These protocols are significant because they provide detailed steps for validation of the potency and selectivity of novel HAT inhibitors, which are important research tools and potential therapeutics. The techniques demonstrated in this video are easy to perform and provide information about the effects of HAT inhibitors on global and regional histone acetylation. They make it possible to understand epigenetic regulation of gene expression.
Attention to the details is critical. It is important to follow the protocols step-by-step. Begin by preparing the enzymatic reaction in a 10 microliter volume inside a 0.2 milliliter PCR tube according to manuscript directions.
Then incubate the complete reaction mixture at 30 degrees Celsius for one hour in a PCR thermal cycler. Meanwhile, add two mercaptoethanol at a one to 10 ratio to the 6X SDS sample buffer. Remove samples from the PCR thermal cycler and add two microliters of the prepared SDS sample buffer to each reaction mix.
Heat the samples to 95 degrees Celsius for five minutes on a heat block, then cool them on ice. Store the samples at minus 20 or minus 80 degrees Celsius or proceed with gel electrophoresis and immunoblotting. Seed 100, 000 MCF-7 cells in one milliliter of cell culture medium into each well of a 12-well plate and allow the cells to grow to 80 to 90%confluency.
When the cells reach the desired confluency, aspirate the cell culture medium from wells four, five, and six, and pipette one milliliter of three micromolar MS275 in medium into each well. Then aspirate the cell culture medium from wells one, two, and three, and pipette one milliliter of diluted DMSO into each well. Return the cells to the incubator for four hours to allow for accumulation of acetylated histones in cells exposed to MS275.
While the cells are incubating, prepare dilutions of A-485 in DMSO according to manuscript directions. When the incubation is finished, aspirate the medium from the wells and add the dilutions. Return the cells to the incubator and culture them for 20 hours, then aspirate the cell culture medium from the wells and wash the cells with one milliliter of PBS.
Aspirate the PBS and add 100 microliters of passive lysis buffer. Store the plate at minus 80 degrees Celsius overnight. Pipette 100 microliters of sonicated chromatin from cells treated with DMSO into two 1.5 milliliter tubes, then pipette 400 microliters of ChIP dilution buffer to each tube to bring the total volume up to 500 microliters.
Remove five microliters of the solution from one of the tubes and store it at minus 20 degrees Celsius as DMSO input. Prepare two tubes with sonicated chromatin from cells treated with A-485 in the same way. Use a pipette to add the IgG and H3K27 acetyl antibodies to the DMSO and A-485 samples.
Then add 20 microliters of protein A magnetic beads to each tube, making sure that the beads are well resuspended. Rotate the samples overnight at four degrees Celsius. Pellet the protein A magnetic beads using a magnetic separator and remove the supernatant without disturbing the beads.
Wash the beads with 500 microliters to one milliliter of low salt wash buffer and rotate them for five minutes at four degrees Celsius. Perform a quick spin down, pellet the beads with a magnetic separator and remove the supernatant. Repeat the wash procedure with high salt wash buffer, lithium chloride wash buffer, and TE buffer.
After the wash with TE buffer, remove the input samples from the freezer and put them on ice. Thaw an aliquot of proteinase K, then pellet the beads using a magnetic separator and remove the TE buffer from the beads. Add 100 microliters of ChIP elution buffer and one microliter of proteinase K to every sample including the input samples and incubate them with shaking at 62 degrees Celsius for two hours using a thermocycler.
After the incubation, heat the samples to 95 degrees Celsius for 10 minutes, then cool them to room temperature. Pellet the magnetic beads with a magnetic separator and transfer the DNA-containing supernatant to a new 1.5 milliliter tube. Purify the DNA using a standard PCR cleanup kit and run qPCR.
The in vitro histone acetyltransferase assay was used to investigate the effect of anacardic acid on p300 HAT activity towards a histone substrate. A concentration range was tested and acetyl-CoA was not added to the negative control reaction. The immunoblot results were quantified with ImageJ, showing a clear reduction in acetyl H3K18 and acetyl H3K9 at 100 micromolar anacardic acid compared to the DMSO control.
In the chromatin hyper-acetylation inhibition assay, treatment of MCF-7 cells with HDACi MS275 strongly upregulated acetylation of histone 3 on several lysine residues. The basal levels of acetyl H3K18 and acetyl H3K27 were low, showing the benefits of adding an HDACi in the ChHAI assay. The addition of A-485 in cells pretreated with MS275 attenuates the increased histone acetylation at H3K18 and H3K27, but not H3K9.
The immunoblot results were also quantified with ImageJ. ChIP qPCR was used to investigate the effects of HAT inhibitors at gene regulatory elements that control oncogene expression. In the DMSO sample, the DNA precipitated by the IgG control antibody produced a higher Ct value than the acetyl H3K27 antibody in the qPCR reaction for the cyclin D1 promoter, indicating that the non-specific IgG control precipitated less DNA histone complexes than the acetyl H3K27 specific antibody at the promoter.
Compared with the DMSO control, A-485 reduced acetyl H3K27 enrichment at the cyclin D1 promoter. Importantly, A-485 is known to significantly reduce acetyl H3K27 in cell culture. When attempting this protocol, keep in mind that the pre-incubation steps of the in vitro HAT and ChHAI assays are essential and should not be forgotten.
It is also important to remember to save the input samples and to avoid loss of the beads during ChIP. After performing these procedures, ChIP-seq is an additional method that can be executed to gain global information about histone acetylation at regulatory elements for the entire genome. These protocols are helpful for scientists to carefully validate novel HAT inhibitors and to avoid publishing low-quality chemical probes in the literature.
The validated HAT inhibitors can undergo further development as potential therapeutics.
