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

Summary

Fluorescence resonance energy transfer (FRET) is an imaging technique for detecting protein interactions in living cells. Here, a FRET protocol is presented to study the association of histone-modifying enzymes with transcription factors that recruit them to the target promoters for epigenetic regulation of gene expression in plant tissues.

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.

Introduction

Plant histone deubiquitinases play an important role in controlling gene expression by post-translational modification of histones, specifically by erasing their monoubiquitylation marks¹. So far, OTLD1 is one of the only few plant histone deubiquitinases characterized at the molecular level in Arabidopsis^2,3. OTLD1 removes monoubiquitin groups from the H2B histone molecules, thereby promoting the removal or addition of euchromatic acetylation and methylation modifications of H3 histones in the target gene chromatin^4,5. Moreover....

Protocol

Nicotiana benthamiana, Agrobacterium tumefaciens strain EHA105, or GV3101 were used for the present study.

1. FRET vector construction

1. Select fluorescent tags for the donor/acceptor FRET pair.
   1. Use EGFP from pPZP-RCS2A-DEST-EGFP-N1^15,28 (see Table of Materials) to generate the donor vector.
   2. Use mRFP from pPZP-RCS2A-DEST-mRFP-N1 (see Table of Materials) to generate the acceptor vector.
2. Generate the donor/acceptor FRET constructs using a site-specific recombin....

Discussion

This FRET protocol is simple and easy to reproduce; it also requires minimal supply investment and utilizes standard equipment for many modern laboratories. Specifically, five main technical features distinguish the versatility of this procedure. First, the FRET constructs are generated using site-specific recombination, a cloning approach that is easy to use, produces accurate results, and saves time compared to traditional restriction enzyme-based cloning. Second, N. benthamiana plants are simple to grow, prod.......

Materials

Name	Company	Catalog Number	Comments
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