Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture (4C)

The precise spatiotemporal regulation of genes is controlled by diverse cis-regulatory elements that interact between them. Our protocol makes it possible to quantitatively interrogate chromatin contacts of selected loci such as promoters. This technique allows us to identify and quantify the interaction frequencies between various regulatory elements simultaneously.

To generate embryoid bodies, begin by culturing mouse embryonic stem cells to 60%confluency. Then remove the culture medium, and wash twice with two milliliters of sterilized PBS. Remove the PBS completely, and add two milliliters of cell detachment medium.

Incubate the culture dish at 37 degrees Celsius. After incubation for five minutes, add eight milliliters of EB differentiation medium to the dish. To dissociate the mESC colonies to obtain a single-cell suspension, pipette the medium up and down 15 to 20 times, and transfer the suspension to a 10-milliliter tube.

Centrifuge the cells at 300 times g at room temperature for five minutes. Then carefully remove the supernatant. After counting the cells, resuspend the cell pellet with EB differentiation medium.

Invert the lid of a 15-centimeter culture dish. Using a 200-microliter multichannel pipette, deposit 20-microliter drops of resuspended cells on the lid. Invert the lid carefully, and place it onto the bottom portion of the culture dish.

Incubate the dish with the hanging drops for three days. To collect the cells after incubation, wash the lid gently with 10 milliliters of PBS. Transfer the PBS, which will contain the EBs, to a 50-milliliter plastic tube.

After letting the tube sit for 30 minutes at room temperatures, carefully remove the supernatant. The EBs will be left at the bottom of the tube. Gently resuspend the EBs in 10 milliliters of fresh EB differentiation medium.

Transfer the suspension to a 10-centimeter bacteriological Petri dish. Three to six days later, use an inverted microscope to check EB formation. The EBs should be round and homogeneous in size.

Collect the EBs from two or three 10-centimeter dishes into a 50-milliliter plastic tube. Centrifuge the EBs at 300 times g for five minutes at room temperature. Carefully remove the supernatant, and resuspend the EBs in 10 milliliters of PBS.

Centrifuge the EBs at 300 times g at room temperature again, this time for three minutes. Remove the supernatant, and add two milliliters of trypsin-EDTA to the pellet. Incubate the tube at 37 degrees Celsius for 15 minutes.

During the incubation, pipette up and down every three minutes to obtain a single-cell suspension. To stop the trypsin reaction, add eight milliliters of EB differentiation medium. After resuspending the cells in fresh EB medium, add paraformaldehyde to a final concentration of 1%Incubate the cells for 10 minutes at room temperature under rotation.

Quench the formaldehyde by adding glycine to a final concentration of 0.125 molar. Complete the fixation process as described in the manuscript, and then snap-freeze the cell pellets in liquid nitrogen. Gently resuspend the cell pellet in freshly prepared ice-cold lysis buffer, and place the tube on ice.

After 15 minutes, centrifuge the cells at 1, 000 times g for five minutes at four degrees Celsius. Discard the supernatant, and wash the pellet with 500 microliters of cold lysis buffer. Resuspend the pellet in a 1.5-milliliter tube with 50 microliters of 0.5%SDS in 1x buffer two, and place the tube in a heating block at 62 degrees Celsius.

After 10 minutes, remove the tube from the heating block, and add digestion buffer. Mix well by pipetting, avoiding excessive foaming, and incubate the tube at 37 degrees Celsius. To ensure efficient restriction enzyme digestion, in this protocol, we have included a repetitive step of digestion.

That's why it's also important to resuspend well the nuclei in the digestion buffer. Add another 25 microliters of digestion buffer, mix by inverting, and take eight microliters as an undigested control. Store the undigested control sample at negative 20 degrees Celsius.

Next, you will add several aliquots of the restriction enzyme Mbol, incubating at 37 degrees Celsius under rotation after each aliquot. Take eight microliters as a digested control sample. To de-crosslink both control samples, add 80 microliters of TE buffer in 10 microliters of proteinase K.Incubate at 65 degrees Celsius for one hour.

To check digestion efficiency, run 20-microliter aliquots on a 0.6%gel. Successful digestions show fragments mostly in the range of 3.0 to 5.0 kilobase pairs. Prepare DNA for shearing as described in the manuscript.

Use a sonicator to shear the DNA to a size of 150 to 700 base pairs. Transfer the sheared DNA to a normal new safe-lock tube, pooling multiple sonications from the same sample. Add warmed DNA purification beads in an amount equal to 1.8 times the existing volume, and mix by resuspending.

After five minutes of incubation, collect the beads with a magnetic rack. Keeping the tubes in the magnetic rack, wash the beads two times with one milliliter of freshly prepared 80%ethanol. After air-drying the beads at room temperature for two to three minutes, elute the DNA by resuspending the beads in 1x Tris buffer.

Using the hanging-drop method, a homogenous population of EBs was obtained six days after the induction of ESC differentiation. Gel electrophoresis was performed throughout the protocol for quality control. Efficient digestion by Mbol results in fragments of less than three kilobase pairs.

Electrophoresis after ligation showed that most fragments were larger than three kilobase pairs. Fragment sizes of 400 to 500 base pairs are expected after sonication. After dephosphorylation and single-end adapter ligation, two rounds of PCR were performed to amplify the targets of interest.

Each target was amplified separately with two different primer pairs, A and B for the Pou5f1 locus and C and D for the T locus. This resulted in a DNA smear around 400 base pairs. Alternatively, multiplex PCR was performed to amplify targets A and C simultaneously, resulting in a similar fragment size after purification.

In these 4C profiles, the light blue boxes indicate the location of enhancers with dynamic changes during differentiation. Chromatin regions contact promoters of Pou5f1 and T genes during EB differentiation. Pou5f1 was downregulated during EB differentiation.

Conversely, T was upregulated during EB differentiation. Efficient restriction enzyme digestion and then proximity ligation of the crossing genomic DNA are key factors to ensure the success of this protocol. Another thing is the primer design to interrogate the contact map of each locus.

This procedure is ideally suited to interrogate chromatin contact at targeted loci. If global changes are required, genome-wide approaches such as Hi-C, promoter capture Hi-C, or HiChIP could be performed.