Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

The overall goal of this chromatin immunoprecipitation procedure is to evaluate TGF beta 1-induced binding of the transcription factor SMAD2 to SMAD binding elements in the C kit promoter. This method can answer key questions in the field of cellular biology such as cytokine induced gene transcription. The main advantage of this technique is that it has the potential to demonstrate direct binding of transcription factors and other transcription regulatory proteins to their DNA binding sites in vivo.

Grow the cells to between 70 and 80%confluence on ten centimeter tissue culture plates. Stimulate the cells per the experimental aims. Remove the tissue culture medium, and add five milliliters of fixation solution.

Then, incubate the cells for 10 minutes at room temperature on a shaking platform. Remove the fixation solution, and wash the cells twice with 10 milliliters of ice cold one X PBS. Remove the one X PBS and add five milliliters of glycine stop fix solution.

After incubating the cells for five minutes at room temperature on a shaking platform, remove the glycine stop fix solution and wash the cells twice with 10 milliliters of ice cold one X PBS. After removing the PBS, add two milliliters of cell scraping solution and harvest the cells using a cell scraper. Then, transfer the harvested cells to a conical tube on ice.

Centrifuge the samples for 10 minutes at 600 times G, and four degrees celsius. Remove the supernatant, re-suspend the pellet in one milliliter of one X cell lysis buffer, and incubate on ice for 30 minutes. Once the pellet is re-suspended in the cell lysis buffer, transfer the cell suspension to an ice cold Dounce homogenizer, and Dounce for 10 strokes using a type B pestle for cell disruption.

Then, transfer the cell suspension to a microcentrifuge tube, and centrifuge the sample for 10 minutes at 2, 400 times G, and four degrees celsius. Discard the supernatant and re-suspend the pellet in 350 microliters of nuclei digestion buffer. After incubating the samples for five minutes at 37 degrees celsius, add micrococcal nuclease and mix by vortexing.

Incubate the samples at 37 degrees celsius for five to 20 minutes, mixing the samples every two minutes by inversion. The time in some concentration, if you're between cell lines, it might require optimization in case of suboptimal enzymatic shearing. An alternative shearing procedure using commercially available sonicare can be found in the text protocol.

Following the incubation with the micrococcal nuclease, add seven microliters of ice cold 0.5 molar EDTA to stop the reaction. Centrifuge the samples with the sheared DNA for 10 minutes at 16, 200 times G and four degrees celsius. Transfer the sheared DNA containing supernatant to a fresh microcentrifuge tube.

Aliquot 50 microliters into a separate microcentrifuge tube, to confirm the successful shearing of the DNA as described in the text protocol. Use the remaining volume immediately for immunoprecipitation or store it at minus 80 degrees celsius. Combine 10 to 25 micrograms of the sheared cross linked chromatin with 25 microliters of chromatin immunoprecipitation grade protein G magnetic beads.

Then, add 10 microliters of immunoprecipitation dilution buffer, and one microliter of protease inhibitor cocktail. After four hours of incubation, add one to 10 micrograms of antibody to a 1.5 milliliter microcentrifuge tube. And add distilled water to a final volume of 100 microliters.

Incubate the samples on an end-to-end rotator for four to 12 hours at four degrees celsius. Following incubation, place the tubes on a magnetic stand. Once the magnetic beads aggregate to the side of the tube, carefully remove and discard the supernatant.

Wash the beads three times with 800 microliters of wash buffer one. For each wash, mix the beads by inverting the tube several times. Then, place the tube on the magnetic stand and carefully remove the wash buffer once the magnetic beads aggregate on the side of the tube.

Next, wash the beads once with 800 microliters of wash buffer two using the same procedure. After washes, re-suspend the beads in 50 microliters of elution buffer and incubate the samples on an end-to-end rotator for 15 minutes at room temperature. Then, transfer the supernatant to a fresh tube with the aid of the magnetic stand.

Add six microliters of five molar sodium chloride to reverse the cross linking followed by two microliters of proteinase K.After mixing the samples, incubate them for two hours at 65 degrees celsius. At this point, the samples can be directly analyzed for protein binding sites as described in the text protocol, or, can be stored at minus 20 degrees celsius. To confirm the TGF beta 1-induced binding of SMAD2 to the SCF promoter, the chromatin immunoprecipitation assay was performed.

The TGF beta 1 treatment resulted in SMAD2 binding to the SCF promoter in human liver cancer lines, HepG2 and Hep3B. In the absence of TGF beta 1 stimulation, no SMAD binding was noted. As a positive control, for TGF beta 1-induced SMAD activation, chromatin immunoprecipitation for PAI-1 was performed.

The PAI-1 promoter is a known target of TGF beta and SMAD. Specificity of the SMAD2 immunoprecipitation was confirmed through use of unspecific IgG antibodies. No SMAD2 binding to the SBE was noted after immunoprecipitation with IgG.

To confirm the TGF Beta 1-induced binding of STAT3 to the TGF beta ligand gene, chromatin immunoprecipitation assays were performed using TGF beta 1 treated HepG2 and Hep3B cells. The TGF beta 1 treatment resulted in STAT3 binding to the second putative STAT3 binding site of the TGF beta gene, but not to the first one. In this example, the positive STAT3 binding to STB-2 serves as an internal positive control.

Similar to the other chromatin immunoprecipitation experiment, STAT3 binding to the ST-2 was not seen after immunoprecipitation using IgG. Once mastered, this technique can be done in one or two days depending on the antibody if it is performed properly. While attempting this procedure, it's important to follow laboratory safety guidelines and be highly accurate at every step of the protocol.

Precautions such as checking MSDS and wearing protective gear such as gloves, white coat and safety glasses should be taken while performing this procedure. After watching this video, you should have a good understanding of how to analyze protein binding to the specific DNA binding sequences. Following this protocol, other methods like promoter assays can be performed in order to confirm the functional relevance of protein DNA binding demonstrated by chromatin immunoprecipitation.