Visualization of the Genomic Loci That Are Bound by Specific Multiprotein Complexes by Bimolecular Fluorescence Complementation Analysis on Drosophila Polytene Chromosomes

Abstract

We have developed a procedure that enables visualization of 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.

Keywords: BiFC complex imaging; Chromatin binding proteins; Fluorescent protein fragments; Genomewide analysis; Polytene chromosome spreads; Transcription factor interaction; Transgene expression in specific tissues.

Fig. 1

(A) Diagram of BiFC complex formation by proteins fused to fluorescent protein fragments (YN and YC) (modified from Deng and Kerppola 2014); (B) Plasmids encoding dKeap1 and CncC fused to fluorescent protein fragments (YN and YC) were constructed using the pUAST vector (Brand and Perrimon, 1993). The pUAST vector contains five tandem GAL4 binding sites (UAS) that mediate transgene activation by GAL4 and sequences that facilitate transcription and P-element sequences that facilitate transgene insertion into the Drosophila genome. (C) BiFC analysis of the specificity of association of FKBP and FRB fusions to fragments of the Venus (VN173) and YFP (YC173) fluorescent proteins in S2 cells. The cells were cultured in the absence (−) and in the presence (+) or rapamycin, which induces FKBP-FRB interaction (Rivera et al., 1996). The fluorescence that is observed in the absence of rapamycin indicates that the fluorescent protein fragments associate spontaneously. Scale bars: 2 µm.

Fig. 2

Comparison of the levels of protein expression and subcellular localization of dKeap1 and CncC fusion proteins that are expressed at different levels with their endogenous counterparts. (A) The salivary glands of larvae that express the fusion proteins indicated above each lane were excised and the levels of protein expression were analyzed by immunoblotting using the antibodies indicated to the right of each image. The endogenous dKeap1 and CncC proteins were not detected in this exposures. The bands representing dKeap1 (blue) and CncC (red) fusion proteins are labeled by arrowheads. Image modified from Deng and Kerppola 2014. (B) The salivary glands of wild type larvae (left column) and of larvae that expressed the fusion proteins indicated at the top of the images (right column) were analyzed by immunofluorescence (yellow) using the antibodies indicated at the bottom of the images. The nuclei were labeled by Hoechst staining (blue). Scale bars: 10 µm.

Fig. 3

Visualization of BiFC complexes on polytene chromosomes. (A) Diagram of BiFC complex formation on polytene chromosomes. The intensity of BiFC complex fluorescence at individual genomic loci is enhanced by the parallel alignment of hundreds of chromatids within the polytene chromosomes. (B) Examples of BiFC complex fluorescence (green) on polytene chromosomes stained using Hoechst (blue). The chromosomes were spread under acid-free conditions. The banding patterns of the polytene chromosomes stained using Hoechst are shown in the images to the right. Note the differences in chromosome spreading and extension that are necessary to map the localization of the BiFC complexes to individual genomic loci. Upper panel: an intact spread allowing mapping of a number of loci, including the two copies of the 55F locus, corresponding to the two homologues that were presumably separated during the squash procedure; Middle panel: a partial spread with broken arms, which are well extended and suitable for mapping a subset of the loci; Lower panel: a poorly extended spread on which the loci that are bound by BiFC complex cannot be easily identified. Scale bars: 10 µm. (C) Comparison of the localization of BiFC complexes and of the individual BiFC fusion proteins using acid-free (upper images) and conventional (middle and bottom images) squash protocols. Polytene chromosomes that prepared using the acid-free protocol have less sharp banding patters than those that are prepared using conventional squash protocols. Both dKeap1-CncC BiFC complexes and each of the fusion proteins bound the 55F locus. Both dKeap1 and CncC fusions bound many other loci, whereas the dKeap1-CncC BiFC complex bound the 55F locus with higher specificity. Scale bars: 5 µm.