 275-283 (2013).
  21. Yoshida, A., et al. TAWAWA1, a regulator of rice inflorescence architecture, functions through the suppression of meristem phase transition. Proceedings of the National Academy of Sciences. 110 (2), 767-772 (2013).
  22. Yoshida, A., Suzaki, T., Tanaka, W., Hirano, H. Y. The homeotic gene long sterile lemma (G1) specifies sterile lemma identity in the rice spikelet. Proceedings of the National Academy of Sciences. 106 (47), 20103-20108 (2009).
  23. Iyer, L. M., Aravind, L. ALOG domains: provenance of plant homeotic and developmental regulators from the DNA-binding domain of a novel class of DIRS1-type retroposons. Biology Direct. 7, 39 (2012).
  24. Peng, P., et al. The rice TRIANGULAR HULL1 protein acts as a transcriptional repressor in regulating lateral development of spikelet. Scientific Reports. 7 (1), 13712 (2017).
  25. Day, R. N., Periasamy, A., Schaufele, F. Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. Methods. 25 (1), 4-18 (2001).
  26. Qian, J., Yao, B., Wu, C. Fluorescence resonance energy transfer detection methods: Sensitized emission and acceptor bleaching. Experimental and Therapeutic. 8 (5), 1375-1380 (2014).
  27. Campbell, R. E., et al. A monomeric red fluorescent protein. Proceedings of the National Academy of Sciences. 99 (12), 7877-7882 (2004).
  28. Tzfira, T., et al. pSAT vectors: a modular series of plasmids for fluorescent protein tagging and expression of multiple genes in plants. Plant Molecular Biology. 57 (4), 503-516 (2005).
  29. Bracha-Drori, K., et al. Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. The Plant Journal. 40 (3), 419-427 (2004).
  30. Citovsky, V., Gafni, Y., Tzfira, T. Localizing protein-protein interactions by bimolecular fluorescence complementation in planta. Methods. 45 (3), 196-206 (2008).
  31. Citovsky, V., et al. Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta. Journal of Molecular Biology. 362 (5), 1120-1131 (2006).
  32. Ohad, N., Shichrur, K., Yalovsky, S. The analysis of protein-protein Interactions in plants by bimolecular fluorescence complementation. Plant Physiology. 145 (4), 1090-1099 (2007).
  33. Gookin, T. E., Assmann, S. M. Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors. The Plant Journal. 80 (3), 553-567 (2014).
  34. Tran, P. T., Citovsky, V. Receptor-like kinase BAM1 facilitates early movement of the Tobacco mosaic virus. Communications Biology. 4 (1), 511 (2021).
  35. Walhout, A. J., et al. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods in Enzymology. 328, 575-592 (2000).
  36. Ashwini, M., Murugan, S. B., Balamurugan, S., Sathishkumar, R. Advances in molecular cloning. Biologie moléculaire. 50 (1), 1-6 (2016).
  37. Tzfira, T., et al. Transgenic Populus: a step-by-step protocol for its Agrobacterium-mediated transformation. Plant Molecular Biology Reporter. 15, 219-235 (1997).
  38. Wise, A. A., Liu, Z., Binns, A. N. Nucleic acid extraction from Agrobacterium strains. Methods in Molecular Biology. 343, 67-76 (2006).
  39. Bal, M., Zhang, J., Hernandez, C. C., Zaika, O., Shapiro, M. S. Ca2+/calmodulin disrupts AKAP79/150 interactions with KCNQ (M-Type) K+ channels. The Journal of Neuroscience. 30 (6), 2311-2323 (2010).
  40. Feige, J. N., Sage, D., Wahli, W., Desvergne, B., Gelman, L. PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs. Microscopy Research & Technique. 68 (1), 51-58 (2005).
  41. Bal, M., Zhang, J., Hernandez, C. C., Zaika, O., Shapiro, M. S. Ca2+/calmodulin disrupts AKAP79/150 interactions with KCNQ (M-Type) K+ channels. Journal of Neuroscience. 30 (6), 2311-2323 (2010).
  42. Taylor, N. J., Fauquet, C. M. Microparticle bombardment as a tool in plant science and agricultural biotechnology. DNA and Cell Biology. 21 (12), 963-977 (2002).
  43. Broussard, J. A., Green, K. J. Research techniques made simple: methodology and applications of Förster resonance energy transfer (FRET) microscopy. Journal of Investigative Dermatology. 137 (11), 185-191 (2017).
  44. Kudla, J., Bock, R. Lighting the way to protein-protein interactions: recommendations on best practices for bimolecular fluorescence complementation analyses. The Plant Cell. 28 (5), 1002-1008 (2016).
  45. Bartel, P., Chien, C. T., Sternglanz, R., Fields, S., Hartley, D. A. . Cellular Interactions in Development: A Practical Approach. , 153-179 (1993).
  46. Fields, S., Song, O. A novel genetic system to detect protein-protein interactions. Nature. 340 (6230), 245-246 (1989).
  47. Phizicky, E. M., Fields, S. Protein-protein interactions: methods for detection and analysis. Microbiological Reviews. 59 (1), 94-123 (1995).
  48. Fields, S. High-throughput two-hybrid analysis. The promise and the peril. The FEBS Journal. 272 (21), 5391-5399 (2005).
  49. Azad, T., Tashakor, A., Hosseinkhani, S. Split-luciferase complementary assay: applications, recent developments, and future perspectives. Analytical and Bioanalytical Chemistry. 406 (23), 5541-5560 (2014).
  50. Wang, L., Yu, G., Macho, A. P., Lozano-Durán, R. Split-luciferase complementation imaging assay to study protein-protein interactions in Nicotiana benthamiana. Bio-protocol. 11 (23), 4237 (2021).
  51. Grefen, C., Lalonde, S., Obrdlik, P. Split-ubiquitin system for identifying protein-protein interactions in membrane and full-length proteins. Current Protocols in Neuroscience. 41 (1), 5-27 (2007).
  52. Thaminy, S., Miller, J., Stagljar, I. The split-ubiquitin membrane-based yeast two-hybrid system. Methods in Molecular Biology. 261, 297-312 (2004).
  53. Lin, J. S., Lai, E. M. Protein-protein interactions: co-immunoprecipitation. Methods in Molecular Biology. 1615, 211-219 (2017).
  54. Jiang, Z., Yang, M., Cao, Q., Zhang, Y., Li, D. A powerful method for studying protein-protein interactions in plants: coimmunoprecipitation (co-IP) assay. Methods in Molecular Biology. 2400, 87-92 (2022).
  55. Magori, S., Citovsky, V. Agrobacterium counteracts host-induced degradation of its F-box protein effector. Science Signaling. 4 (195), (2011).

Tags

Keywords: FRETProtein-protein InteractionAgroinfiltrationNicotiana BenthamianaHistone Modifying EnzymesTranscription FactorsLiving CellFluorescence Resonance Energy Transfer
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Vo Phan, M. S., Tran, P. T., Citovsky, V. Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer. J. Vis. Exp. (188), e64656, doi:10.3791/64656 (2022).
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