Currently there are no drugs that are well-established in promoting heterochromatin formation. Here we present a simple and efficient method for identifying small molecule compounds that promote this process. Our screening protocol provides a simple, inexpensive, easy to follow in vivo system for heterochromatin-promoting drug identification.
Identifying compounds that promote heterochromatin formation may lead to the discovery of mechanisms of cancer that mend and cancer therapy. This cost effective has been measured for identifying heterochromatin-promoting compounds in Drosophila could also be used to count form hits as it is a preventative measure to a highest cell pool of cell screening. Our screening protocol could also be useful for quick pilot screens as this Drosophila TranSheet system produces a variegated eye color phenotype that is inversely correlated to the amount of heterochromatin present in the fly.
Helping to demonstrate the procedure will be Kenny Dao, an undergrad student from our laboratory. After identifying and preparing a drug library for a screening using an appropriate solvent, and the desired concentration, cross three male flies with three or more virgin female flies in a 25 by 95 millimeter food vial. With nine millimeters of standard Drosophila food medium.
Set up three replica vials for each drug. Add one control vial for the screen. And allow the flies to lay eggs for two days at room temperature.
On day two of the cross, anesthetize the flies with a short burst of carbon dioxide. And discard the parent flies in a bottle containing 70%ethanol. Next, dilute each drug compound from the initial stock to attend micro molar final concentration in 33%DMSO in water.
And add 60 microliters of the drug, or the control 33%DMSO alone, on top of the fly food. Then confirm that no parent flies are present within the vials. And add the drug solution to the appropriate vials.
Two days after the F1 flies emerge, transfer anesthetized flies from one vial at a time onto a porous dissecting pad with carbon dioxide filtering through from underneath the filter paper. Under a dissecting microscope, examine each whole fly, identifying all of the heterozygous males by their lack of the curly o-dominant curly wing phenotype. Score the eye color of each heterozygous male on a scale of one to five.
With a one score being assigned to a less than 5%red scattered eye total surface area. Add a two score to flies with a 6 to 25%red spot eye total surface area. A three to flies with a 26 to 50%red spot eye total surface area.
A four to flies with a 51 to 75%red spot eye total surface area. And a five flies with a greater than 75%red spot eye total surface area. Then calculate the mean color index of all of the males from each vial scored in triplicate.
To confirm that the compounds indeed promote heterochromatin formation, a different validation technique, such as western blotting, can be performed. In this representative experiment, a small molecule drug library, oncology set 3, which is composed of 97 FDA authorized oncology drugs, was screened using the DX1 strain of Drosophila Melanogaster. According the screening assay, methotrexate, coded as E7 in the library, caused the most variation within the Dx1 strain.
Suggesting that methotrexate could be the most promising heterochromatin-promoting drug for future cancer targeted therapy. To further confirm that this compound promotes heterochromatin transformation, western blot data was performed. Demonstrating the up regulation of heterochromatin formatin associated protein, H3K9 trimethylation, and validating the screening results.
The critical step is scoring the eye color of each of the white 11-18 DX1 heterozygous male flies on a scale of one to five according to a predetermined study route. We can use western blots or immunostaining to confirm the hits. The discovery of compounds that are suspected to promote heterochromatin formation may also lead to the discovery of IV genetic mechanism of cancer development and clinical trials in human patients.
