The overall goal of applying these chromatin immunoprecipitation approaches to differentiating embryonic stem cells or cardiac embryonic tissues, is to uncover the epigenetic regulation of cardiac development. Since fine tuning of gene transcription during embryonic cord development is regulated epigenetic components. Chromatin immunoprecipitation is a suitable approach to understanding this process.
The main advantage of this technique is that we can specifically target a locus and look at the epigenetic marks that sit on that specific piece of DNA. This method can provide insight into epigenetic regulation of cardiac development. It can also be applied to other organs or any cell types.
This is Imen. She is a PhD student in the lab and she would do the experiment today. This procedure utilizes embryonic stem or ES cells.
Embryoid bodies generated from ES cells and embryonic heart tissues dissected from E9.5 mouse embryos. Fix the cells and tissues. Use 1%formaldehyde in PBS for the cells and PB2 permeabilization buffer for the embryonic tissues.
Place the tubes on an orbital shaker at a speed of 60 RPM at room temperature for exactly 10 minutes. Stop the cross-linking reaction by adding glycine to a final concentration of 125 millimolar and incubate for five minutes at room temperature on the orbital shaker at a speed of 60 RPM. To begin this procedure, resuspend the pellet in 10 milliliters of PBS.
And the embryonic tissues in one milliliter of PB2. Centrifuge at 1000 times G for five minutes at four degrees Celsius. Discard the supernatant and add the respective buffer for a second wash.
Add protease inhibitors to buffers PB1 and PB2 on ice. Add one milliliter of buffer PB1 supplemented with protease inhibitors to the cell pellet and pipette up and down to resuspend the cells. In the same way, resuspend the embryonic tissues in one milliliter of buffered PB2 supplemented with protease inhibitors.
After that use a one milliliter syringe with a 21-gauge needle to homogenize the cells or tissues. Transfer the cell suspension to a clean 1.5 milliliter tube. Incubate the homogenized cells and tissues at four degrees Celsius for 10 minutes.
Spin down at 3000 times G for five minutes at four degree Celsius. Discard the supernatant and resuspend each pellet in 300 microliters of the respective sonication buffer, supplemented with protease inhibitors. Transfer the cell suspension to a certified RNase, DNase, and pyrogen-free tube to prevent any DNA degradation and incubate for 15 to 30 minutes on ice.
To sheer the chromatin, sonicate the samples at four degree Celsius using the appropriate sonication program for each material. Adjust the sonication duration depending on whether the DNA will be used for PCR or sequencing. Next, centrifuge the sonicated cell lysates for 10 minutes.
Transfer the supernatant which is the chromatin in solution from each sample to a clean 1.5 milliliter tube and discard the pellet. Take a 1.5 microliter aliquot of each sample and dilute it 1:10 with water. Assess the optical density of all samples in a nano spectrophotometer to determine protein concentration.
Prior to the chromatin immunoprecipitation, prepare protein A conjugated beads by washing them as described in the protocol text. In a clean 1.5 milliliter tube combine the antibody raised against modified histones or transcription factors, 20 microliters of the washed protein A conjugated beads and one milliliter of buffer C supplemented with protease inhibitors. Incubate the samples on a rotator at 40 RPM for at least two hours at four degree Celsius.
Wash the antibody bead complexes three times with one milliliter of buffer C as described in the protocol text. Prepare two tubes, one containing 150 micrograms of chromatin and antibody bead complexes and the other containing 150 micrograms of chromatin and 20 microliters of washed beads. Add one milliliter of buffer C supplemented with protease inhibitors to each tube and incubate the samples on a rotating wheel at 40 RPM overnight at four degree Celsius.
When the overnight immunoprecipitation is complete, set the samples in the magnetic rack and recover the supernatant containing unbound antibody chromatin. Elute the antibody bound protein by adding 150 microliters of buffer D1 to the washed materials. Incubate the samples for 20 minutes at 50 degree Celsius in a heating block.
After 20 minutes, remove the samples from the heating block and place them in the magnetic rack. Recover the supernatant in a clean 1.5 milliliter tube and discard the beads. To reverse the crosslink, add five molar sodium chloride to the supernatant to obtain a final concentration of 200 millimolar.
Prepare an input sample of chromatin in a clean 1.5 milliliter tube. Use the same amount of chromatin as that of the immunoprecipitation sample in a final volume of 150 microliters of RNase and DNase free water and five molar sodium chloride to a final concentration of 200 millimolar. Incubate the samples overnight at 65 degree Celsius.
On the following day, remove the samples from the heating block. Add EDTA to a final concentration of 12.5 millimolar and Proteinase K to a final concentration of 250 micrograms per milliliter and digest at 55 degree Celsius for two hours. Begin this procedure by preparing the beads.
First bring the beads to room temperature for at least 30 minutes. Then vortex the beads very thoroughly and quickly pipette one milliliter of beads to a clean 1.5 milliliter tube. Set the tube of beads on the magnet for two to three minutes for the solution to clarify and discard the supernatant.
Remove the tube from the magnet. Add one milliliter of 0.5 molar EDTA and mix by brief vortexing. Wash the beads twice in this manner and discard the supernatant after the final wash.
Remove the tube from the magnet. Add one milliliter of buffer E and slowly resuspend the beads. Transfer the entire mixture into 50 milliliters of buffer E.Place on a shaker and let it mix slowly at room temperature for one hour.
Subsequently, test the DNA binding beads using a one KB DNA ladder and three different ratios of volume of beads to volume of DNA. The 2.5/1 ratio was determined to work best for DNA binding. To use the beads for DNA purification, add 425 microliters or 2.5 volumes of the beads to each DNA sample and resuspend slowly by pipetting up and down about 10 times.
Incubate at room temperature for 10 minutes. Place on the magnet for five minutes. Then aspirate and discard the supernatant.
Wash twice with freshly made 80%ethanol by adding 600 microliters to each tube. Waiting for one minute, and then aspirating and discarding the supernatant. Give the tubes a quick spin.
Place the tubes on the magnet. Remove the last drops of ethanol and let dry for one minute at room temperature. Remove the sample from the magnet and elute the DNA by adding 20 microliters of DNase and RNase free water.Vortex.
Agarose gel electrophoresis revealed the sizes of DNA fragments obtained after reverse cross-link of whole sonicated DNA extracted from mouse ES cells, embryoid bodies, and embryonic cardiac tissue. A chromatin immunoprecipitation experiment was designed to look at the epigenetic regulation of epithelial-mesenchymal transition genes in wild type or genetically mutated cells, when ES cells differentiate toward a mesodermal cell fate. Binding of modified histones was monitored by PCR of the promoters or enhancers of E-Cadherin and Twist genes.
Sequential chromatin immunoprecipitation was performed on chromatin extracted from four human ES cell lines using antibodies to a chromatin activating mark followed by antibodies to a repressed mark. These marks are modified when cells are challenged by morphogens but are variable depending upon the human ES cell lines. Likely because of the original blastocysts from which they are derived.
Chromatin immuno precipitation can also be applied to chromatin extracted from specific embryonic cardiac regions. This figure shows genomic regions of the cardiac genes and Nkx2.5 and TBx5 enriched in the modified histone and one genomic region of the Shox2 pacemaker specific gene not enriched and not expressed in the ventricle. Once mastered, this technique can be done in three hours if it is performed properly.
While attempting this procedure, it is important to remember to take a sonication efficiency before starting the IP protocol. Following this procedure, other methods like Eth-ek-se-ek can be performed in order to answer additional questions like interrogating the old state of chromatin whether open or closed or monitoring nucleosome spacing. After its development this technique paved the way for researchers in the field of epigenetics to explore any epigenetic regulation of cell fate decisions in any cell type or embryonic organ.
After watching this video, you should have a good understanding of how chromatin immuno precipitation assays can be performed.
