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

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?