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

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.

Waveform Plasticity under Entrainment to 12-h T-cycles in Drosophila melanogaster: Behavior, Neuronal Network, and Evolution.

A crucial property of circadian clocks is the ability to regulate the shape of an oscillation over its cycle length (waveform) appropriately, thus enhancing Darwinian fitness. Many studies over the past decade have revealed interesting ways in which the waveform of rodent behavior could be manipulated, one of which is that the activity bout bifurcates under environments that have 2 light/dark cycles within one 24-h day (LDLD). It has been observed that such unique, although unnatural, environments reveal acute changes in the circadian clock network. However, although adaptation of waveforms to different photoperiods is well studied, modulation of waveforms under LDLD has received relatively less attention in research on insect rhythms. Therefore, we undertook this study to ask the following questions: what is the extent of waveform plasticity that Drosophila melanogaster exhibits, and what are the neuronal underpinnings of such plasticity under LDLD? We found that the activity/rest rhythms of wild-type flies do not bifurcate under LDLD. Instead, they show similar but significantly different behavior from that under a long-day LD cycle. This behavior is accompanied by differences in the organization of the circadian neuronal network, which include changes in waveforms of a core clock component and an output molecule. In addition, to understand the functional significance of such variations in the waveform, we examined laboratory selected populations that exhibit divergent eclosion chronotypes (and therefore, waveforms). We found that populations selected for predominant eclosion in an evening window (late chronotypes) showed reduced amplitude plasticity and increased phase plasticity of activity/rest rhythms. This, we argue, is reflective of divergent evolution of circadian neuronal network organization in our laboratory selected flies.