Summary

Tahmin ve Gen Düzenleme Elementlerin Doğrulama Retinoik Asit Kaynaklı Embriyonik sırasında Aktif Hücre Farklılaşma Kök

Published: June 21, 2016
doi:

Summary

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Abstract

Embriyonik gelişim, birçok genlerin aktivasyonunu ve baskıyı içeren çok aşamalı bir süreçtir. genomu içinde güçlendirici elemanlar hücre farklılaşması esnasında gen sentezlenmesinin doku ve hücre tipi özel düzenlemeye katkıda bulunduğu bilinmektedir. Böylece, kendi kimlik ve daha fazla araştırma hücre kaderi nasıl belirlendiğini anlamak için önemlidir. Gen ifadesi verileri (örneğin, mikrodizi veya RNA-seq) ve kromatin immunoprecipitation (ChIP) tabanlı genom çalışmaları (ChIP-seq) sonuçlarının entegrasyonu bu düzenleyici bölgelerin büyük ölçekli belirlenmesini sağlar. Ancak, hücre türüne özgü arttırıcılar işlevsel doğrulama in vitro ve in vivo deney prosedürü de gerektirir. Burada aktif artırıcılar tespit ve deneysel olarak doğrulanmış nasıl açıklar. ChIP-seq veri analizi, 2) klonlama ve exper tarafından düzenleyici bölgelerin 1) tanımlanması: Bu protokol içeren bir adım-adım iş akışı sağlarimental bir raportör analizinde tanımlanmış olan genom dizilerinin varsayımsal düzenleme potansiyeli doğrulama ve güçlendirici RNA transkript seviyesinin ölçülmesiyle in vivo güçlendirici aktivitesinin 3) belirlenmesi. sunulan protokol laboratuarında bu iş akışını kurmak için kimseye yardımcı olmak için yeterli ayrıntılı. Önemli olarak, iletişim kuralı, kolayca adapte ve herhangi bir hücresel model sistemde kullanılabilir.

Introduction

Development of a multicellular organism requires precisely regulated expression of thousands of genes across developing tissues. Regulation of gene expression is accomplished in large part by enhancers. Enhancers are short non-coding DNA elements that can be bound with transcription factors (TFs) and act from a distance to activate transcription of a target gene1. Enhancers are generally cis-acting and most frequently found just upstream of the transcription start site (TSS), but recent studies also described examples where enhancers were found much further upstream, on the 3′ of the gene or even within the introns and exons2.

There are hundreds of thousands of potential enhancers in the vertebrate genomes1. Recent methods based on chromatin immunoprecipitation (ChIP) provide high-throughput data of the whole genome that can be used for enhancer analysis3-9. Though data obtained by ChIP-seq experiments greatly increases the likelihood to identify cell and tissue-specific enhancers, it is important to keep in mind that detected binding sites do not necessarily identify direct DNA binding and/or functional enhancers. Thus, further functional analysis of newly identified enhancers is indispensable. In this work, we present a basic three-step process of putative active enhancer identification and validation. This includes: 1) selection of putative transcription factor binding sites by bioinformatics analysis of ChIP-seq data, 2) cloning and validation of these regulatory sequences in reporter constructs, and 3) measurement of enhancer RNA (eRNA).

Exposure of embryonic stem (ES) cells to retinoic acid (RA) is frequently used to promote neural differentiation of the pluripotent cells 10. RA exerts its effects by binding to RA receptors (RARα, β, γ) and retinoid X receptors (RXRα, β, γ). RARs and RXRs in a form of heterodimer bind to DNA motifs called RA-response elements, that is typically arranged as direct repeats of AGGTCA sequence (called as half site) and regulate transcription. Ligand-treatment experiments allowed the identification of several retinoic acid regulated genes in ES cells 11,12. However, enhancer elements for many of these genes has not been described yet. To demonstrate how the here-described workflow can be used for enhancer identification and validation we show step-by-step the selection and characterization of two retinoic acid-dependent enhancers in embryonic stem cells.

Protocol

Chip-seq Analiz dayanarak 1. Artırıcı Seçimi http://ngsdebftp.med.unideb.hu/bioinformatics/ gelen RXR ChIP seq ham veri fastq dosyası (mm_ES_RXR_24h_ATRA.fastq.gz) now Indirin ve bizim durumumuzda (uyum için gerekli BWA dizin dosyası ayıklamak: Mus_musculus_UCSC_mm10).(ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Mus_musculus/UCSC/mm10/Mus_musculus_UCSC_mm10.tar.gz NOT: biyoinformatik analiz aşamaları hakkında daha fazla bilgi için https://github.com/ahorvath/Bioinformatics_scrip…

Representative Results

Biz genom hangi RA-düzenlenen genler kendi yakın reseptör zenginleştirme var tanımlamak amacıyla bir pan-spesifik RXR antikor kullanıldı. Retinoik asit ile tedavi ES hücrelerinden elde RXR ChIP seq veri Biyoinformatik analiz RXR sitesi işgal altında nükleer reseptör yarım ekibi (AGGTCA) (Şekil 1) zenginleştirme saptandı. Bir biyoinformatik algoritması kullanarak biz RXR ChIP-seq verilere yarım sitesinden (Şekil 2) için motif arama so…

Discussion

In recent years, advances in sequencing technology have allowed large-scale predictions of enhancers in many cell types and tissues 7-9. The workflow described above allows one to perform primary characterization of candidate enhancers chosen based on ChIP-seq data. The detailed steps and notes will help anyone to set up a routine enhancer validation in the lab.

The most critical step in the luciferase reporter assay is the transfection efficiency. It is recommended to include a GFP…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

The authors would like to acknowledge Dr. Bence Daniel, Matt Peloquin, Dr. Endre Barta, Dr. Balint L Balint and members of the Nagy laboratory for discussions and comments on the manuscript. L.N is supported by grants from the Hungarian Scientific Research Fund (OTKA K100196 and K111941) and co-financed by the European Social Fund and the European Regional Development Fund and Hungarian Brain Research Program – Grant No. KTIA_13_NAP-A-I/9.

Materials

KOD DNA polymerase Merck Millipore 71085-3 for PCR amplification of enhancer from gDNA
DNeasy Blood & Tissue kit  Qiagen 69504 for genomic DNA isolation
QIAquick PCR Purification kit Qiagen 28106 for PCR product purification
Gel extraction kit  Qiagen 28706 for gel extraction if there are more PCR product
HindIII NEB R3104L restriction enzyme
BamHI NEB R3136L restriction enzyme
FastAP Thermo Scientific EF0651 release of 5'- and 3'-phosphate groups from DNA
T4 DNA ligase NEB M0202 for ligation
QIAprep Spin Miniprep kit Qiagen 27106 for plasmid isolation
DMEM Gibco 31966-021 ES media
FBS Hyclone SH30070.03 ES media
MEM Non-Essential Amino Acid Sigma M7145 ES media
Penicillin-Streptomycin Sigma P4333 ES media
Beta Mercaptoethanol Sigma M6250 ES media
FuGENE HD  Promega E2311 transfection reagent
Opti-MEM® I Reduced Serum Medium Life Technologies 31985-062 for transfection
All-trans retinoic acid Sigma R2625 ligand, for activation of RAR/RXR
96-well clear plate Greiner 655101 for Beta galactosidase assay
96-well white plate Greiner 655075 for Luciferase assay
D-luciferin, potassium salt Goldbio.com 115144-35-9 for Luciferase assay
ATP salt Sigma A7699-1G for Luciferase assay
MgSO4x 7H2O Sigma 230391-25G for Luciferase assay
HEPES Sigma H3375-25G for Luciferase assay
Na2HPO4 x 7H2O Sigma 431478-50G for Beta galactosidase assay
NaH2PO4 x H2O Sigma S9638-25G for Beta galactosidase assay
MgSO4 x 7H2O Sigma 230391-25G for Beta galactosidase assay
KCl Sigma P9541-500G for Beta galactosidase assay
ONPG (o-nitrophenyl-β-D-galactosidase) Sigma N1127-1G for Beta galactosidase assay
TRIzol® Life Technologies 15596-026 RNA isolation
High-Capacity cDNA Reverse Transcription Kit Life Technologies 4368814 reverse transcription of eRNA
Rnase-free Dnase Promega M6101 Dnase treatment
SsoFast Eva Green BioRad 750000105 RT-qPCR mastermix
CFX384 Touch™ Real-Time PCR Detection System BioRad qPCR machine
BioTek Synergy 4 microplate reader BioTek luminescent counter

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Citar este artículo
Simandi, Z., Horvath, A., Nagy, P., Nagy, L. Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation. J. Vis. Exp. (112), e53978, doi:10.3791/53978 (2016).

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