A potential screening method for epigenetic drugs: uncovering stress-induced gene silencing in .

Histone modifications and DNA methylation together govern promoter availability, thereby influencing gene expression. This study queries the unicellular chlorophyte, using a three step "epigenetic assay" design to phenotypically track the variegation of a randomly integrated Paromomycin resistance transgene(s) (). Based on its position of integration, the gene expression hinged on two epigenetic hallmarks: the spreading of heterochromatin, and the transmissible memory of epigenetic states across generations. Using a spot-dilution analysis, the loss of antibiotic resistance phenotype was scored from 0 to 4, four being maximally silenced. Appropriate construct designs were used to demonstrate that the -spread of heterochromatin could be interfered with a stronger euchromatic barrier ( promoter). When assayed for metal ion stress, a combination of Mn deficiency with excess Cu or Zn stress was shown to induce gene silencing in . Cu stress resulted in the accumulation of intracellular ROS, while Zn stress elevated the sensitivity to ROS. As proof of functional conservation, mammalian epigenetic drugs demonstrably interfered with stress-induced gene silencing. Finally, a selected group of transgenic clones responsive to HDACi sodium butyrate, when tested in a gradient plate format showed similarity in phenotype to the plant-derived compound cinnamic acid. This indicated a possible commonality in their mode of action, unlike curcumin which might have a different mechanism. Thus, using binned libraries, based on a common set of responses to known drugs, a cost-effective high-throughput screening strategy for epigenetically active compounds from plants or other sources is described.