The overall goal of this procedure is to characterize the proteomic composition of functionally distinct chromatin domains in order to understand how the different elements synergize to determine the transcriptional state.Eight. This is accomplished by first preparing nuclei from cultured cells and digesting chromatin into fragments of optimal length through enzymatic or mechanical means. The second step is to purify a distinct functional domain from bulk chromatin by immunoprecipitation using bait antibodies against either a histone post-translational modification or a protein known to specifically mark the region of interest.
Next, the immuno purified chromatin proteins are separated by SDS page and ingel digested prior to mass spectrometry. The following step is the liquid chromatography mass spectrometry analysis on a high resolution instrument. Ultimately, mass spectrometry raw data are analyzed by ad hoc software, followed by manual inspection of the spectra to identify and quantify histone modifications and coen enrich proteins based respectively on either the calculation of the relative abundance in immuno precipitated material relative to input or on the SLAC ratio and competition experiments using soluble peptide as competitor to the bait Through crop, we can gain insight into the interplays between histo modification pattern and variance and reach that specific chromatin regions and the nuclear interac recruited thereby, which all act in a concerted manner.
To define a specific chromatin landscape with regulatory effect on gene expression, the main advantage of crop over existing method based on in vitro screening for soluble interact or histo modification, such ass pull down. Using either histone peptide or reconstituted chromatin templates is the possibility to investigate locus specific chromatin composition in more native, less artificial condition. The application of crop can also be extended to other model system or in time dependent study.
For instance, we can use crop in a cell line that respond to certain perturbation eliciting a global transcriptional response. Demonstrating this procedure will be Monica Sodi. A postdoc in my lab Nuclei for the N chip procedure are prepared from unlabeled.
He A S3 cells use one to two times 10 to the eighth cells per experiment. To harvest cells, make four aliquots of five times 10 to the seven cells in 50 milliliter tubes and centrifuge at 340 times G for 10 minutes at four degrees Celsius. Wash cells with ice cold PBS discard the supernatant and resus.
Suspend each cell pellet in eight milliliters, lysis, buffer, and transfer in 15 milliliter tubes. Incubate for 10 minutes at four degrees Celsius on a rotating wheel. Next, carefully pour each cellular lysate on a sucrose cushion and centrifuge in a swing out rotor at 3, 270 times G for 20 minutes at four degrees Celsius to separate nuclei from the cytoplasm.
Discard the supernatants and wash the nu nuclear pellets twice in ice. Cold PBS at the second wash. Pool the nuclei in one 50 milliliter tube after discarding the supernatant from the second wash.
Resuspend a nuclear pellet in one milliliter of digestion buffer. Divide each sample into two aliquots of 500 microliters and keep on ice. Add to each aliquot five microliters of MNA corresponding to a final concentration of 0.005 units of enzyme per microliter of nuclei.
Mix gently and incubate at 37 degrees Celsius for 60 minutes. Add one millimolar EDTA to stop the reaction and keep on ice. Pellet the digested nuclei by centrifugation at 7, 800 times G at four degrees Celsius for 10 minutes.
Transfer the supernatants to two tubes and store at four degrees Celsius. This is the S one fraction which contains the first soluble fraction of chromatin comprising mononucleo zones carefully resuspend pellets in one milliliter of dialysis, buffer and dialyze overnight at four degrees Celsius in three liters of dialysis Buffer with constant mild stirring on the following day. Collect dialyzed material and centrifuge at 7, 800 times G for 10 minutes at four degrees Celsius.
Transfer the super natin to a new einor tube. This is the S two fraction, which dye to tta nucleosomes depending on the extent of M a's digestion. Store at four degrees Celsius prior to immunoprecipitation.
Check the quality and deficiency of MN a's digestion by aros gel electrophoresis and visual inspection of the nucleosomes ladder as depicted in this example, fraction S one is highly enriched in mononucleo zones while fraction S two contains mainly nucleosome for chromatin immunoprecipitation. First add one volume of chip dilution buffer to fraction S one. Then add the antibody against the histone post-translational modification or protein of interest and incubate overnight on a rotating wheel at four degrees Celsius on the following day.
Isolate the chromatin using protein G coupled magnetic beads. Add 100 microliters of blocked beads to the S one chromatin sample and incubate on a rotating wheel at four degrees Celsius for three hours. After three hours.
Centrifuge at 340 times G for one minute. Put the einor tubes in a magnetic rack to pellet the beads. Save the supernatant, which is the flow through containing unbound nucleosome.
Add wash buffer to the beads and incubate for five minutes at four degrees Celsius on a rotating wheel before centrifugation. In this manner, wash the beads a total of four times with increasing salt concentration. Two washes at 75 millimolar sodium chloride followed by one wash at 125 millimolar and another at 175 millimolar sodium chloride to elute.
The amino precipitated chromatin. Incubate the beads in 30 microliters of LDS Sample buffer supplemented with 50 millimolar dihi. Three etol for five minutes at 70 degrees Celsius.
Alluded proteins are subsequently separated by SDS page to begin this procedure, cross-link the cells with formaldehyde. Add 0.75%formaldehyde to SLAC labeled cells. Mix briefly and incubate for 10 minutes At room temperature.
Point the formaldehyde by adding 125 millimolar glycine and incubating for five minutes at room temperature. Next, divide cells into two to four aliquots of about five times 10 to the seven cells per aliquot. Rinse cells three times with ice cold PBS after the last wash.
Discard the supernatants and keep the pellets resuspend each cell pellet in 10 milliliters of lysis buffer. Incubate for 10 minutes at four degrees Celsius with rotation centrifuge at 430 times G for five minutes at four degrees Celsius. Discard the supernatants and keep the nuclear pellets.
After washing the nuclear pellets with washing buffer Resus, suspend each nuclear pellet in three milliliters of chip incubation buffer and keep on ice to fragment chromatin sonicate in a cooled bio rupture at 200 watts. Remove 2%of sonicate chromatin and reverse the cross-linking by incubation at 65 degrees Celsius for a minimum of one hour. In chip incubation buffer.
This is the input material. Subsequently, the DNA is extracted using a PCR purification kit and chromatin fragmentation is evaluated by aros gel electropheresis to immuno precipitate the cross-linked chromatin. Add the antibody of choice to light and heavy labeled sonicated chromatin.
In the light channel, add an excess fold of the soluble peptide in addition to the antibody aliquot both light and heavy samples to three micro centrifuge tubes each and incubate overnight at four degrees Celsius on a rotating wheel on the following day, add 100 microliters of blocked protein G coupled magnetic beads to each chromatin sample and incubate on a rotating wheel for three hours at four degrees Celsius. Centrifuge at 340 times G For one minute. Put the einor tubes in the magnetic rack to pellet the beads.
The supernatant is the flow through containing unbound nucleosomes, which is saved for subsequent controls. Wash the beads four times in washing buffer with increasing salt concentrations. Two washes at 150 millimolar sodium chloride followed by two washes at 300 millimolar sodium chloride.
Incubate the beads for 25 minutes at 95 degrees Celsius and 30 microliters of SDS page. Loading sample buffer to elute and de cross link the immuno precipitated proteins. Lastly, pool the EITs and separate the proteins by SDS page prior to mass spectrometry.
The histones enriched from NIP are digested to begin this procedure. Gel slices corresponding to the core histone bands from the colloidal kumasi stained four to 12%BIS CITRIS acrylamide gradient gel are cut out and then des stain with 50%acetyl nitrile in and deionized distilled water alternating with 100%aceto nitrile to shrink the gel when the gel pieces are completely des stain. Dry them in a vacuum centrifuge.
Add D six acetic anhydride one to nine volume per volume in one molar ammonium bicarbonate and three microliters of sodium acetate as a catalyzer to the gel pieces. Incubate for three hours at 37 degrees Celsius with strong shaking. Shrink the gel pieces in 100%acetyl nitrile and then dehydrate them for about five minutes in a vacuum centrifuge next rehydrate the gel pieces with ice cold.
100 nanogram per microliter tryin solution in 50 millimolar ammonium bicarbonate overnight at 37 degrees Celsius. The combination of chemical modification of lysines using deuterated, acetic anhydride and trypsin digestion generates an RC like in gel digestion pattern of histone collects soluble digested peptides in a new einor tube and dry the peptides for about one hour. Resuspend lyophilized peptides in 0.5%acetic acid 0.1%Tri Fluor acetic acid desalt and concentrate peptides on a reversed phase C 18 carbon sandwich and ion exchange chromatography on handmade micro columns.
Load 50%of peptides onto the C 18 carbon sandwich stage tip and 50%onto the SCX stage tip Elute peptides from the SCX tips using 5%ammonium hydroxide, 30%methanol and from the C 18 carbon tips using 80%aceto nitrile, 0.5%acetic acid. Lastly, lyophilize, the eluded peptides and res suspend them in 0.1%formic acid in the native chromatin immunoprecipitation and crop approach. Chromatin is digested with MNA as a first step to purify a distinct functional domain from bulk chromatin.
The panel on the left shows a small scale MNA test in which DNA is resolved on a 1%aros gel after chromatin digestion with MNA at different time lapses. A large scale MNA digestion is shown on the right. DNA is resolved on a 1%aros gel after 60 minutes of MNAs chromatin digestion and after separation of the S one fraction containing mononucleo zones and from the S two fraction containing poly nucleosomes.
The digested chromatin and riched in mono nucleosomes is incubated with an antibody that recognizes a specific hisone post-translational modification. For example, trimethylation of lysine nine on histone H three K nine ME three, a marker of silent chromatin. The immuno purified proteins as well as the chromatin input are then separated by SDS page and colloidal kumasi staining shows core histones H three, H four, H two A, and H two B around and below the 17 kilodalton band with H three and H two B co migrating the comparison between the amount of trimm methylated glycine nine on H three present in the flow through and input indicates that about 50%of the region of interest is immuno purified, excluding the risk of a bias due to the enrichment of a minor subpopulation of chromatin.
This histogram represents the average plus minus SEM from three independent experiments for each modification mass spectrometry MS is then employed to characterize the post-translational modifications co associated within the enriched nucleosomes. All histone post-translational modifications are verified by manual inspection of the corresponding ms ms spectra in this representative Ms m Ms spectra using CID fragmentation. The B ion and Y ion series allow the definition of the sequence of the H three nine to 17 peptide and localization of the trimethylation on the lysine nine residue label-free quantification is achieved by first constructing the extracted ion chromatogram or XIC for each corresponding to every modified peptide based on the mass charge value and calculating the area under the curve or a UC for each peak.
Next, the relative abundance of the different degrees of methylation on lysine nine in the H three nine to 17 peptide is estimated by dividing the A UC of each modified peptide by the sum of the areas corresponding to all observed unmodified and modified forms of that peptide. Thirdly, the relative enrichment of each modification in the chipped material is expressed as a log two ratio between the relative abundance of each methylation in the chipped peptide over the input. The analysis of the H three nine to 17 peptide shows the enrichment of D and TRIMM methylated lysine nine with the corresponding depletion of the unmodified and monomethyl forms.
With these results as positive control for antibody specificity, the cos association or depletion of all other modifications can be assessed both at the intramolecular level on the same H three and at the intermolecular level on other coen enriched histones within the same nucleosome. This representative heat map summarizes the enrichment of all cos associating H ptms identified on histone H three, H four and H two A.Each row corresponds to a different modification and ND indicates not detected modifications to screen for all proteins. Co associating within hetero chromatin chip of cross-linked chromatin fragmented by sonication is used in combination with stable isotope labeling by amino acids in cell culture or lac.
First, the labeling efficiency is evaluated by calculating the incorporation of heavy lysine and heavy arginine into proteins. For accurate protein quantification incorporation superior to 95%for both heavy amino acids is required as observed here, the median of lysine and arginine peptide density distribution is equal to 0.974 and 0.964 respectively. Second, the ratio of soluble peptide to antibody must be optimized.
This figure shows a zoomed mass spectra at the corresponding mass charge value of the two plus charge trimm methylated lysine nine K nine ME three in the H three nine to 17 peptide, both for light and heavy forms in input and chipped octr. While in input, the intensities of heavy and light peptide are close to one in the forward SLAC chip. The intensity of light peptide is much lower than that of the heavy counterpart, thus indicating an efficient competition.
A representative result of SDS page of light L and heavy H labeled chromatin input and co amino precipitated material chip is shown here. The lane corresponding to the chipped material was cut into 10 slices while only two slices of input were analyzed. For incorporation test analysis, slac competition experiments are typically performed in duplicate, in so-called forward and reverse formats where the competition with the excess soluble peptide is switched from the heavy H to the light L chromatin samples.
This representative full spectra shows the SLAC pair corresponding to peptides from HP one and macro two A proteins a ratio of heavy to light protein greater than one. In the forward experiment mirrored by a ratio of less than one in the reverse replica, demonstrate the specific enrichment of these proteins in hetero chromatin proteins whose intensity in the heavy and light forms are similar in both forward and reverse experiments produce a constant ratio close to one and are classified as background. The intersection of the forward and reverse X chipp experiments leads to the identification of 635 proteins present in both experiments and quantified with at least two ratio counts.
The log two plot of their heavy to light ratios represents the so-called hetero chromosome. The red dotted lines in this scatterplot represent the top 40%and 30%of protein ratios. As indicated, the genuine trimm methylated lysine nine interactors are unambiguously identified as the proteins present within the top 40%of the protein ratio distributions.
Here, the protein ratio distributions from input forward and reverse X crop experiments are shown in black, blue, and orange respectively. After watching this video, you should have a good understanding on how to prepare chromatin for E and end crop to process the mono precipitated proteins pre to mass spectrometry analysis. How to identify and quantify histon modification in mass spectra and how to define a specific interac on their protein ratios.
