Voorspelling en Validatie van Gene Regulatory Elements geactiveerd tijdens retinoïnezuur Induced embryonale stamcellen Differentiatie
Summary
In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.
Abstract
Embryonale ontwikkeling is een meerstaps proces waarbij activering en onderdrukking van vele genen. Enhancer elementen in het genoom is gekend dat ze weefsel en celtype-specifieke regulatie van genexpressie tijdens de celdifferentiatie. Dus, de identificatie en verder onderzoek is belangrijk om te begrijpen hoe lot van de cel wordt bepaald. Integratie van genexpressie data (bijvoorbeeld microarray of RNA-volgende) en de resultaten van chromatine immunoprecipitatie (ChIP) gebaseerde genoomwijde studies (ChIP-seq) maakt het mogelijk op grote schaal identificatie van deze regulerende gebieden. Functionele validatie van celtype enhancers vereist verdere in vitro en in vivo experimentele procedures. Hier beschrijven we hoe actief enhancers kunnen worden geïdentificeerd en experimenteel gevalideerd. Dit protocol voorziet in een stap-voor-stap workflow die bestaat uit: 1) de identificatie van regulerende gebieden van Chip-seq data-analyse, 2) het klonen en experimental validatie van putatieve regulerende potentieel van de geïdentificeerde genomische sequenties in een reporter assay, en 3) bepaling van enhancer activiteit in vivo door het meten enhancer RNA transcript niveau. De gepresenteerde protocol is gedetailleerd genoeg om iedereen te helpen bij het opzetten van deze workflow in het lab. Belangrijk, kan het protocol eenvoudig aangepast worden aan en gebruikt in een cellulair modelsysteem.
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 |
References
- Wamstad, J. A., Wang, X., Demuren, O. O., Boyer, L. A. Distal enhancers: new insights into heart development and disease. Trends in cell biology. 24, 294-302 (2014).
- Pennacchio, L. A., Bickmore, W., Dean, A., Nobrega, M. A., Bejerano, G. Enhancers: five essential questions. Nat Rev Genet. 14, 288-295 (2013).
- Mardis, E. R. ChIP-seq: welcome to the new frontier. Nature methods. 4, 613-614 (2007).
- Muller, F., Tora, L. Chromatin and DNA sequences in defining promoters for transcription initiation. Biochim Biophys Acta. 1839, 118-128 (2013).
- Ounzain, S., Pedrazzini, T. The promise of enhancer-associated long noncoding RNAs in cardiac regeneration. Trends Cardiovasc Med. , (2015).
- Lam, M. T., Li, W., Rosenfeld, M. G., Glass, C. K. Enhancer RNAs and regulated transcriptional programs. Trends Biochem Sci. 39, 170-182 (2014).
- Dickel, D. E., et al. Function-based identification of mammalian enhancers using site-specific integration. Nature methods. 11, 566-571 (2014).
- Blum, R., Vethantham, V., Bowman, C., Rudnicki, M., Dynlacht, B. D. Genome-wide identification of enhancers in skeletal muscle: the role of MyoD1. Genes Dev. 26, 2763-2779 (2012).
- Vermunt, M. W., et al. Large-scale identification of coregulated enhancer networks in the adult human brain. Cell reports 9. , 767-779 (2014).
- Bain, G., Kitchens, D., Yao, M., Huettner, J. E., Gottlieb, D. I. Embryonic stem cells express neuronal properties in vitro. Dev Biol. 168, 342-357 (1995).
- Mahony, S., et al. Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis. Genome Biol. 12 (R2), (2011).
- Simandi, Z., Balint, B. L., Poliska, S., Ruhl, R., Nagy, L. Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies. FEBS Lett. 584, 3123-3130 (2010).
- Li, H., Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 25, 1754-1760 (2009).
- Heinz, S., et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell. 38, 576-589 (2010).
- Robinson, J. T., et al. Integrative genomics viewer. Nat Biotechnol. 29, 24-26 (2011).
- Daniel, B., et al. The active enhancer network operated by liganded RXR supports angiogenic activity in macrophages. Genes Dev. 28, 1562-1577 (2013).
- Zhu, Y., et al. Predicting enhancer transcription and activity from chromatin modifications. Nucleic Acids Res. 41, 10032-10043 (2013).
- Visel, A., et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature. 457, 854-858 (2009).
- Heintzman, N. D., et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet. 39, 311-318 (2007).
- Rada-Iglesias, A., et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature. 470, 279-283 (2010).
- Bonn, S., et al. Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development. Nat Genet. 44, 148-156 (2012).
- Hollenberg, S. M., Giguere, V., Segui, P., Evans, R. M. Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor. Cell. 49, 39-46 (1987).
- Untergasser, A., et al. Primer3–new capabilities and interfaces. Nucleic Acids Res. 40, e115 (2012).
- Rhead, B., et al. The UCSC Genome Browser database: update 2010. Nucleic Acids Res. 38, D613-D619 (2010).
- Kim, T. K., et al. Widespread transcription at neuronal activity-regulated enhancers. Nature. 465, 182-187 (2010).
- Wang, D., et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature. 474, 390-394 (2011).
- Simandi, Z., et al. PRMT1 and PRMT8 regulate retinoic acid-dependent neuronal differentiation with implications to neuropathology. Stem Cells. 33, 726-741 (2014).
- Zhou, H. Y., et al. A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential. Genes Dev. 28, 2699-2711 (2014).
