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Biology

Undersøgelse af interaktioner mellem histonmodificerende enzymer og transkriptionsfaktorer in vivo ved fluorescensresonansenergioverførsel

Published: October 14, 2022
doi:
10.3791/64656
, ,
1Department of Biochemistry and Cell Biology,State University of New York

Summary

Fluorescensresonans energioverførsel (FRET) er en billeddannelsesteknik til påvisning af proteininteraktioner i levende celler. Her præsenteres en FRET-protokol for at studere sammenhængen mellem histonmodificerende enzymer med transkriptionsfaktorer, der rekrutterer dem til målpromotorerne til epigenetisk regulering af genekspression i plantevæv.

Abstract

Epigenetisk regulering af genekspression påvirkes almindeligvis af histonmodificerende enzymer (HME’er), der genererer heterokromatiske eller eukromatiske histonmærker til henholdsvis transkriptionel undertrykkelse eller aktivering. HME’er rekrutteres til deres målkromatin ved transkriptionsfaktorer (TF’er). Således er detektering og karakterisering af direkte interaktioner mellem HME’er og TF’er afgørende for at forstå deres funktion og specificitet bedre. Disse undersøgelser ville være mere biologisk relevante, hvis de blev udført in vivo inden for levende væv. Her beskrives en protokol til visualisering af interaktioner i planteblade mellem en plante histon deubiquitinase og en plantetranskriptionsfaktor ved hjælp af fluorescensresonansenergioverførsel (FRET), som muliggør påvisning af komplekser mellem proteinmolekyler, der er inden for <10 nm fra hinanden. To variationer af FRET-teknikken præsenteres: SE-FRET (sensibiliseret emission) og AB-FRET (acceptorblegning), hvor energien overføres ikke-strålingsagtigt fra donoren til acceptoren eller udsendes radiativt af donoren ved fotoblegning af acceptoren. Både SE-FRET og AB-FRET tilgange kan let tilpasses til at opdage andre interaktioner mellem andre proteiner i planta.

Introduction

Plantehistondeubiquitinaser spiller en vigtig rolle i at kontrollere genekspression ved posttranslationel modifikation af histoner, specifikt ved at slette deres monoubiquityleringsmærker1. Indtil videre er OTLD1 en af de eneste få plante histon deubiquitinaser karakteriseret på molekylært niveau i Arabidopsis 2,3. OTLD1 fjerner monoubiquitingrupper fra H2B-histonmolekylerne og fremmer derved fjernelse eller tilsætning af eukromatisk acetylering og methyleringsmodifikationer af H3-histoner i målgenetkromatin 4,5. Desuden interagerer OTLD1 med et andet kromatinmodificerende enzym, histonlysindemethylase KDM1C, for at påvirke transkriptionel undertrykkelse af målgenerne 6,7.

De fleste histonmodificerende enzymer mangler DNA-bindingsevner og kan derfor ikke genkende deres målgener direkte. En mulighed er, at de samarbejder med DNA-bindende transkriptionsfaktorproteiner, der binder disse enzymer og leder dem til deres kromatinmål. Specifikt i planter vides flere større histonmodificerende enzymer (dvs. histonmethyltransferaser8,9, histonacetyltransferaser 10, histondemethylaser 11 og polycomb-undertrykkende komplekser12,13,14) at blive rekrutteret af transkriptionsfaktorer. I overensstemmelse med denne idé blev der for nylig foreslået en mulig mekanisme til OTLD1-rekruttering til målpromotorerne, som er baseret på specifikke protein-protein-interaktioner af OTLD1 med en transkriptionsfaktor LSH1015.

LSH10 tilhører en familie af planten ALOG (Arabidopsis LSH1 og Oryza G1) proteiner, der fungerer som centrale udviklingsregulatorer 16,17,18,19,20,21,22. Det faktum, at medlemmerne af ALOG-proteinfamilien indeholder DNA-bindende motiver23 og udviser kapaciteten til transkriptionel regulering22, nuklear lokalisering19 og homodimerisering24, understøtter yderligere forestillingen om, at disse proteiner, herunder LSH10, kan fungere som specifikke transkriptionsfaktorer under epigenetisk regulering af transkription. En af de vigtigste eksperimentelle teknikker, der anvendes til at karakterisere LSH10-OTLD1-interaktionen in vivo, er fluorescensresonansenergioverførsel (FRET)15.

FRET er en billeddannelsesteknik til direkte påvisning af nærmiljøinteraktioner mellem proteiner inden for <10 nm fra hinanden25 inde i levende celler. Der er to hovedvariationer af FRET-metoden26: sensibiliseret emission (SE-FRET) (figur 1A) og acceptorblegning (AB-FRET) (figur 1B). I SE-FRET overfører de interagerende proteiner – hvoraf det ene er mærket med et donorfluorkrom (f.eks. Grønt fluorescerende protein, GFP) og det andet med et acceptorfluorkrom (f.eks. Monomært rødt fluorescerende protein, mRFP27,28) – ikke-strålingsenergi fra donoren til acceptoren. Fordi der ikke udsendes fotoner under denne overførsel, produceres et fluorescerende signal, der har et strålingsemissionsspektrum svarende til acceptorens. I AB-FRET detekteres og kvantificeres proteininteraktioner baseret på forhøjet strålingsemission af donoren, når acceptoren er permanent inaktiveret ved fotoblegning og dermed ikke er i stand til at modtage den ikke-strålingsenergi, der overføres fra donoren (figur 1). Det er vigtigt, at den subcellulære placering af FRET-fluorescensen er vejledende for lokaliseringen af de interagerende proteiner i cellen.

Evnen til at anvende FRET i levende væv og bestemme den subcellulære lokalisering af de interagerende proteiner samtidig med påvisning af denne interaktion i sig selv, gør FRET til den valgte teknik til undersøgelser og indledende karakterisering af protein-protein-interaktioner in vivo. En sammenlignelig in vivo fluorescensbilleddannelsesmetode, bimolekylær fluorescenskomplementering (BiFC)29,30,31,32, er en god alternativ tilgang, selvom BiFC i modsætning til FRET kan producere falske positiver på grund af spontan samling af de autofluorescerende BiFC-journalister 33, og kvantificeringen af dens data er mindre præcis.

Denne artikel deler den vellykkede erfaring med implementering af både SE-FRET og AB-FRET teknikker og præsenterer en protokol for deres implementering for at undersøge interaktionerne mellem OTLD1 og LSH10 i planteceller.

Protocol

Nicotiana benthamiana, Agrobacterium tumefaciens stamme EHA105 eller GV3101 blev anvendt til denne undersøgelse. 1. FRET vektor konstruktion Vælg fluorescerende tags til donor / acceptor FRET-parret.Brug EGFP fra pPZP-RCS2A-DEST-EGFP-N115,28 (se materialetabel) til at generere donorvektoren. Brug mRFP fra pPZP-RCS2A-DEST-mRFP-N1 (se Materialetabe…

Representative Results

Figur 2 illustrerer de typiske resultater af et SE-FRET-eksperiment, hvor cellekernerne samtidig blev registreret i tre kanaler (dvs. donor GFP, acceptor mRFP og SE-FRET). Disse data blev brugt til at generere billeder af SE-FRET-effektivitet kodet i en pseudofarveskala. På denne skala svarer overgangen fra blå til rød til en stigning i FRET-effektivitet, et mål for protein-protein-nærhed fra 0% til 100%. I dette repræsentative eksperiment blev SE-FRET-signalet registreret i cellekerne…

Discussion

Denne FRET-protokol er enkel og nem at gengive; Det kræver også minimal forsyningsinvestering og bruger standardudstyr til mange moderne laboratorier. Specifikt skelner fem vigtigste tekniske træk alsidigheden af denne procedure. For det første genereres FRET-konstruktionerne ved hjælp af stedspecifik rekombination, en kloningsmetode, der er nem at bruge, giver nøjagtige resultater og sparer tid sammenlignet med traditionel restriktionsenzymbaseret kloning. For det andet er N. benthamiana-planter enkle at …

Disclosures

The authors have nothing to disclose.

Acknowledgements

Arbejdet i V.C.’s laboratorium er støttet af tilskud fra NIH (R35GM144059 og R01GM50224), NSF (MCB1913165 og IOS1758046) og BARD (IS-5276-20) til V.C.

Materials

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
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Tags

Keywords: FRETProtein-protein InteractionAgroinfiltrationNicotiana BenthamianaHistone Modifying EnzymesTranscription FactorsLiving CellFluorescence Resonance Energy Transfer
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Vo Phan, M. S., Tran, P. T., Citovsky, V. Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer. J. Vis. Exp. (188), e64656, doi:10.3791/64656 (2022).
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