Abstract
Histone modifications and DNA methylation together govern promoter availability, thereby influencing gene expression. This study queries the unicellular chlorophyte, Chlamydomonas reinhardtii using a three step “epigenetic assay” design to phenotypically track the variegation of a randomly integrated Paromomycin resistance transgene(s) (PmR). Based on its position of integration, the PmR 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 cis-spread of heterochromatin could be interfered with a stronger euchromatic barrier (TUB2 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 Chlamydomonas. 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.
Acknowledgments
The authors thank John James, Minoshka Jones, Narendra Yadav, Nizam Ahmed, Sarthak Gupta, Shyam Nair, Neelima P. V., and Mohit Virdi for help during the study. The authors thank the Ramalingaswami Fellowship (Department of Biotechnology, D.O. NO BT/HRD/35/02/2006) for funding this study.
Author contributions
SS conceived and executed the experiments with SK, SP, and USJD provided the plasmid pCC1G and strains used in the study. SK contributed to , SP contributed to and US contributed to . SS wrote the paper with inputs from all the authors.
Author agreement
The authors are in informed agreement about the authenticity of the data presented, authorship, and necessary permissions for peer review submission.
Disclosure statement
The authors have no competing personal or financial conflicts of interest to declare that would influence this study.
Statement of informed consent
No conflicts, informed consent, human or animal rights applicable.