Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors <em>in vivo</em> 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.