Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Abstract

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone marks for transcriptional repression or activation, respectively. HMEs are recruited to their target chromatin by transcription factors (TFs). Thus, detecting and characterizing direct interactions between HMEs and TFs are critical for understanding their function and specificity better. These studies would be more biologically relevant if performed in vivo within living tissues. Here, a protocol is described for visualizing interactions in plant leaves between a plant histone deubiquitinase and a plant transcription factor using fluorescence resonance energy transfer (FRET), which allows the detection of complexes between protein molecules that are within <10 nm from each other. Two variations of the FRET technique are presented: SE-FRET (sensitized emission) and AB-FRET (acceptor bleaching), in which the energy is transferred non-radiatively from the donor to the acceptor or emitted radiatively by the donor upon photobleaching of the acceptor. Both SE-FRET and AB-FRET approaches can be adapted easily to discover other interactions between other proteins in planta.

Figure 1:. Schematic summary of the SE-FRET and AB-FRET techniques.

(A) The basic principle of SE-FRET. One of the tested proteins is tagged with GFP, which acts as a donor fluorochrome, and the other with mRFP, which acts as an acceptor fluorochrome. The donor molecule is excited, and the acceptor emission is recorded. If the tested proteins interact with each other such that they are positioned within 10 nm of each other, the energy from the excited donor is transferred non-radiatively to the acceptor, which then becomes excited and emits fluorescence in the FRET emission channel. If no interaction occurs, no energy is transferred from the donor to the acceptor, and no FRET emission by the acceptor is detected. (B) The basic principle of AB-FRET. The tested proteins are tagged as described in (A) for SE-FRET. The donor molecule is excited, and if the interaction between the tested proteins occurs, the donor excites the acceptor in a non-radiative fashion, resulting in FRET. Then, the acceptor is permanently inactivated by photobleaching, thereby losing its ability to accept non-radiative energy from the donor and emit the FRET fluorescence in the FRET emission channel; the fluorescence emitted by the donor, on the other hand, is increased because the donor loses less energy by the non-radiative transfer.

Figure 2:. Specific interaction of LSH10 with OTLD1 in N. benthamiana leaves detected by SE-FRET.

Images from three detection channels (donor, acceptor, and SE-FRET) are shown for the indicated protein combinations. The SE-FRET efficiency images were calculated by the subtraction of spectral bleed-through (SBT) and are shown in pseudo-color, with the colors red and blue signifying the highest and the lowest signal, respectively. (A) High SE-FRET efficiency signal produced by the mRFP-GFP positive control. (B) Positive SE-FRET efficiency signal produced by the interacting LSH10-GFP and OTLD1-mRFP proteins. (C) Coexpression of the negative control protein LSH4-GFP and OTLD1-mRFP produced no SE-FRET efficiency signal. (D) Coexpression of the negative control-free mRFP protein and LSH10-GFP produced no SE-FRET efficiency signal. Scale bars = 10 μm.

Figure 3:. Specific interaction of LSH10 with OTLD1 in N. benthamiana leaves detected by AB-FRET.

Images from two detection channels (donor and acceptor) before and after photobleaching are shown for the indicated protein combinations. The circle indicates the photobleached region. AB-FRET, visualized as an increase in GFP fluorescence after mRFP photobleaching, is displayed using pseudo-color with the colors red and blue, signifying the highest and lowest signal, respectively. (A) An increase in the GFP donor fluorescence produced by the mRFP-GFP positive control. (B) An increase in the GFP donor fluorescence produced by the interacting LSH10-GFP and OTLD1-mRFP proteins. (C) Coexpression of the negative control protein LSH4-GFP and OTLD1-mRFP produced negligible changes in the GFP donor fluorescence. (D) Coexpression of the negative control free mRFP protein and LSH10-GFP produced negligible changes in the GFP donor fluorescence. Scale bars = 10 μm.

Figure 4:. A Quantification of AB-FRET.

The percentage increase in the GFP donor fluorescence after mRFP photobleaching (%AB-FRET) is shown for the indicated protein combinations. Error bars represent the mean for n = 13 cells for each measurement. The two-tailed t-test determined that differences between mean values are statistically significant for the p-values *p < 0.05, **p < 0.01, and ***p < 0.001; p ≥ 0.05 are not statistically significant (ns).