Name: investigating interactions between histone modifying enzymes and transcription factors in vivo by fluorescence resonance energy transfer
Uploaded: 2022-10-14T15:00:00
Description: Watch this Scientific Journal Video about Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer at JoVE.com

The work in V.C.&#39;s laboratory is supported by grants from NIH (R35GM144059 and R01GM50224), NSF (MCB1913165 and IOS1758046), and BARD (IS-5276-20) to V.C.

Nicotiana benthamiana, Agrobacterium tumefaciens strain EHA105, or GV3101 were used for the present study.
1. FRET vector construction
<ol>
	<li>Select fluorescent tags for the donor/acceptor FRET pair.
	<ol>
		<li>Use EGFP from pPZP-RCS2A-DEST-EGFP-N115,28 (see Table of Materials) to generate the donor vector.</li>
		<li>Use mRFP from pPZP-RCS2A-DEST-mRFP-N1 (see Table of M...

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...

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 Arabidopsis2,3. 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 chromatin4,5. Moreover, OTLD1 interacts with another chromatin-modifying enzyme, the histone lysine demethylase KDM1C, to affect transcriptional suppression of the target genes6,7.
Most histone-modifying enzymes lack DNA binding capabilities, and thus cannot recognize their target genes directly. One possibility is that they cooperate with DNA-binding transcription factor proteins which bind these enzymes and direct them to their chromatin targets. Specifically, in plants, several major histone-modifying enzymes (i.e., histone methyltransferases8,9, histone acetyltransferases10, histone demethylases11, and Polycomb repressive complexes12,13,14) are known to be recruited by transcription factors. Consistent with this idea, recently, one possible mechanism for OTLD1 recruitment to the target promoters was proposed which is based on specific protein-protein interactions of OTLD1 with a transcription factor LSH1015.
LSH10 belongs to a family of the plant ALOG (Arabidopsis LSH1 and Oryza G1) proteins that function as central developmental regulators16,17,18,19,20,21,22. The fact that the members of the ALOG protein family contain DNA binding motifs23 and exhibit the capacities for transcriptional regulation22, nuclear localization19, and homodimerization24 lends further support to the notion that these proteins, including LSH10, may act as specific transcription factors during epigenetic regulation of transcription. One of the main experimental techniques used to characterize the LSH10-OTLD1 interaction in vivo is fluorescence resonance energy transfer (FRET)15.
FRET is an imaging technique for directly detecting close-range interactions between proteins within &#60;10 nm from each other25 inside living cells. There are two main variations of the FRET approach26: sensitized emission (SE-FRET) (Figure&#160;1A) and acceptor bleaching (AB-FRET) (Figure 1B). In SE-FRET, the interacting proteins-one of which is tagged with a donor fluorochrome (e.g., green fluorescent protein, GFP) and the other with an acceptor fluorochrome (e.g., monomeric red fluorescent protein, mRFP27,28)-non-radiatively transfer the excited state energy from the donor to the acceptor. Because no photons are emitted during this transfer, a fluorescent signal is produced that has a radiative emission spectrum similar to that of the acceptor. In AB-FRET, protein interactions are detected and quantified based on elevated radiative emission of the donor when the acceptor is permanently inactivated by photobleaching, and thus is unable to receive the non-radiative energy transferred from the donor (Figure&#160;1). Importantly, the subcellular location of the FRET fluorescence is indicative of the localization of the interacting proteins in the cell.
The ability to deploy FRET in living tissues and determine the subcellular localization of the interacting proteins simultaneously with detecting this interaction per se, makes FRET the technique of choice for studies and initial characterization of protein-protein interactions in vivo. A comparable in vivo fluorescence imaging methodology, bimolecular fluorescence complementation (BiFC)29,30,31,32, is a good alternative approach, although, unlike FRET, BiFC may produce false positives due to spontaneous assembly of the autofluorescent BiFC reporters33, and quantification of its data is less precise.
This article shares the successful experience in implementing both SE-FRET and AB-FRET techniques and presents a protocol for their deployment to investigate the interactions between OTLD1 and LSH10 in plant cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetosyringone (3&#8242;,5&#8242;-Dimethoxy-4&#8242;-hydroxyacetophenone)</td>
			<td>Sigma-Aldrich</td>
			<td>#D134406-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>#214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto trypton</td>
			<td>BD Biosciences</td>
			<td>#211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto yeast extract</td>
			<td>BD Biosciences</td>
			<td>#212750&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope (CLSM)</td>
			<td>Zeiss</td>
			<td>LSM900</td>
			<td>Any CLSM with similar capabilities is suitable</td>
		</tr>
		<tr>
			<td>EHA105</td>
			<td>VWR</td>
			<td>104013-310</td>
			<td>We use the stock in the Citovsky bacterial lab stock collection</td>
		</tr>
		<tr>
			<td>Gateway BP Clonase II&#160;</td>
			<td>Invitrogen</td>
			<td>#11789100</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway LR Clonase II</td>
			<td>Invitrogen</td>
			<td>#11791020</td>
			<td></td>
		</tr>
		<tr>
			<td>GV3101</td>
			<td>VWR</td>
			<td>104013-296</td>
			<td>We use the stock in the Citovsky bacterial lab stock collection</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td>https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Sigma-Aldrich</td>
			<td>#69889-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>#63068-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>#S5886-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotiana benthamiana seeds</td>
			<td>Herbalistics Pty</td>
			<td>RA4 or LAB</td>
			<td>We use the stock in the Citovsky seed lab stock collection</td>
		</tr>
		<tr>
			<td>pDONR207</td>
			<td>Invitrogen</td>
			<td>#12213013</td>
			<td></td>
		</tr>
		<tr>
			<td>pPZP-RCS2A-DEST-EGFP-N1&#160;</td>
			<td>N/A</td>
			<td></td>
			<td>Refs. 15, 28</td>
		</tr>
		<tr>
			<td>pPZP-RCS2A-DEST-mRFP-C1</td>
			<td>N/A</td>
			<td></td>
			<td>Generated based on the pPZP-RCS2A-DEST-EGFP-C1 construct (see refs. 15, 28)</td>
		</tr>
		<tr>
			<td>pPZP-RCS2A-DEST-mRFP-N1&#160;</td>
			<td>N/A</td>
			<td></td>
			<td>Generated based on the pPZP-RCS2A-DEST-EGFP-N1 construct</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Sigma-Aldrich</td>
			<td>#R7382-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Sigma-Aldrich</td>
			<td>#S4014-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes without needles</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Zen software for CLSM imaging</td>
			<td>Zeiss</td>
			<td>ZEN 3.0 version</td>
			<td>The software should be compatible with the CLSM used</td>
		</tr>
	</tbody>
</table>

investigating interactions between histone modifying enzymes and transcription factors in vivo by fluorescence resonance energy transfer

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 &#60;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 2 illustrates the typical results of a SE-FRET experiment, in which the cell nuclei were simultaneously recorded in three channels (i.e., donor GFP, acceptor mRFP, and SE-FRET). These data were used to generate images of SE-FRET efficiency coded in a pseudo-color scale. On this scale, the transition from blue to red corresponds to an increase in FRET efficiency, a measure of protein-protein proximity from 0% to 100%. In this representative experiment, the SE-FRET signal was recorded i...

Watch this Scientific Journal Video about Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer at JoVE.com

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

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.

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

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone ...

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