Formaldehyde-bijgewoonde isolatie van regelgevende elementen maatregel chromatine toegankelijkheid in zoogdiercellen

The overall goal of this experiment is to determine the chromatin accessibility of a genetic locus by covalently cross-linking the DNA to the associated proteins, followed by purification and quantification of the free DNA. This method can help to answer key questions in the chromatin field as DNA accessibility can be determined, regardless of the cell type in untreated cells and also in cells undergoing chromatin remodeling. The main advantage of this technique is that it does not require the use of enzymes to cleave the DNA or antibodies that need to be titrated for each experimental setting.

This protocol has the additional advantage that it does not require any special equipment other than a Sonicator. Demonstrating the procedure will be Olesja Ritter and Tabea Riedlinger, who are PhD students from my laboratory. To begin the protocol, add formaldehyde directly to the growth medium of cells that were seeded one day before, to reach a final concentration of 1%volume by volume.

Incubate the medium for 10 minutes at room temperature and slew the plates manually every two minutes. Next, add glycine to a final concentration of 0.125 molar to quench the formaldehyde. Incubate the solution for five minutes at room temperature and slew it every two minutes.

Aspirate the medium and then carefully add ice-cold PBS to the side of the dish to wash the cells. Aspirate the medium and carefully repeat the wash twice. After the third wash, re-suspend the cells in one milliliter of ice-cold PBS and use a cell scraper to detach the cells, if necessary.

Transfer them to a 1.5 milliliter reaction tube on ice. Centrifuge the cells for five minutes at 300g and four degrees Celsius in a cooled tabletop centrifuge. Carefully aspirate and discard the supernatant.

Re-suspend the cell pellet in one milliliter of FAIRE lysis buffer by carefully pipetting up and down several times. Incubate the cells for 20 minutes on ice. After incubation, sonicate the cross-linked DNA to share it to an average size of 200 to 300 base pairs and cool the samples during sonication to avoid heating the sample.

The fragmentation of DNA is critical for this experiment since the size of the fragments is affecting the sensitivity of the solution of the whole technique, so it's important to establish sonication conditions carefully beforehand and to check the size of the chromatin fragments in every single experiment. Centrifuge the sample for 15 minutes and 13, 000g at four degrees Celsius. Then, transfer the supernatant to a new reaction tube.

Split the samples into 100 microliter aliquots. Use a 100 microliter aliquot to reverse the crosslink and to be used as a reference for the total DNA. Add 10 microliters of RNase A and incubate the sample for one hour at 37 degrees Celsius.

Next, add 10 microliters of proteinase K.Use a programmable thermoblock to incubate the sample for four hours at 37 degrees Celsius and then for six hours at 65 degrees Celsius to cleave the proteins and to reverse the crosslinks. Obtain a new, 100 microliter aliquot, add 10 microliters of RNase A and incubate the sample for one hour at 37 degrees Celsius. After incubation, proceed with the non de-crosslinked sample and the de-crosslinked sample from the previous section in parallel.

Add H2O to reach a final volume of 300 microliters. Then, add 300 microliters of phenol chloroform isoamyl alcohol in a fume hood and vortex vigorously. Centrifuge the samples for 10 minutes at 13, 000g and four degrees Celsius.

Next, carefully transfer 280 microliters of the upper aqueous phase to a new reaction tube. Make sure not to take any debris from the interphase, which also contains proteins, and discard the remaining lower phase as organic solvent waste. Repeat the treatment and carefully transfer 270 microliters of the upper aqueous phase to a new reaction tube.

Add 270 microliters of chloroform in a fume hood and vortex vigorously. Centrifuge the sample. After centrifugation, carefully transfer 250 microliters of the upper aqueous phase to a new reaction tube, taking care not to carry any debris from the interphase.

Discard the remaining sample as organic solvent waste. Then, add 25 microliters of five molar sodium chloride, 250 microliters of isopropanol and add five microliters of glycogen as the DNA carrier. Invert the tubes and incubate them at room temperature.

After incubation, centrifuge the sample to precipitate the DNA. Next, wash the DNA pellet with 400 microliters of 70%volume by volume ethanol and centrifuge the sample. Aspirate the supernatant carefully and let the pellet dry for 10 minutes at room temperature.

Once the pellet is dry, re-suspend it in 100 microliters of TE buffer. Incubate the non de-crosslinked free DNA sample for four hours at 65 degrees Celsius to remove inter-DNA crosslinks. Finally, quantify the amount of DNA using a spectrophotometer and run a small aliquot to check the fragment size on a 2%weight-by-volume agarose gel.

HeLA cells were stimulated with TNF for one hour and analyzed by FAIRE. The ratio between free versus total DNA was determined for the promoter of ACTB, the gene that codes for beta-actin and the positive control, a heterochromatin region on Chromosome 12, the negative control, and the IL8-Promoter. An ethidium bromide stained gel featuring different sonication times was generated and indicates the optimal chromatin size of 200 to 300 base pairs was obtained after 20 minutes of sonication.

Once mastered, this technique can be done in two days. Following this procedure, other methods such as deep sequencing can be performed in order to determine the chromatin accessibility at a genome-wide level. Don't forget that working with formaldehyde and phenol chloroform can be extremely hazardous and precautions such as working in a fume hood, wearing gloves, and appropriate disposal of the waste should be ensured by performing this procedure.