Name: Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation (Video) | JoVE Journal
Uploaded: 2016-06-21T13:00:00.000+00:00
Duration: 9 min 7 s
Description: Learn to identify and validate gene regulatory enhancers using ChIP-seq analysis and reporter assays in embryonic stem cells. This workflow enables functional characterization of enhancers during cell differentiation.

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5 minutes

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.

1. Enhancer Selection Based on Chip-seq Analysis
<ol>
	<li>Download the RXR ChIP-seq raw data fastq file (mm_ES_RXR_24h_ATRA.fastq.gz) from http://ngsdebftp.med.unideb.hu/bioinformatics/</li>
	<li>Download and extract the required BWA index file for the alignment (in our case: Mus_musculus_UCSC_mm10).(ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Mus_musculus/UCSC/mm10/Mus_musculus_UCSC_mm10.tar.gz 
	NOTE: Visit to https://github.com/ahorvath/Bioinformatics_scripts for more information regarding the steps of bioinformatics analysis and to download the scripts used below.</li>
	<li>Align the example fastq file to mm10 genome (use the script: perform_alignment.sh). This will create a folder with a .bam file and statistics of the alignment. Aligned RXR ChIP-seq data (mm_ES_RXR_24h_ATRA.bam), and the corresponding index file (.bai) are available here: 
	http://ngsdebftp.med.unideb.hu/bioinformatics/ 
	NOTE: BWA is a software package that aligns relatively short sequences (e.g., ChIP-seq results) to a sequence database, such as the mouse reference genome (e.g., mm10) 13.</li>
	<li>Run the script (callpeaks.sh) for peak calling and de novo motif analysis. Use the .bam file as the input. The script is based on Homer findPeaks 14. 
	NOTE: The output files give us information about the total number of peaks, enrichment of the motifs, the percentage of the background, and the target sequences with motifs, etc. (Figure 1). Typically several motifs are listed in order of their significance.</li>
	<li>Remap the #1 ranked motif (NR half `AGGTCA`) (use the script: remap_motif.sh) 
	NOTE: As the result, the script generates a .bed file that will show which ChIP-peaks are covering genomic regions that contain the canonical binding site of the transcription factor of interest.</li>
	<li>Download and visualize results of the aligned RXR ChIP-seq reads (mm_ES_RXR_24h_ATRA.bam (also download the .bai)) and the AGGTCA motif occurrences (mm_ES_RXR_24h_ATRA_homerpeaks_motif1_mm10s_200_remaped_mbed.bed) by Integrative Genomics Viewer (IGV)15 to identify putative RAR/RXR binding sites (Figure 2 and Figure 3). 
	NOTE: Integration of various ChIP-seq data will help to more precisely predict good candidate enhancers16,17. Recent studies revealed that active enhancers are typically enriched for P300 and correlate with H3K4me1 and H3K27ac, and are located in open chromatin regions, thereby displaying DNase-I hypersensitivity is also recommended 18-21.</li>
	<li>Use the &#34;Define a region of interest&#34; in IGV 15 to obtain 200 - 400 bp sequence of the selected genomic region and use these sequences for subsequent primer designs.</li>
</ol>
2. Reporter Assay
NOTE: The luciferin/luciferase system is used as a very sensitive reporter assay for transcriptional regulation. Depending on the enhancer activity luciferase enzyme is produced that will catalyze the oxidation of luciferin to oxyluciferin resulting in bioluminescense, which can be detected. As a first step, identified putative enhancer sequences should be subcloned into a reporter vector (e.g., TK-Luciferase 22, pGL3 or NanoLuc).
<ol>
	<li>Primer Design
	<ol>
		<li>Design PCR primers for amplification of 200 - 400 bp of putative enhancer regions by Primer3 (available here: http://bioinfo.ut.ee/primer3/)23</li>
		<li>Copy-paste the genomic sequence from IGV that includes the putative binding site into Primer3 (see step 1.7). Set product size range to 250 - 300 bp in Primer3.</li>
		<li>Validate the PCR primers using the UCSC Genome Browser&#39;s In silico PCR tool (https://genome.ucsc.edu/cgi-bin/hgPcr)24 
		NOTE: This protocol in a later step uses HindIII and BamHI restriction enzymes for cloning. Check whether the PCR amplified sequence as predicted by the UCSC In-silico PCR tool will contain AAGCTT or GGATCC restriction sites (e.g., &#160;http://nc2.neb.com/NEBcutter2/).</li>
		<li>Obtain the required primers from a commercial vendor.</li>
	</ol>
	</li>
	<li>PCR Amplification of Enhancer Sequence from Genomic DNA
	<ol>
		<li>Purify template genomic DNA from primary cells (e.g., primary embryonic fibroblasts). Use commercial kit for gDNA isolation and follow the manufacturer&#39;s instructions. 
		NOTE: High quality of gDNA is essential for successful PCR amplification. Appropriate BAC clones can be used if PCR is not easy from gDNA.</li>
		<li>Prepare 50 &#181;l PCR reaction containing 100 ng gDNA, 5 &#181;l 10x buffer, 3 &#181;l MgSO4 (25 mM), 5 &#181;l dNTP (2 mM each), 1.5 &#181;l Fwd primer (10 &#181;M), 1.5 &#181;l Rev primer (10 &#181;M), 1 &#181;l DNA polymerase 
		NOTE: Use high-fidelity DNA polymerase with a low error rate for the PCR reactions.</li>
		<li>Set the PCR cycle (2 min 95 &#176;C, (20 sec 95 &#176;C, 10 sec 58 - 65 &#176;C, 18 sec 70 &#176;C) x25 repeat, 2 min 70 &#176;C, forever 4 &#176;C). Set annealing temperature with the consideration of the primer&#39;s predicted Tm values.</li>
		<li>Purify the PCR product with a commercial PCR purification kit and follow the manufacturer&#39;s instructions.</li>
		<li>Introduce the restriction sites for cloning by repeating the PCR with primers as used above, but adding overhangs on their 5&#39; ends (e.g., Fwd: 5&#39;-ATATAAGCTTxxxxxxxxxxxxxxxxxxxx-3&#39; (HindIII), Rev: 5&#39;-TATAGGATCCxxxxxxxxxxxxxxxxxxxx-3&#39; (BamHI)).</li>
		<li>Run 5 &#181;l of the PCR product on agarose gel to check for unspecific PCR products. 
		NOTE: If more bands are detected, run the entire PCR product and purify the appropriate band by a commercial gel extraction kit.</li>
	</ol>
	</li>
	<li>Restriction 
	NOTE: To clone insert upstream of TK promoter22 the purified PCR product and the vector (e.g., TK-Luc) should be digested with HindIII and BamHI.
	<ol>
		<li>Prepare a 50 &#181;l reaction mixture containing 1 &#181;g purified PCR product, 1 &#181;l HindIII (20 U/&#181;l), 1 &#181;l BamHI (20 U/&#181;l) and 5 &#181;l 10x buffer.</li>
		<li>Prepare a 250 &#181;l reaction mixture containing 5&#181;g vector (TK Luc, or alternative reporter vector), 5 &#181;l HindIII (20 U/&#181;l), 5 &#181;l BamHI (20 U/&#181;l), 5 &#181;l thermosensitive Alkaline Phosphatase (1 U/&#181;l) and 25 &#181;l 10x buffer. 
		NOTE: AP treatment of the vector greatly decreases the self-ligation of single-digested vector and thus the background.</li>
		<li>Incubate the reaction mixtures for 4 hr at 37 &#176;C, then purify the digested PCR products and the vector with a commercial PCR purification kit.</li>
	</ol>
	</li>
	<li>Ligation
	<ol>
		<li>Set up the reaction for ligation in PCR tubes including 2 &#181;l 10x T4 DNA ligase buffer, 10 ng TK-Luc vector, 50 ng insert (digested PCR product), 1 &#181;l T4 DNA ligase (400 U/&#181;l) and nuclease-free water up to 20 &#181;l. 
		NOTE: Prepare a negative control where the reaction does not contain insert.</li>
		<li>Gently mix the reaction by pipetting up and down, spin down briefly the PCR tubes using mini-centrifuge and then incubate at RT for 10 min.</li>
		<li>Heat inactivate at 65 &#176;C for 10 min then chill on ice and use 5 &#181;l of the product for transformation into 50 &#181;l competent cells (e.g., DH5&#945;).</li>
		<li>Use standard heat shock procedure to transform bacteria, pick up colonies and isolate plasmid DNA with a commercial kit. Validate all constructs by sequencing prior to the next steps.</li>
	</ol>
	</li>
	<li>Transfection of ES Cells
	<ol>
		<li>Use 48-well plates. Add 200 &#181;l 0.1% gelatin solution/well 30 min prior to cell plating.</li>
		<li>Plate embryonic stem (ES) cells a day before transfection. Use feeder-free ES cells and plate at a density of 3 x 104 cells per well in 250 &#181;l ES media/well.</li>
		<li>Prepare the plasmid mixes (each mix is calculated for 8 wells, 4 untreated and 4 retinoic acid-treated). Mix 1: 1,250 ng TK-Luc Empty (negative control), 950 ng &#946;-Galactosidase coding vector (&#946;Gal), Mix 2: 1,250 ng TK-Luc Hoxa1 enhancer (positive control), 950&#160;ng &#946;Gal, Mix 3: 1,250 ng TK-Luc PRMT8 enhancer (region of interest), 950 ng &#946;Gal. 
		NOTE: ES cells express the desired transcription factors (RAR/RXR). Leave wells untransfected to determine base values later during the luciferase and &#946;-galactosidase measurements.</li>
		<li>Add transfection quality reduced serum media to each plasmid mix up to 106 &#181;l total volume (enough for 8 wells).</li>
		<li>Add 4.5 &#181;l ES quality transfection reagent then mix carefully by pipetting 15 times up and down. Incubate the transfection mix for 10 min at RT.</li>
		<li>Add 13 &#181;l of the transfection mix to the cells per well, mix thoroughly by pipetting. Place the cells back into the incubator (37 &#176;C) O/N before ligand treatment.</li>
		<li>Remove media carefully by aspiration from cells and add 250 &#181;l fresh media/well containing 0.01 - 1 &#181;M all-trans retinoic acid (RA) as final concentration or DMSO as vehicle. Incubate cells for 24 - 48 hr. 
		NOTE: Retinoic acid is light sensitive.</li>
	</ol>
	</li>
	<li>Preparation of Cell Lysates
	<ol>
		<li>Dilute 5x Lysis Buffer (1.25 ml 0.5 M Tris pH = 7.8, 1 ml 1 M dithiothreitol (DTT) (in H2O), 10 ml 0.1 M EDTA pH = 8.0, 50 ml glycerol, 5 ml Triton X-100, sterile water up to 100 ml) to 1x using sterile water, add 20 &#181;l 1 M DTT/10 ml and equilibrate to RT before use. Prepare 10 ml for a 48-well plate on the day of the assay.</li>
		<li>Remove media carefully by aspiration from cells. Rinse cells once with 1x PBS. Add 200 &#181;l 1x Lysis Buffer/well directly to the cells.</li>
		<li>Shake for 2 hr at RT (chemical lysis). Freeze the cell lysates on the plate at -80 &#176;C (mechanical lysis). Lysates can be stored at -80 &#176;C for several days.</li>
		<li>Thaw the lysates. After complete thawing, transfer cell lysates to a 96-well plate using an electronic multichannel pipette. Transfer 80 &#181;l of the cell lysate to 96-well clear plate for &#946;-galactosidase assay and 40 &#181;l of the cell lysate to a white plate for luciferase measurement. Avoid forming any bubbles.</li>
	</ol>
	</li>
	<li>&#946;-galactosidase Assay 
	NOTE: Beta galactosidase or alternative second reporters should be used as an internal control to account for non-specific experimental variations.
	<ol>
		<li>Prepare &#946;-galactosidase substrate buffer: 80.0 g Na2HPO4&#8226;7H2O, 3.8 g NaH2PO4&#8226;H2O, 30.0 g KCl, 1.0 g MgSO4&#8226;7H2O, make up to 1,000 ml with sterile water. Adjust pH to 7.4. Filter Sterilize (0.2 micron) and store at 4 &#176;C</li>
		<li>Mix 1 ml &#946;-galactosidase substrate buffer with 4 mg ONPG (o-nitrophenyl-&#946;-D-galactosidase). Add 3.5 &#181;l of 2-Mercaptoethanol (&#946;ME) per 1 ml of buffer just before use. Add 100 &#181;l &#946;-galactosidase substrate buffer to 80 &#181;l cell lysate.</li>
		<li>Incubate the reactions at 37 &#176;C until a faint yellow color has developed. Read absorbance at OD 420 nm on a plate reader and export data. 
		NOTE: The time varies depending on the transfection efficiency, usually no longer than 30 min.</li>
	</ol>
	</li>
	<li>Luciferase Assay
	<ol>
		<li>Prepare 50 ml luciferin substrate reagent: 15.1 mg D-luciferin salt, 82.7 mg ATP salt, 185 mg MgSO4&#8226;7H2O, add to 50 ml polypropylene tube, then add 1.5 ml 1 M HEPES buffer, 48.5 ml ATP free water, vortex, make sure all compounds dissolve completely. Keep 5 - 10 ml aliquots at -80 &#176;C.</li>
		<li>Warm 5 ml luciferin substrate reagent to RT before starting. Pipette 100 &#181;l substrate reagent to each well of the white plate containing the 40 &#181;l of the cell lysate and measure the signal immediately using a luminescent counter machine.</li>
		<li>Obtain activities from at least three independent experiments in triplicate transfections.</li>
		<li>Calculate first the base normalized luciferase activity and base normalized &#946;-galactosidase values for each well by subtracting the average values measured for non-transfected cells from each value measured. Then calculate the ratio of luciferase/&#946;-galactosidase activity.</li>
	</ol>
	</li>
</ol>
3. Characterization of Enhancer RNA
NOTE: A more direct indicator of enhancer activity has emerged from recent genome-wide studies that identified many short non-coding RNAs, ranging in size from 50 to 2,000 nt, which are transcribed from enhancers, and are termed enhancer RNAs (eRNAs)16,25,26 (Figure 4). eRNA induction highly correlates with the induction of adjacent exon-coding genes. Thus, signal-dependent enhancer activity can be quantified in vivo by comparing eRNA production between various conditions by RT-qPCR.
<ol>
	<li>Guidelines for Primer Design for eRNA&#160;detection by RT-qPCR
	<ol>
		<li>Select genomic regions for eRNA measurements that are at least 1.5 - 2 kb away from annotated transcription start sites. 
		NOTE: Importantly, high levels of mRNA transcription across intragenic enhancers prevent accurate quantification of eRNA in the sense orientation, antisense eRNAs at intragenic enhancers are detectable and are similar in level to eRNAs at extragenic enhancers.</li>
		<li>As a general rule, use regions 200 - 1,000 bp from the center of the transcription factor binding site of interest for primer design either on the sense or anti-sense strand (Figure 4 and Figure 6). 
		NOTE: If GRO-seq, DNase-seq, H3K4me1, H3K4me3 or H3K27ac ChIP-seq data are available from the desired cell types and conditions it can be used for more accurate primer design. eRNAs are transcribed from putative enhancer regions characterized by high levels of H3K4me1 and me3. Expression of eRNAs positively correlates with the enrichment of activated enhancer histone mark H3K27ac.</li>
		<li>Design PCR primers for fluorescent dye-based RT-qPCR measurement of eRNAs by Primer3 (http://bioinfo.ut.ee/primer3/)23Generate anti-sense sequence (reverse complement of the sense strand) for primer design as per: http://www.bioinformatics.org/sms/rev_comp.html. Insert 200 - 300 bp of sense or anti-sense sequence for primer design.</li>
	</ol>
	</li>
	<li>Measurement of eRNA Transcription
	<ol>
		<li>Isolate total RNA from treated cells with commercial acidic phenol/chloroform-based extraction reagent according to the manufacturer&#39;s recommendations.</li>
		<li>Use DNaseI to treat isolated RNA prior to using them in reverse transcription reaction. Inactivate DNase according to the manufacturer&#39;s recommendation prior to reverse transcription.</li>
		<li>Measure RNA concentration after DNase I treatment and use 1,000 ng per RT reaction. Use high quality reverse transcriptase. 
		NOTE: As large number of eRNAs may not be polyadenylated, reverse transcription requires the use of random primers.</li>
		<li>Detect reverse transcribed eRNA by RT-qPCR using standard procedure. Measure a non-treatment-dependent mRNA for normalization (e.g., 36B4, Ppia, Gapdh, Actb).</li>
	</ol>
	</li>
</ol>

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<li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reprogramming+transcription+by+distinct+classes+of+enhancers+functionally+defined+by+eRNA%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA.</a> Nature. 474, 390-394 (2011).</li>
<li>Simandi, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PRMT1+and+PRMT8+regulate+retinoic+acid-dependent+neuronal+differentiation+with+implications+to+neuropathology%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">PRMT1 and PRMT8 regulate retinoic acid-dependent neuronal differentiation with implications to neuropathology.</a> Stem Cells. 33, 726-741 (2014).</li>
<li>Zhou, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Sox2+distal+enhancer+cluster+regulates+embryonic+stem+cell+differentiation+potential%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential.</a> Genes Dev. 28, 2699-2711 (2014).</li>
</ol>

The authors have nothing to disclose.

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...

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 (RAR&#945;, &#946;, &#947;) and retinoid X receptors (RXR&#945;, &#946;, &#947;). 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.

<table><tbody><tr><td>KOD DNA polymerase</td><td>Merck Millipore</td><td>71085-3</td><td>for PCR amplification of enhancer from gDNA</td></tr><tr><td>DNeasy Blood &amp;amp; Tissue kit&amp;nbsp;</td><td>Qiagen</td><td>69504</td><td>for genomic DNA isolation</td></tr><tr><td>QIAquick PCR Purification kit</td><td>Qiagen</td><td>28106</td><td>for PCR product purification</td></tr><tr><td>Gel extraction kit&amp;nbsp;</td><td>Qiagen</td><td>28706</td><td>for gel extraction if there are more PCR product</td></tr><tr><td>HindIII</td><td>NEB</td><td>R3104L</td><td>restriction enzyme</td></tr><tr><td>BamHI</td><td>NEB</td><td>R3136L</td><td>restriction enzyme</td></tr><tr><td>FastAP</td><td>Thermo Scientific</td><td>EF0651</td><td>release of 5&#039;- and 3&#039;-phosphate groups from DNA</td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202</td><td>for ligation</td></tr><tr><td>QIAprep Spin Miniprep kit</td><td>Qiagen</td><td>27106</td><td>for plasmid isolation</td></tr><tr><td>DMEM</td><td>Gibco</td><td>31966-021</td><td>ES media</td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30070.03</td><td>ES media</td></tr><tr><td>MEM Non-Essential Amino Acid</td><td>Sigma</td><td>M7145</td><td>ES media</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td>ES media</td></tr><tr><td>Beta Mercaptoethanol</td><td>Sigma</td><td>M6250</td><td>ES media</td></tr><tr><td>FuGENE HD&amp;nbsp;</td><td>Promega</td><td>E2311</td><td>transfection reagent</td></tr><tr><td>Opti-MEM&amp;reg; I Reduced Serum Medium</td><td>Life Technologies</td><td>31985-062</td><td>for transfection</td></tr><tr><td>All-trans retinoic acid</td><td>Sigma</td><td>R2625</td><td>ligand, for activation of RAR/RXR</td></tr><tr><td>96-well clear plate</td><td>Greiner</td><td>655101</td><td>for Beta galactosidase assay</td></tr><tr><td>96-well white plate</td><td>Greiner</td><td>655075</td><td>for Luciferase assay</td></tr><tr><td>D-luciferin, potassium salt</td><td>Goldbio.com</td><td>115144-35-9</td><td>for Luciferase assay</td></tr><tr><td>ATP salt</td><td>Sigma</td><td>A7699-1G</td><td>for Luciferase assay</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>230391-25G</td><td>for Luciferase assay</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-25G</td><td>for Luciferase assay</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4 &lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>431478-50G</td><td>for Beta galactosidase assay</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;&amp;#8226;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>S9638-25G</td><td>for Beta galactosidase assay</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>230391-25G</td><td>for Beta galactosidase assay</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541-500G</td><td>for Beta galactosidase assay</td></tr><tr><td>ONPG (o-nitrophenyl-&amp;beta;-D-galactosidase)</td><td>Sigma</td><td>N1127-1G</td><td>for Beta galactosidase assay</td></tr><tr><td>TRIzol&amp;reg;</td><td>Life Technologies</td><td>15596-026</td><td>RNA isolation</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Life Technologies</td><td>4368814</td><td>reverse transcription of eRNA</td></tr><tr><td>Rnase-free Dnase</td><td>Promega</td><td>M6101</td><td>Dnase treatment</td></tr><tr><td>SsoFast Eva Green</td><td>BioRad</td><td>750000105</td><td>RT-qPCR mastermix</td></tr><tr><td>CFX384 Touch&amp;trade; Real-Time PCR Detection System</td><td>BioRad</td><td></td><td>qPCR machine</td></tr><tr><td>BioTek Synergy 4 microplate reader</td><td>BioTek</td><td></td><td>luminescent counter</td></tr></tbody></table>

prediction and validation of gene regulatory elements activated during retinoic acid induced embryonic stem cell differentiation

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

We used a pan-specific RXR antibody in order to identify genome-wide which RA-regulated genes have receptor enrichment in their close proximity. Bioinformatics analysis of RXR ChIP-seq data obtained from ES cells treated with retinoic acid revealed the enrichment of the nuclear receptor half site (AGGTCA) under the RXR occupied sites (Figure 1). Using a bioinformatics algorithm we mapped back the motif search result for the half site to the RXR ChIP-seq data (Figu...

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

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

The overall goal of this video is to provide an integrated set of methods to identify and experimentally validate geneomic regulatory elements so called enhancers. This method can help answer key questions in the transcription and regulation of biologic events, including enhancer selection and usage, using cell differentiation, or in response to external stimuli. The main advantage to the presented work-flow is that a protocol can be easily adapted and used in any cellular model systems.I will demonstrate the steps of the procedure. To begin, choose a candidate enhancer based on pre-existing chip seq analysis. Use a genome browser to view the results of the desired chip seq experiment, and identify binding sites in the proximity of the gene of interest.Choose Define a Region of Interest to obtain a 200-400 base pair sequence of the selected genomic region, and use the sequences for subsequent primer design. With standard molecular biology techniques, amplify and clone the selected genomic region into a reporter plasmid construct. To test enhancer activity, transfect the reporter constructs into mass embryonic stem cells by first adding 200 micro liters of 0.1%gelatin solution per well to 48 well plates.Incubate the plates for 30 minutes prior to plating. The day before transfection, plate feeder-free embryonic stem, or ES cells, in 250 micro liters of ES medium at a density of three times 10 to the fourth cells per well. On the day of transfection, prepare the plasmid mixes, making enough for eight wells from each mix.Add transfection quality reduced serum medium to each plasmid mix to bring the volume up to 106 micro liters. Then, add 4.5 micro liters of ES quality transfection reagent, and carefully mix by pipetting 15 times up and down. Incubate the transfection mix for 15 minutes at room temperature.After the incubation, add 13 micro liters per well of the transfection mix to the cells and mix thoroughly by pipetting. Then, place the cells back into the incubator over night before lichen treatment. Transfection efficiency is a critical step.If another cell type is used, this step requires optimization. Use the most relevant cell type to test enhancer activity to minimize the context-dependent effects. The following day, carefully remove the medium by aspiration, and add 250 micro liters of fresh medium per well containing one micro molar all trans retinoic acid, or DMSO as a vehicle.Incubate the cells for 24-48 hours. After preparing lysis buffer according to the text protocol, carefully aspirate the medium from the cells. Use 1X PBS to rise the cells once, then add 200 micro liters of 1X lysis buffer per well.Shake the cells at room temperature for two hours. Then, for complete cell lysis, freeze the cell lysates in the plate at 80 degrees Celsius. After completely thawing the lysates, use an electronic multi-channel pipet to transfer 80 micro liters of the cell lysate to 96 well clear plate for the beta galactosidase, and 40 micro liters of the lysate to a white plate for luciferase measurement.To carry out the beta galactosidase, mix one milliliter of the beta gal substrate buffer with four milligrams of OMPG. Then, add 3.5 micro liters of beta mercaptoethanol to the mixture and immediately add 100 micro liters of the buffer to the 80 micro liters of cell lysate. Incubate the reactions at 37 degrees Celsius until the faint yellow color develops.Read the absorbence at 420 nano meters on a plate reader, and export the data. To perform the luciferase assay, after preparing the substrate reagent according to the text protocol, warm the luciferase substrate reagent to room temperature before starting. Then, pipet 100 micro liters into each well of the white plate containing the 40 micro liters of cell lysate.Immediately use a luminescent counter machine to measure the signal. Obtain the activities from at least three independent transfections and experiments. Perform calculates for both assays according to the text protocol.To design the primers for eRNA detection by RTCPR, select a genomic region that is at least 1.5 to two kilo bases away from any annotated gene transcripts. Identify the relative direction of the gene, and determine the position of the sense and anti-sense transcripts. To design primers for the enhancer RNA quantification, choose regions 200-1, 000 base pairs from the center of the transcription factor binding site.If the gene is located on the minus strand, copy 200-300 base pairs of the positive strand and convert the sequence to get the reverse complement sequence. Then, use this sequence for subsequent primer design. Set the Product Size Range between 80-150 base pairs.To measure eRNA transcription, after isolating total RNA from treated cells according to the text protocol, detect reverse transcribed eRNA by RTCPR using a standard procedure. Measure a non-treatment dependent mRNA for normalization. As shown here, bioinformatics analysis of RXR chip seq data obtained from ES cells treated with retinoic acid revealed the enrichment of the nuclear receptor half site under the RXR occupied sites.Using a bioinformatics algorithm, the motif search results for the half-site was mapped back to the RXR chip seq data. This analysis identified chip peaks overlapping with canonical nuclear receptor binding sites. Visualization of the sites in IGV indicated enrichment of the transcription factors close to Hoxa1.A previously characterized RAR RXR target. A punitive enhancer for a novel RA target chain, PRMT8, was also identified. The identified enhancer regions were cloned into TK luciferase reporter vector, and ES cells were transfected with these constructs.Luciferase activity was measured in the absence and presence of RA.The constructs containing the RA response element, or RARE, showed increased luciferase signal intensity upon RA treatment, while the construct without the RARE was not induced. To ascertain if the sense and anti-sense eRNA expression of the Hoxa1 RAR RXR bound enhancer correlates with the mRNA expression of the gene, the level of Hoxa1 eRNA was also measured. The results confirmed that the enhancer activity is induced by short term RA treatment.Once mastered, this workload can be done in a few days by carrying out the described enhancer trap experiment and enhancer quantification, one can provide strong functional evidence for enhancer activity. After watching this video, you should have a good understanding of how to identify and characterize punitive enhancers. Once an enhancer is identified and validated, other methods, such as chromosomal confirmation capture, or CCC, can be performed in order to answer additional important questions, like which gene is regulated by the validated enhancer?

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Learn to identify and validate gene regulatory enhancers using ChIP-seq analysis and reporter assays in embryonic stem cells. This workflow enables functional characterization of enhancers during cell differentiation.

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation (Video) | JoVE Journal

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

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