Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

The overall goal of the modified chromatin immunoprecipitation and DNA supercoiling experiments is to study the association and subsequent topological modifications induced by architectural proteins on site-specific DNA damage induced by chemotherapeutic or carcinogenic agents in human cells. This methodology will allow us to address key questions about DNA damage and repair which should facilitate discovery of new pharmacological targets for cancer therapy. Our technology allows us to direct site-specific DNA damage, and facilitates evaluation of critical DNA repair processes relevant to human cancer.

To begin, 24 hours before transfection, plate 400, 000 mammalian cells per 60 millimeter dish in DMEM supplemented with 10%FBS without antibiotics. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide overnight. In a 14-milliliter round bottom tube, combine 30 microliters of room temperature transfectionary agent with 500 microliters of 1X growth medium that has been warmed to 37 degrees Celsius.

In a second tube, combine two micrograms of TFO-directed ICL-containing plasmids in 500 microliters of 1X growth medium. Incubate the tubes at room temperature for 10 minutes. Combine mix one with mix two, and pipette up and down to thoroughly mix.

Then incubate the solution at room temperature for 25 to 30 minutes. Next, use warm PBS to wash the cells twice. Then, add the transfection mix in a drop-wise fashion, distributing the solution evenly throughout the plate of cells.

Add one milliliter of growth medium to the plate, and bring the final volume to two milliliters. Mix well, and place the culture plates at 37 degrees Celsius and 5%carbon dioxide. Four hours later, replace the medium with three milliliters of growth medium with 10%FBS, and incubate an additional 16 hours.

Following the incubation, add 80 microliters of fresh 37%formaldehyde per plate to the cells, and incubate at room temperature in the dark for 10 minutes. Quench the formaldehyde cross-linking by adding 300 microliters of chilled glycine per plate, and incubate for five minutes. Then remove the medium, and use chilled PBS to wash the plates twice.

Next, after adding one millimeter of chilled PBS, collect the cells by scraping into a microcentrifuge tube on ice, keeping samples on ice at all times. Prepare cell pellets by centrifuging at 13, 400 times G, and four degrees Celsius for five minutes. Then, remove the supernatant, and place the pellets on ice.

Use one milliliter of chilled Buffer A to homogeneously re-suspend the pellets, and incubate on ice for 10 minutes. Then, pellet the cells as before. Now, with one milliliter of chilled Buffer B, homogeneously re-suspend the pellets, and incubate on ice for 10 minutes with occasional mixing by inverting.

Then pellet the cells as before. After re-suspending the cells again, in 200 microliters of chilled Buffer B, add two microliters of micrococcal nuclease, or MNase, and incubate the reaction at room temperature for 10 minutes. Then, stop the MNase reaction by placing the tubes on ice, and adding 40 millimolar EDTA.

The level of digestion by micrococcal nuclease is very critical, because overdigestion may cause loss of signal, whereas underdigestion may cause a lot of nonspecific background. After pelleting the cells, re-suspend the pellets in 100 microliters of chromatin immunoprecipitation, or ChIP buffer, supplemented with protease inhibitor cocktail. Then, transfer the cell suspension to a thin-walled microcentrifuge tube.

Sonicate the samples by floating them on a water bath sonicator for 20 seconds, followed by 20 seconds on ice. Repeat the sonication steps nine times to generate approximately 800 to 1, 200 base-pair fragments. To 20 microliters of the sonicated samples, add three microliters of five molar sodium chloride, and one microliter of RNase A.Vortex the tubes, and incubate them at 37 degrees Celsius for 30 minutes.

Then, add two microliters of proteinase K, and incubate at 65 degrees Celsius for two hours. After purifying the samples, according to the text protocol, run them on 1%agarose gel, and visualize with ethidium bromide. In three separate, pre-chilled siliconized tubes, add 30 microliters of lysate, and 70 microliters of chilled 1X ChIP buffer with added protease inhibitors.

Add one microgram of anti-IgG, anti-histone H3, or anti-HMGB1 antibodies to the respective tubes. Incubate overnight in a cold room with continuous rotation. In the cold room, after re-suspending Protein G beads, add four microliters of the beads to each reaction tube, and incubate in a cold room with rotation for another two to four hours.

Place the tubes on a magnetic rack to pellet the beads, and remove the supernatant. Add 200 microliters of 1X ChIP buffer with protease inhibitor cocktail to the pellets, and wash in the cold with rotation for five minutes. Next, with high salt 1X ChIP buffer, perform an additional wash.

Then, use 160 microliters of elution buffer to re-suspend the pellets, and incubate at 37 degrees Celsius with rotation for 30 minutes. Pellet the beads, and collect the supernatant in a fresh tube. Add six microliters of five molar sodium chloride, and two microliters of proteinase K, and incubate overnight at 65 degrees Celsius.

After purifying the DNA, according to the text protocol, perform PCR reactions using primer sets to the region or regions of interest, and resolve the products on a 1%agarose gel, stained with ethidium bromide. Prior to use, thaw previously prepared DNA repair synthesis buffer on ice, and add 40 millimolar phosphocreatine, and 2.5 micrograms of creatine phosphokinase. After preparing 10X supercoiling buffer, according to the text protocol, use one microliter of vaccinia topoisomerase 1 with 1X supercoiling buffer in a 10 microliter reaction to linearize 300 nanograms of supercoiled plasmid, PCA FG1.

To the relaxed plasmids, add 25 microliters of freshly-thawed HeLa cell extract, and DNA synthesis buffer. Mix well, then add one microliter of vaccinia topoisomerase 1 to the reaction, and incubate at 37 degrees Celsius for one hour. Terminate the reactions by adding 1%SDS, and 0.25 milligrams per milliliter of proteinase K, and incubate at 65 degrees Celsius for two hours.

After casting a 1%agarose gel with 1X TPE buffer, add DNA-loading dye to the reactions, and load the samples directly onto the gel at least four lanes apart. Run the gel for eight to 10 hours at two volts per centimeter in 1X TBE for the first dimension at room temperature to resolve the topoisomers. Prepare two liters of 1X TBE with three micrograms per milliliter of chloroquine phosphate.

Soak the gel in 200 milliliters of the solution, covered in aluminum foil for 20 minutes. To electrophorese the gel in the second dimension, turn the gel 90 degrees in a clockwise fashion, and run it for four to six hours at two volts per centimeter in chloroquine containing 1X TBE to resolve the positive and negative supercoils. The amount of chloroquine in the electrophoresis buffer, and in the gel, is very critical, as well as running the gel at a very low voltage for the second dimension is very critical to separate the positive and the negative supercoils.

After staining and destaining the gel, according to the text protocol, use an imager to visualize the thermodistribution of the topoisomers. As seen in this gel, approximately 79%of the plasmids harboring TFO-directed ICLs migrate with a slower mobility through the agarose gel matrix when compared to the uncross-linked control plasmids. ChIP assays performed on lysates from human U2OS cells transfected with either control plasmid or plasmids containing TFO-directed ICLs show enrichment of HMGB1 at the P-ICL region compared to the control region when immunoprecipitated with anti-HMGB1 antibody.

When HMGB1 was depleted from the cells via siRNA treatment, the P-ICL-specific enrichment was diminished. When the same samples were immunoprecipitated using an anti-IgG antibody, minimal PCR amplification was detected. Finally, in this figure, two-dimensional agarose gel electrophoresis demonstrates architectural modification of the TFO-directed ICL-containing plasmids as negative supercoiling by HMGB1 in HeLa cell extracts.

Once mastered, this technique can be performed in approximately five days, if performed correctly. While attempting this procedure, it's very important to remember that all the washes should be done in the cold room, as room temperature washes may increase the background signal. Following this procedure, other methods, such as siRNA-mediated protein depletion, followed by mutation screening assays, can be performed in order to answer questions, such as, is the protein of interest involved in DNA repair?

Or, does it significantly alter the mutation spectra during the process? This technique paved the way to study association between proteins and site-specific DNA damage. After watching this video, you should have a good idea of how to study the association of architectural proteins to target a DNA cross-links, and subsequently, determine the effect of such association on the topology of the DNA.

Don't forget that working with ethidium bromide can be extremely hazardous, and therefore, proper protective gear should be always worn during performing this experiment.