Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The eukaryotic nucleus is very crowded with many biological processes occurring simultaneously. The scope of our research is to understand how all of these processes are coordinated on our genomes without major accidents that would otherwise lead to genomic instability and human diseases. This protocol allows us to purify a specific locus of the genome in its native state, allowing both the compositional and functional characterization of the isolated material such as the measurements of protein-DNA interactions.

This protocol enables an enormous level of enrichment of a targeted locus, which overcomes many current limitations of locus specific chromatin isolation. The material has not been cross-linked. That also allows many downstream functional analysis, such as in vitro transcription or in vitro replication.

This protocol enabled us to excise and purify distinct replication origins from yeast chromosomes, allowing us to perform a proteomic analysis and identify novel origin interacting chromatin factors, and therefore advance the research field of DNA replication. This protocol enables in vitro replication studies using the purified origin material to examine chromatin states, histone modifications, and perform structural analysis on the native nucleosomal templates from endogenous chromosomes, advancing chromatin research. Begin by inoculating the control and recombination competent yeast strains from YPD plates into five milliliters of YPR medium, and grow the culture overnight at 30 degrees celsius and 200 RPM.

Next, scale up the culture by inoculating two milliliters into 100 milliliters of fresh YPR medium and growing it overnight at 30 degrees celsius and 200 RPM. For each strain, dispense 1, 800 milliliters of autoclaved yeast peptone medium and 200 milliliters of autoclaved 20%raffinose solution into two five liter erlenmeyer flasks. Inoculate each yeast strain with 0.2 optical density at 600 nanometers in the respective medium, and incubate for about six hours at 200 RPM or until the cells reach the desired 1.0 optical density.

Add 200 milliliters of 2%galactose and 110 microliters of yeast mating factor alpha, or YMFA, simultaneously to arrest cells in the G1 phase and incubate it for two hours. Transfer the cell suspension into one liter centrifuge buckets and centrifuge the bucket at 6, 000 G and four degrees celsius for 10 minutes. Discard the supernatant and resuspend the cell pellets in 10 to 15 milliliters of distilled water.

Next seal a 25 milliliter syringe with a lure plug and place it inside a 50 milliliter conical tube filled with water. Transfer the cell suspension to the syringe assembly. Wash the centrifuge buckets with five milliliters of distilled water to collect the remaining cells.

Then, centrifuge the assembly at 2, 397 G for 10 minutes at room temperature and discard the supernatant. Then remove the lure plug from the syringe and extrude the cells into liquid nitrogen to form cell spaghetti. Lastly, transfer the frozen cell spaghetti into a 50 milliliter conical tube and storing at minus 80 degrees celsius until further use.

Begin cooling a commercial coffee grinder by grinding approximately 30 to 50 grams of dry ice twice for 30 seconds each time. Discard the dry ice powder and mix three grams of frozen yeast cell spaghetti with approximately 30 to 50 grams of dry ice in the pre-cooled coffee grinder. Seal the junction between the cap and the grinder with Parafilm.

Mix the yeast cell, dry ice mix 10 times for 30 seconds each, allowing 30 second intervals between every round. Transfer the resulting yeast cell, dry ice powder to a plastic beaker using a clean and dry spatula. After dry ice evaporation, add 2.25 milliliters of magnetic bead or MB buffer with protease and phosphatase inhibitors to the yeast cell powder.

After vigorously pipetting the cell buffer mixture, transfer the cell suspension to a 15 milliliter conical tube. Next, store 0.1%samples from the crude cell extracts for DNA and 0.05%samples for protein analysis at minus 20 degrees celsius. Transfer the remaining cell suspension into low binding two milliliter reaction tubes and separate cell debris by centrifuging the tubes.

Once done, pool all supernatants into one 15 milliliter conical tube. Again, store 0.1%of the supernatant at minus 20 degrees celsius for DNA and 0.05%of the supernatant for protein analysis. Next, wash 500 microliters of immunoglobulin G coupled magnetic bead slurry with 500 microliters of cold MB buffer containing protease and phosphatase inhibitors two times for five minutes each at four degrees celsius on rotation at 20 RPM.

Incubate the magnetic beads in 500 microliters of cold MB buffer for one hour at four degrees celsius on rotation at 20 RPM before removing the supernatant from the beads using a magnetic rack. Mix the equilibrated magnetic bead slurry with the cell lysate obtained earlier and pooled into a 15 milliliter conical tube. After incubating for two hours at four degrees celsius and 20 RPM, transfer the complete magnetic bead cell lysate suspension to low binding reaction tubes.

Separate the magnetic beads carrying the chromatin rings of interest from the cell lysate using a magnetic rack. Transfer the remaining flow through from each tube to a fresh 15 milliliter conical tube before storing some samples at minus 20 degrees celsius for DNA and protein analysis. Next, suspend the magnetic bead from each 15 milliliter conical tube in 300 microliters of cold MB buffer and combine the beads into one reaction tube.

Perform five washes of 10 minutes each, using 750 microliters of cold MB buffer containing protease and phosphatase inhibitors at four degrees celsius while rotating at 20 RPM. Next, conduct the final wash with 750 microliters of cold MB buffer without protease and phosphatase inhibitors. Suspend the prepared beads in 40 microliters of cold MB buffer without protease and phosphatase inhibitors.

Release the LexA CBP chromatin ring complexes from the magnetic beads used to purify chromatin single copy gene locus by incubating the beads with two microliters of 6x hist-tagged recombinant tobacco etch virus or TEV protease. After separating the beads from the final eluate using a magnetic rack, transfer the eluate containing cleaved chromatin rings to a new 1.5 milliliter reaction tube. Again, place the tubes on a magnetic rack to separate residual beads from the final eluate.

Resuspend the beads in 750 microliters of cold ammonium carbonate buffer before storing some samples at minus 20 degrees celsius for DNA and protein analysis. From the final eluate, take out the samples and store them at minus 20 degrees celsius for DNA and protein analysis. Further, take samples from the residual beads.

Begin the denaturation eluation by washing the magnetic beads two times in 750 microliters of cold ammonium carbonate buffer for 20 minutes each, four degrees celsius rotating at 20 revolutions per minute. Next, add 500 microliters of 0.5 molar ammonium hydroxide to the beads to extract bound LexA chromatin complexes and incubate for 30 minutes at room temperature. Separate the beads from the suspension using a magnetic rack and transfer the eluate to a low binding reaction tube.

Incubate the low binding reaction tube at room temperature for 30 minutes and separate the beads from the suspension using a magnetic rack. Once done, transfer the eluate to a low binding reaction tube. Resuspend the beads in 750 microliters of deionized water and collect samples for DNA and protein analysis.

Lastly, obtain DNA and protein analysis samples from the final eluate. In the study, the purification efficiency of the ARS316 locus in the recombination strain was quantified and compared to the control locus PDC1. The ARS316 locus showed lower recovery in the flow through compared to PDC1.

Although a fraction of chromatin rings remained bound to the TEV beads due to incomplete TEV protease cleavage, denaturation elution resulted in 80 to 90%ARS316 locus recovery. This corresponds to the high enrichment of ARS316 molecules in TEV beads and denaturation elution fractions when factoring in the size of the yeast genome compared to the control strain, that showed no enrichment of ARS316 locus in any of the quantified fractions. After TEV elution, the beads showed a higher recovery percentage of the ARS316 locus as the TEV cleavage efficiency was not 100%This resulted in a fraction of chromatin rings remaining bound to the beads.

However, the denaturing elution showed 80 to 90%recovery of the ARS316 locus in the recombination strain, corresponding to the high enrichment of ARS316 molecules when factoring in the size of the yeast genome.