Name: genome-wide mapping of protein-dna interactions with chec-seq in saccharomyces cerevisiae
Uploaded: 2017-06-03T15:00:00
Description: Watch this Scientific Journal Video about Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae at JoVE.com

Vi takker Moustafa Saleh og Jay Tourigny for kritisk læsning af manuskriptet og Steven Hahn og Steven Henikoff til mentorskab og support under udviklingen af ​​ChEC-seq og dens anvendelse til Mediator-komplekset. SG understøttes af NIH tilskud R01GM053451 og R01GM075114 og GEZ understøttes af Indiana University startup funds.

1. Frembringelse af gærstammer <ol><li> Generer en gærstamme, der bærer den faktor af interesse, der er mærket med MNase. <ol><li> PCR amplificerer MNase-mærkningskassetten fra den ønskede vektor ( tabel 1 ) under anvendelse af den specificerede reaktionsblanding ( tabel 2 ) og cykliske betingelser ( tabel 3 ). Bland 5 μL af PCR-reaktionen og 1 μl 6X DNA-fyldstof. Kør hver PCR-alikvot på en 0,8% agarosegel ved 120 V i 40 minutter. Den forven...

<ol>
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Vi har vist, at ChEC kan kortlægge forskellige klasser af gærproteiner på kromatin og forudse, at det vil være bredt anvendeligt for forskellige familier af TF&#39;er og andre kromatinbindende faktorer i gær. ChEC-seq er fordelagtig, idet den ikke kræver tværbinding, chromatinopløseliggørelse eller antistoffer. Således undgår ChEC artefakter, der potentielt er til stede i X-ChIP-seq, såsom hyper-ChIPable artefact 3 , 4 og native ChIP, såsom falske n...

Kortlægning af bindingsstederne for transkriptionsfaktorer (TF&#39;er), chromatin-remodelerer og andre kromatinassocierede regulatoriske faktorer er nøglen til forståelsen af ​​alle kromatinbaserede processer. Mens chromatinimmunpræcipitation og høj gennemstrømningssekvensering (ChIP-seq) fremgangsmåder er blevet brugt til at opnå mange vigtige indsigter i genombiologi, har de bemærkelsesværdige begrænsninger. Vi introducerede for nylig en alternativ metode, kaldet chromatin endogen spaltning og high-throughput sekventering (ChEC-seq) 1 , for at omgå disse ulemper. ChIP-seq udføres oftest med et indledende formaldehydtværbindelsestrin (X-ChIP-seq) for at bevare protein-DNA-interaktioner. Imidlertid har en række nyere undersøgelser vist, at X-ChIP-seq fanger transient eller ikke-specifikke protein-DNA-interaktioner 2 , 3 , 4 , 5 &lt;Sup&gt;, 6 , 7 , 8 , hvilket giver anledning til falske positive bindingssteder. Hertil kommer, at sonikering, der almindeligvis anvendes til fragmentering af chromatin i X-ChIP-seq-eksperimenter, fortrinsvis saksregioner af åben chromatin, hvilket fører til forspændt opsving af fragmenter fra disse regioner 9 , 10 . Sonikering giver også en heterogen blanding af fragmentlængder, der i sidste ende begrænser bindingsstedets opløsning, selvom tilsætningen af ​​et exonukleasefordøjelsestrin i høj grad kan forbedre opløsningen 11 , 12 . Native ChIP-metoder såsom besatte regioner af genomer fra affinitetsrenset naturligt isoleret kromatin (ORGANISK) 13 bruger ikke tværbinding og fragmentkromatin med mikrokokernuklease (MNase), lindrende potentielle biaser forbundet med formaldehyd-tværbinding og sonikering. Imidlertid,Opløseligheden af ​​mange kromatinbundne proteiner under de forholdsvis milde betingelser, der kræves til nativ kromatinekstraktion er dårlig, hvilket potentielt fører til reduceret dynamisk område og / eller falske negativer 14 . Mens forskellige iterationer af ChIP-seq mest anvendes til genomfattende kortlægning af protein-DNA-interaktioner, er der også implementeret adskillige kortlægningsteknikker baseret på fusion af proteiner af interesse for forskellige DNA-modificerende enzymer. En sådan fremgangsmåde er DNA-adeninmethyltransferaseidentifikation (DamID) 15 , hvor et chromatinbindende protein af interesse er genetisk fusioneret til Dam, og denne fusion udtrykkes i celler eller dyr, hvilket resulterer i methylering af GATC-sekvenser, der er proximale til bindingssteder for proteinet. DamID er fordelagtig, fordi den ikke er afhængig af immunpræcipitation og således undgår tværbinding, antistoffer eller chromatinopløseliggørelse. Det udføres også in vivo . Imidlertid, Er opløsningen af ​​DamID begrænset til kilobaseskalaen og den methylerende aktivitet af Dam-fusionsproteinet er konstitutiv. En anden metode baseret på enzymatisk fusion er Calling Card-seq 16 , der anvender fusion af en faktor af interesse for en transposase, der dirigerer stedsspecifik integration af transposoner. Som DamID er Calling Card-seq ikke baseret på immunpræcipitation og har dermed tilsvarende fordele, med den ekstra fordel ved forøget opløsning. Calling Card-seq kan imidlertid begrænses af sekvensforspændinger af transposaser og er også afhængig af tilstedeværelsen af ​​restriktionssteder tæt på transposon-insertionssteder. En tredje enzymatisk fusionsmetode, der er udviklet i Laemmli lab, er chromatin endogen spaltning (ChEC) 17 . I ChEC udtrykkes en fusion mellem et chromatinassocieret protein og MNase i celler, og efter calciumtilsætning til aktivering af MNase spaltes DNA proximalt til bindingssteder for den mærketFaktor ( figur 1 ). I forbindelse med Southern blotting er ChEC blevet anvendt til at karakterisere chromatinstruktur og proteinbinding på et antal individuelle loci i gær 17 , 18 og er blevet kombineret med lavopløsnings-mikroarrayanalyse for at undersøge interaktionen mellem nukleare pore-komponenter med gæren Genom 19 . ChEC tilbyder fordele svarende til DamID og Calling Card-seq, og dens opløsning er næsten single-base pair, når den analyseres ved primer forlængelse 19 . ChEC er også kontrollerbar: Robust DNA spaltning ved MNase afhænger af tilsætning af millimolar calcium, hvilket sikrer, at MNase er inaktiv ved de lave frie calciumkoncentrationer observeret i levende gærceller 20 . Tidligere postulerede vi at kombinere ChEC med high-throughput-sekventering (ChEC-seq) ville tilvejebringe kort med høj opløsning af TF-bindingssteder. Faktisk, ChEC-seq genererede kort med høj opløsning af de spirende gær generelle regulatoriske faktorer (GRF&#39;er) Abf1, Rap1 og Reb1 på tværs af genomet 1 . Vi har også med succes anvendt ChEC-seq til det modulære Mediator-kompleks, en bevaret, essentiel global transkriptionel coaktivator 21 , der udvider anvendeligheden af ​​ChEC-seq til megadalton-størrelse komplekser, der ikke direkte kontakter DNA og kan være vanskelige at kortlægge ved ChIP- Baserede metoder. ChEC-seq er en stærk metode både til uafhængig validering af ChIP-seq-resultater og generering af nye indsigter i reguleringen af ​​chromatin-residente processer. Her præsenterer vi en trin-for-trin protokol til implementering af denne metode i spirende gær.

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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">dNTPs</td>
			<td style="width:159px;">NEB</td>
			<td style="width:119px;">N0447</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Q5 high-fidelity DNA polymerase</td>
			<td style="width:159px;">NEB</td>
			<td style="width:119px;">M0491L</td>
			<td>Other high-fidelity DNA polymerases, such as Phusion, may be used for cassette amplification.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">TrackIt 1 Kb Plus DNA ladder</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10488085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Taq DNA polymerase</td>
			<td style="width:159px;">NEB</td>
			<td style="width:119px;">M0273L</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:307px;">cOmplete Mini EDTA-free protease inhibitor cocktail</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:119px;">11836170001</td>
			<td>It is important that an EDTA-free protease inhibitor mix is used, so as not to inhibit Mnase cleavage by chelation of Ca2+.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">PMSF</td>
			<td style="width:159px;">ACROS Organics</td>
			<td style="width:119px;">AC215740010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Digitonin, High Purity</td>
			<td style="width:159px;">EMD Millipore</td>
			<td style="width:119px;">300410-250MG</td>
			<td>Make a 2% stock by dissolving 20 mg digitonin in 1 mL DMSO with vigorous vortexing.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Proteinase K, 20 mg/mL</td>
			<td style="width:159px;">Invitrogen</td>
			<td style="width:119px;">25530049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">RNase A, 10 mg/mL</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:119px;">EN0531</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Ampure XP beads</td>
			<td style="width:159px;">Beckman Coulter</td>
			<td style="width:119px;">A63880</td>
			<td>Ampure-like beads can be generated using a published protocol (ref 24).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">MagneSphere magnetic rack</td>
			<td style="width:159px;">Promega</td>
			<td style="width:119px;">Z5342</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide mapping of protein-dna interactions with chec-seq in saccharomyces cerevisiae

Genomfattende kortlægning af protein-DNA-interaktioner er kritisk for forståelse af genregulering, chromatin-remodeling og andre chromatin-residente processer. Formaldehyd-tværbinding efterfulgt af chromatinimmunpræcipitation og høj-gennemstrømningssekvensering (X-ChIP-seq) er blevet anvendt til at opnå mange værdifulde indsigter i genombiologi. Imidlertid har X-ChIP-seq bemærkelsesværdige begrænsninger forbundet med tværbinding og sonikering. Native ChIP undgår disse ulemper ved at udelade tværbinding, men resulterer ofte i dårlig genopretning af kromatinbundne proteiner. Derudover er alle ChIP-baserede metoder underlagt antistofkvalitets overvejelser. Enzymatiske metoder til kortlægning af protein-DNA-interaktioner, der involverer fusion af et protein af interesse for et DNA-modificerende enzym, er også blevet anvendt til kortlægning af protein-DNA-interaktioner. Vi har for nylig kombineret en sådan metode, chromatin endogen spaltning (ChEC), med høj gennemstrømningssekvensering som ChEC-seq. ChEC-seq er afhængig af fusion af en chromatin-assocIeret protein af interesse for mikrokokernuklease (MNase) til dannelse af målrettet DNA-spaltning i nærværelse af calcium i levende celler. ChEC-seq er ikke baseret på immunpræcipitation og omgår således potentielle problemer med tværbinding, sonikering, chromatinopløseliggørelse og antistofkvalitet, samtidig med at der tilvejebringes højopløsningsmapping med minimalt baggrundssignal. Vi forestiller os, at ChEC-seq vil være en stærk modstykke til ChIP, hvilket giver et selvstændigt middel til at både validere ChIP-seq-fund og opdage nye indblik i genomisk regulering.

I tilfælde af et vellykket ChEC-forsøg vil analyse af DNA ved agarosegelelektroforese afsløre en calciumafhængig forøgelse i DNA-fragmentering over tid som angivet ved udtværing og eventuel fuldstændig fordøjelse af genomisk DNA. I nogle tilfælde observeres en stige af bånd, der ligner den, der ses med en traditionel MNase-fordøjelse efter udvidet fordøjelse. Dette er tilfældet for ChEC analyse af Reb1, en generel regulatorisk faktor, der binder nukleosomudtømte regioner (N...

Watch this Scientific Journal Video about Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae at JoVE.com

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Vi beskriver chromatin endogen spaltning kombineret med høj gennemstrømningssekvensering (ChEC-seq), en chromatinimmunpræcipitation (ChIP) -orthogonal metode til kortlægning af proteinbindingssteder, som er genomsamlede med mikrokocelle nuklease (MNase) fusionsproteiner.

Genom-bred kortlægning af protein-DNA-interaktioner med ChEC-seq in<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

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JoVE Journal

Genetics

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Genom-dækkende Bestemmelse af pattedyr Replication Timing ved DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Kortlægning af RNA-RNA-interaktioner globalt ved anvendelse af biotinyleret psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Kortlægning Genome-wide tilgængelige kromatin i primære humane T lymfocytter af ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Bestemmelse af Genome-wide udskrift henfald priser i prolifererende og inaktiv humane fibroblaster

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Generation af Genome-wide kromatin kropsbygning Capture biblioteker fra stramt iscenesatte tidlige Drosophila embryoner

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Kromatin Immunoprecipitation (ChIP) af Histon ændringer fra Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

Promotor Capture Hej-C: Høj opløsning, Genome-wide profilering af promotor interaktioner

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Genom-dækkende overvågning af transskription fejl i Eukaryote organismer

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Saccharomyces cerevisiae Metaboliske mærkning med 4-thiouracil og kvantificering af nyligt syntetiserede mRNA som en Proxy for RNA Polymerase II aktivitet

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Genetisk kortlægning af termo Toleranceforskelle mellem arter af Saccharomyces gær via Genome-Wide gensidig Hemizygosity analyse

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...