A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Summary

Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation.

Abstract

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a cost-effective in vivo method to identify heterochromatin-promoting compounds by using Drosophila. The Drosophila's DX1 strain, having a variegated eye color phenotype that reflects the extents of heterochromatin formation, thereby providing a tool for a heterochromatin-promoting drug screen. In this screening method, eye variegation is quantified based on the surface area of red pigmentation occupying parts of the eye and is scored on a scale from 1 to 5. The screening method is straightforward and sensitive and allows for testing compounds in vivo. Drug screening using this method provides a fast and inexpensive way for identifying heterochromatin-promoting drugs that could have beneficial effects in cancer therapeutics. Identifying compounds that promote the formation of heterochromatin could also lead to the discovery of epigenetic mechanisms of cancer development.

Introduction

Heterochromatin is a condensed form of DNA that plays a central role in gene expression, in regulating chromosome segregation during cell division, and in protecting against genome instability¹. Heterochromatin has been considered to be a gene repression regulator and to protect chromosome integrity during cell mitosis^2,3. It is associated with the di- and tri-methylation of histone H3 lysine 9 (H3K9me) during lineage commitment^4,5. Moreover, recruitment of Heterochromatin Protein 1 (HP1) chromodomain proteins is also considered to be associated with heterochromatin and epigenetic repression of gene expression⁶. These proteins are essential components and markers of heterochromatin formation.

Since genomic instability enables cells to acquire genetic alterations that promote carcinogenesis, heterochromatin is becoming more recognized in cancer development and may be targeted for cancer treatment^7,8. Currently, there are no drugs that are well-established in assisting heterochromatin formation. Here, we present a simple and quick yet efficient method for screening small-molecule compounds that promote heterochromatin formation. The screening is done by treating Drosophila with a library of small-molecule drugs. This method takes advantage of a variegated eye color phenotype in the DX1Drosophila strain that is influenced by heterochromatin levels. DX1 flies contain a tandem array of seven P[lac-w] transgenes, which have the variegated expression/depression depending on heterochromatinization, therefore, the extent of variegation in the eye color reflect the heterochromatin level. Specifically, increasing heterochromatin could be detected by the rising proportion of variegated eye color (white eye). On the contrary, decreasing heterochromatin would be detected by the rising proportion of the P[lac-w] transgene expression (red eye)^9,10,11,12.

Therefore, we take advantage of this Drosophila transgene system that produces a variegated eye color phenotype since its expression is directly correlated to the amount of heterochromatin present. Upon discovery of compounds that are suspected to promote heterochromatin formation, we may confirm this suspicion using other methods such as western blot. These heterochromatin-promoting substances may be further developed for clinical trials in patients in the future.

Protocol

1. Drug library preparation

1. Identify and prepare a drug library to be screened using appropriate solvent at a desired concentration (e.g., 10 mM in DMSO).
   NOTE: A specific example for a drug library is the Oncology Set III from National Cancer Institute (NCI) Developmental Therapeutics Program (DTP). Compounds in this set are provided as 20 µL at 10 mM in 100% DMSO in two 96-well PP U-bottom plates and stored at -20 °C. The NCI Plate maps, and the basic chemical data of each drug could be found at the following websites:
   NCI plate number 4740
   NCI plate number 4741

2. Screen for heterochromatin-promoting drugs using Drosophila

1. Drosophila Breeding strategy (Figure 1A)
   1. Day 1: Cross three w¹¹¹⁸/Y; DX1/CyO male flies and three or more virgin w¹¹¹⁸ female flies in a 25 mm x 95 mm food vial with 9 mL of standard Drosophila food media (Bloomington recipe). Set up three replicate vials for each drug and one control for the screen.
      NOTE: DX1 flies were kindly provided by James Birchler (University of Missouri)^9,13.
   2. Day 2 and Day 3: Allow the flies to lay eggs for 2 days at room temperature (22 °C).
   3. Day 4: Use a short burst of CO₂ gas to anesthetize the parent flies and dispose of the parent flies in a "fly morgue" (a bottle containing 70% alcohol).
2. Feed drugs to Drosophila for screening.
   1. Dilute each drug compound from the original stock (20 µL at 10 mM in 100% DMSO in 96-well PP U-bottom plates) to a 10 µM final concentration with 33% DMSO in water. Use 33% DMSO in water without any drug as the control.
      NOTE: Some drugs are poorly soluble in water and were dissolved in DMSO. 100% DMSO is toxic to flies. 33% is tolerable.
   2. Day 4: Pipette 60 µL of a 10 µM drug solution or control onto the top of fly food. At this point, ensure that the parent flies have been removed and there are fly eggs and crawling first-instar larvae on the food.
   3. Day 6: Repeat pipetting 60 µL of the same 10 µM drug solution to the food vial.
      NOTE: Since drugs were added on to the fly food, the top surface food contains nearly 10 µM concentration and the bottom food could be not be fully penetrated. All the flies lay eggs on the food top and eat the top surface food.
3. Observe and score eye color changes.
   1. Two days after the F1 flies emerge (approximately on day 14), examine the flies. Use a short burst of CO₂ to anesthetize the F1 flies and remove the flies from their vial onto a porous dissecting pad with CO₂ sipping through from underneath a filter paper.
   2. Inspect the flies on this pad under a dissection microscope. Examine the whole fly and identify all w¹¹¹⁸/Y; DX1/+ heterozygous males by their lacking the CyO dominant curly wing phenotype.
   3. Score the eye color of each of the w¹¹¹⁸/Y; DX1/+ heterozygous male flies on a scale of 1 to 5 (Figure 1B). The percentage of white eye color was rated as follows:
      1. <5% red scattered eye total surface area
      2. 6% - 25% red spots eye total surface area
      3. 26% - 50% red spots eye total surface area
      4. 51% - 75% red spots eye total surface area
      5. >75% red spots eye total surface area
      NOTE: Alternatively, homogenize fly heads in 100% methanol and use a spectrometer to measure eye pigmentation at OD 450 nm. Select only males because PEV is more pronouced in DX1 males. Score more than 10 males in each vial.
   4. Calculate the mean color index of all the males from each vial scored. Perform triplicates for each compound (Figure 1C).
      NOTE: Since eye color varies with age, this is a time-sensitive step. The eye color should be calculated at the same age – 2 days old. Usually >10 flies should be scored in each vial.
   5. To confirm that the compounds indeed promote heterochromatin formation, use a different technique such as western blotting to validate (Figure 1G).

Results

This protocol was successfully used to screen compounds that promote heterochromatin formation in Drosophila, which is an efficient and low-cost in vivo system for drug development (Figure 1). We screened a small-molecule drug library, Oncology Set III, which is composed of 97 FDA authorized oncology drugs, using the DX1 strain of Drosophila melanogaster (Figure 1B). The results for a significant drug were represented ...

Discussion

Heterochromatin is a condensed form of DNA that plays a central role in regulating gene expression. It is becoming increasingly more recognized in cancer and may serve as a potential target for cancer therapy^15,16,17,18. Small-molecule compounds are commonly used in drug development due to advantages in manufacture, preservation, and metabolism in human bodies. To identify heterochromatin-promo...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Drosophila		DX1 strain	DX1 flies were kindly provided by James Birchler (University of Missouri)
Drosophila food media	UCSD fly kitchen
Methotrexate	NCI drug library
Dimethyl sulfoxide (DMSO)	Sigma	D2650
Ethyl alcohol	Sigma	E7023-500ML