Visualization of the Genomic Loci That Are Bound by Specific Multiprotein Complexes by Bimolecular Fluorescence Complementation Analysis on Drosophila Polytene Chromosomes.
Visualization of the Genomic Loci That Are Bound by Specific Multiprotein Complexes by Bimolecular Fluorescence Complementation Analysis on Drosophila Polytene Chromosomes.
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DOI：
10.1016/bs.mie.2017.02.003
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发表时间：
2017
影响因子：
--
通讯作者：
Kerppola TK
中科院分区：
文献类型：
--
作者：
Deng H;Kerppola TK
我们已经开发了一种程序，可以可视化由染色质结合蛋白的特定组合形成的复合物结合的基因组位点。这个程序是基于成像的双分子荧光互补（BiFC）复合体上的果蝇多染色体。BiFC复合物是由两个荧光蛋白片段的促进结合而形成的，这两个荧光蛋白片段融合到相互作用或彼此接近的蛋白质上。在单个基因组位点的BiFC复合体荧光强度大大增强了平行排列的数百个染色单体在多线染色体。通过比较BiFC复合体荧光的位置与染色体的典型带带模式来绘制由复合体结合的位点。我们描述了融合蛋白的设计、表达和验证策略，用于分析多染色体上的BiFC复合物结合。我们详细的协议为制备多烯染色体扩散已优化为目的的BiFC复合体可视化。最后，我们为解释多染色体上BiFC复合物结合的研究结果以及比较由BiFC复合物结合的基因组位点与由相应内源复合物亚基结合的基因组位点提供了指导。多肽染色体上多蛋白复合物结合的可视化提供了一种独特的方法，可以可视化整个基因组中单个细胞中特定位点上的多蛋白复合物结合。
We have developed a procedure that visualizes the genomic loci that are bound by complexes formed by a specific combination of chromatin-binding proteins. This procedure is based on imaging bimolecular fluorescence complementation (BiFC) complexes on Drosophila polytene chromosomes. BiFC complexes are formed by the facilitated association of two fluorescent protein fragments that are fused to proteins that interact with, or are in close proximity to, each other. The intensity of BiFC complex fluorescence at individual genomic loci is greatly enhanced by the parallel alignment of hundreds of chromatids within the polytene chromosomes. The loci that are bound by the complexes are mapped by comparing the locations of BiFC complex fluorescence with the stereotypical banding patterns of the chromosomes. We describe strategies for the design, expression and validation of fusion proteins for the analysis of BiFC complex binding on polytene chromosomes. We detail protocols for the preparation of polytene chromosome spreads that have been optimized for the purpose of BiFC complex visualization. Finally, we provide guidance for the interpretation of results from studies of BiFC complex binding on polytene chromosomes and for comparison of the genomic loci that are bound by BiFC complexes with those that are bound by subunits of the corresponding endogenous complexes. The visualization of BiFC complex binding on polytene chromosomes provides a unique method to visualize multiprotein complex binding at specific loci, throughout the genome, in individual cells.
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