[
International Worm Meeting,
2015]
The high number of compounds to be tested, the diversity of toxicity endpoints, concentrations, and combinations of chemicals, all provide a challenge in our ability to assess the safety of chemicals. Of particular importance is the potential effect of chemicals on the germline epigenome, which can alter biological processes over several generations. While the cases of DES, vinclozolin and BPA provide evidence of epigenetic effects, there is a great need to explore the influence of environmental compounds on the epigenome and, especially, to develop methods that allow us to quickly and efficiently examine this question. We have established the use of the genetic model system, the worm Caenorhabditis elegans, as a relevant model for epigenetic and reproductive toxicity assessment. By taking advantage of the C. elegans genetic tools, we propose to comprehensively identify chemicals for their ability to disrupt the germline chromatin. To this aim, we are making use of a worm strain where GFP is specifically epigenetically silenced in the germline. We previously showed that valproic acid, a well-known mammalian histone deacetylase inhibitor, disrupts the germline epigenetic state leading to the de-silencing of the transgene in the germline. Our current experiments test this concept further by exposing the worms to environmental compounds. We tested vinclozolin and BPA to analyze a disruption in maintenance and/or establishment of epigenetic marks. Young adult worms were exposed to the compounds for 48 hours, and three subsequent generations were followed and analyzed. Results indicate that de-silencing effects last for at least three generations (up to three-fold compared to our DMSO control). Furthermore, most worms showing de-silencing in the first, second and third generation originate from worms showing de-silencing in the parental exposed generation indicating that the effect is inherited. Thus, we conclude that valproic acid, vinclozolin, and BPA disrupt the germline epigenome over several generations in a heritable fashion. Additionally, in concomitant assays, we are working with the lab of Dr. Amander Clark, using mice primordial germ cell-like cells (PGCLCs) in a mammalian validation approach. Together, we hope to establish a novel in vitro germline differentiation assay to screen for chemicals that affect germline quality.
[
International Worm Meeting,
2019]
How an epigenome's sensitivity to chemical exposure changes with age is not fully understood. This is significant for the germline where the genome is kept relatively silent through repressive histone marks such as H3K9me3 (histone 3 lysine 9 trimethylation) and H3K27me3 (histone 3 lysine 27 trimethylation). Understanding how epigenetic sensitivity changes with age will inform how age should be accounted for in chemical risk assessments. To investigate the relationship between age and epigenetic sensitivity we used immunofluorescence to quantify changes in H3K9me3 and H3K27me3 levels with age in C. elegans germlines. We performed immunofluorescence for H3K27me3 and H3K9me3 at days 1, 3, 5, and 7 of adulthood, with 5-7 replicates and fluorescence quantified in 3-4 gonads per replicate. Variation in H3K27me3 levels increases with age, suggesting a decrease in epigenetic homeostatic control with germline age. Next, we asked whether maternal age affects transmission of a chemical exposure to future generations. We used a green fluorescent protein (GFP) reporter C. elegans strain, NL2507, where GFP expression indicates levels of chromatin accessibility in C. elegans germlines. Under healthy conditions the GFP reporter is silenced in the germline however, with exposure to bisphenol A (BPA), transcriptional regulation is lost and GFP is expressed (Camacho et al., 2018). We exposed C elegans to 500uM BPA during different 48hr windows in their adult lifespan and analyzed GFP levels in F1 and F3 progeny (30 worms per condition). F3 BPA descendants had GFP expression that was double that of their respective control group, indicating that BPA affects the epigenetics of descendants not directly exposed. Furthermore, the F3 from older mothers had higher levels of GFP expression than the F3 from younger mothers. This suggests that older germlines may be more sensitive to chemical exposures because they are more likely to transmit chemically induced epigenetic perturbations to future generations. The epigenetic sensitivity of older germlines is relevant to humans as we tend to have children later in life. Furthermore, understanding how age impacts epigenetic sensitivity will allow us to more accurately assess the safety of chemicals for both exposed individuals and their descendants.
[
European Worm Meeting,
2006]
Maaike C. W. van den Berg1,2, Jessica Z. Woerlee2, Hansong Ma1,2 & Robin C. May1. Cryptococcus neoformans is the causative agent of cryptococcosis, a fatal fungal disease of immunocompromised patients. Our group and others have made use of C. elegans as an alternative host for Cryptococcus, in order to investigate the molecular basis of host-pathogen interactions. Using this system, we now report that male C. elegans show greater resistance to killing by Cryptococcus than hermaphrodite animals and that this resistance can be induced in hermaphrodite animals by inappropriate activation of the male sex-determination pathway. Resistance is molecularly determined, rather than resulting from behavioural changes or reproductive differences, and requires the activity of the stress-response transcription factor DAF-16. Finally, we demonstrate that resistance to Cryptococcus neoformans correlates broadly with longevity within the Caenorhabditis genus. Our results suggest that many of the molecular determinants of longevity and immunity are the same and that differential regulation of these determinants may underlie much sex-dependent and species-dependent variation.