Previsione e Validazione del gene regolatore elementi attivato durante retinoico Acid Induced embrionali cellule staminali Differenziazione
Summary
In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.
Abstract
Lo sviluppo embrionale è un processo a più fasi che coinvolge l'attivazione e la repressione di molti geni. Elementi Enhancer nel genoma sono noti per contribuire al tessuto e cellula-tipo regolazione specifica dell'espressione genica durante la differenziazione cellulare. Pertanto, la loro identificazione e un'ulteriore indagine è importante per comprendere come il destino della cellula è determinata. Integrazione dei dati di espressione genica (ad esempio, microarray o RNA-Seq) e risultati di immunoprecipitazione della cromatina (ChIP) basata su studi genome-wide (ChIP-Seq) permette l'identificazione su larga scala di queste regioni regolatorie. Tuttavia, validazione funzionale di esaltatori specifici di tipo a cella richiede ulteriori in vitro e in vivo procedure sperimentali. Qui si descrive come esaltatori attivi possono essere identificati e validati sperimentalmente. Questo protocollo fornisce un flusso di lavoro passo-passo che comprende: 1) l'identificazione di regioni regolatorie attraverso l'analisi, 2) la clonazione e exper dati ChIP-Seqconvalida imental del potenziale normativo putativo delle sequenze genomiche identificate in un saggio giornalista, e 3) determinazione dell'attività enhancer in vivo misurando il livello trascrizione di RNA enhancer. Il protocollo presentato è abbastanza dettagliata per aiutare chiunque a creare questo flusso di lavoro nel laboratorio. Soprattutto, il protocollo può essere facilmente adattato e utilizzato in qualsiasi sistema modello cellulare.
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
Representative Results
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…
Disclosures
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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