The overall goal of this procedure is to determine the genome-wide binding profiles of polyamides and other DNA-targetting small molecules in cells. This method can help advance the field of drug DNA chemistry by using the genome to guide the design of DNA-targeting molecules. The main advantage of this technique is that direct ligand DNA interactions are measured to provide insight into the mechanism of action for ligands that act on the genome.
Some of most successful therapeutic agents are small molecules that bind to DNA and interfere with genomic transactions. Though this method was developed to study polyamides it can also be applied to other classes of DNA-targeting molecules. The cells for this experiment are grown in E8 Media on 10 cm dishes, with the surface coat that supports pluripotent stem cells at 37 degrees Celsius in a humidified atmosphere of 5%carbon dioxide.
Prior to adding the polyamide, remove the spent culture media and add 8ml of fresh media per 10 cm dish. From this point onward, protect the cells from light to avoid premature photo-cross-linking. Using a pipette add the polyamide directly to the culture media of each dish.
Swirl the dish to disperse the polyamide evenly into the media. Incubate the cells at 37 degrees Celsius in a humidified atmosphere of 5%carbon dioxide for 24 hours and ensure they are protected from light. On the following day, wash each 10 cm dish with 4 ml of PBS using a serological pipette and a pipette dispenser.
Aspirate the PBS and add 3 ml of E8 culture media. With the lights dimmed, place the cells on a flat surface outside of the hood and remove the lids of the five culture dishes. Place a glass filter over the five culture dishes to filter out light with lambda less than 300 nanometers.
Place the UV source on top of the filter. Cross-link the samples for 30 minutes with 365 nanometers UVA radiation. After the UVA radiation, transfer the culture dishes back to the hood.
Aspirate the media with a pasteur pipette attached to a vacuum trap and wash each 10 cm dish with 4 ml of PBS. Aspirate the PBS and add 3 ml of enzyme for cell dissociation per 10 cm dish. Incubate for five minutes at room temperature.
Quench the enzyme with 3 ml of E8 Media per dish. Transfer the dissociated cells into one 15 ml conical tube. Place the cells on ice from this point onward.
Centrifuge the dissociated cells at 500 x g and 4 degrees Celsius for five minutes. Aspirate the supernatant to remove the enzyme and media. To begin the procedure for Chromatin isolation prepare the cosmic buffer with fresh protease inhibitors.
Add 12.1 microliters each of 100 millimolar PMSF, 100 millimolar benzamidine and 150 micromolar pepstatin protease inhibitors fresh to the basal cosmic buffer. Add 1.2 ml of the cosmic buffer to the cell pellet and pipette up and down several times to re-suspend it. Then split the cell solution into two amber 1.7 ml micro-centrifuge tubes.
Sonicate with a sonicator to fragment the genome to sizes between 100 and 500 base pairs. Keep the level of the Chromatin solution in the micro-centrifuge tube parallel to the level of water in the reservoir. Confirm this level by visual inspection.
The sonication time has been optimized imperically, sonicate at 60%power, 10 seconds on and 10 seconds off for 35 minutes. Use a minimal amount of ice in the reservoir to chill the samples, and ensure the ice is not interfering between the samples and the cup horn. When sonication is complete, centrifuge the sample at 12 000 g's for 10 minutes.
Save the aqueous solution containing soluble Chromatin by transferring it to a new amber micro-centrifuge tube. Discard the pellet. Transfer 110 microliters or 10%of the sample to a new micro-centrifuge tube.
Label it as input DNA'and store at 80 degrees Celsius. Save the rest of the Chromatin sample on ice for use in the next steps of this protocol. To capture ligand-DNA cross-links add the remainder of the Chromatin sample to the streptavidin coated magnetic beads and re-suspend the mixture.
Incubate the Chromatin with the beads for at least four hours on a rotating rocking mixer at 4 degrees Celsius. The required wash buffer should be prepared in distilled de-ionised water and filtered with a 0.2 micrometer filter before use. Buffers 1 and 2 can be stored at 4 degrees Celsius for several months, but Buffer 3 must be prepared fresh each day.
When the incubation of the Chromatin with the streptavidin coated magnetic beads is complete wash the sample for seven minutes with wash buffer 1. Place the sample in a magnetic separation rack to capture the beads and remove wash buffer 1. Add wash buffer 2 and wash for another 7 minutes.
Then wash twice with wash buffer 3 for seven minutes per wash. Lastly, wash twice with TE Buffer. After the final TE Buffer wash, re-suspend the beads in 200 microliters of TE Buffer.
This sample of captured DNA is referred to as affinity purified or AP DNA. Next, supplement the input DNA and AP DNA with 10x cross-link reversal buffer to a final concentration of 1x. Incubate for 30 minutes at 90 degrees Celsius.
Place the micro-centrifuge tube containing the AP DNA on the magnetic separation rack for two minutes at room temperature, then transfer the liquid containing the AP DNA to a new amber micro-centrifuge tube. After neutralizing the Input and AP DNA add RNase A to both DNA samples to a final concentration of 0.2 micrograms per microliter. Incubate for one hour at 37 degrees Celsius.
After one hour add Proteinase K to both the Input DNA and AP DNA to a final concentration of 0.2 micrograms per microliter. Incubate for one hour at 55 degrees Celsius. Subsequently, the Input and AP DNA are purified with a DNA column clean up kit and eluted in DNA grade water.
Store the DNA at 20 or 80 degrees Celsius until ready for analysis. The Affinity Purified DNA should always be normalized against a reference of Input DNA. Representative results from quantitative PCR with locus specific primers show a greater than 100 fold increase in polyamide occupancy upon irradiation with 365 nanometer light.
Enriched DNA can also be analysed by next generation sequencing. Once sequenced, raw DNA reads are aligned to the genome and density tracks are prepared. This example shows the density track for Linear Polyamide 4 designed to target AAG repeats.
An analysis of polyamide distributions in cells revealed that clustered binding sites spanning a broad range of affinities best predict occupancy in cells. An algorithm was developed to score the entire genome for binding with invitro cognate site identification data. These violin plots show predicted scores for Polyamide 2 and Polyamide 4 binding across the entire genome.
Representative genomescapes for Polyamide 4 are also shown. This scoring method revealed that loci with multiple low and medium affinity sequences show similar polyamide occupancy to loci with few high affinity sequences. Once mastered, this technique can be completed in two days if it is performed properly.
While attempting this procedure, it's important to remember to protect the samples from light until the DNA is purified. Following this procedure, other methods like RT-PCR and RNase C can be performed in order to answer additional questions like the effect of binding on gene expression. After watching this video, you should have a good understanding on how to use cosmic to map the genome-wide binding profiles of polyamides.
