Epigenetic regulation of behavior in Astatotilapia burtoni, a cichlid fish [electronic resource]
Keywords: Biology.

The ability of an animal to change quickly in response to its surroundings is essential to its survival and in some species, individuals respond by changing phenotype. The studies in this thesis focus on the molecular mechanisms through which environmental information can affect changes in phenotype in an African cichlid fish, Astatotilapia burtoni. This species is particularly useful for this study, as adult male burtoni assume one of two distinct, reversible, behavioral and physiological phenotypes. Dominant (D) males are brightly colored, reproductively capable and engage in mating and aggressive territorial behaviors while non-dominant (ND) males are drably colored, reproductively incapable, and are behaviorally passive. Importantly, the transition from ND to D or the reverse can occur in a matter of minutes and is triggered solely by external social cues. How such external information is translated into phenotypic changes on the molecular level is the focus of these experiments and my data suggest an epigenetic mechanism may regulate this transition. The specific epigenetic mechanism assessed here is DNA methylation, which is the covalent attachment of a methyl group to the cytosine nucleotide of DNA, which can lead to changes in expression. First, I showed that DNA methylation is present in A. burtoni, as not all animals utilize this mechanism. I used immunohistochemistry to demonstrate that high levels of global methylation are present in the nuclei of all cells examined, most likely bound with the highest frequency to heterochromatin, to suppress transcription of specific transcribed regions. However, there were no differences detected between behavioral states using these methods. Next I demonstrated that epigenetic mechanisms play a role during the determination of social status by treating juvenile males with epigenetic modifiers that either promote or interfere with DNA methyltransferase (DNMT). Animals injected with zebularine, which blocks the activity of DNMT, were statistically unlikely to ascend to D status, while those injected with methionine, which acts as a methyl donor, were statistically likely to become D males. Once a potential role for DNA methylation in social status determination was found, I surveyed potential sites of action on the GnRH1 gene, a key regulator of reproductive behavior, for variations in methylation on the single nucleotide level. I found that fully established Ds and NDs do not have differences in methylation levels in any on the individual nucleotides assayed on the GnRH1 promoter or coding region; however, juvenile males have lower levels of methylation than Ds at some sites on the promoter, while males transitioning to D status have lower average promoter methylation, but higher average coding region methylation than D males. Furthermore, ND animals injected with zebularine have higher average levels of methylation on both the promoter and coding region than control ND males. In order to better understand the context of the methylation changes during sexual maturation, methylation levels on the GnRH1 gene were measured during normal development. GnRH1 methylation levels remain constant between two and four weeks of age but increase significantly at several sites between 4 and 6 weeks of age. Furthermore, crowding mothers during the brooding stage and raising young in crowded tanks results in lower methylation levels at both 2 and 6 weeks of age, as well as causing delayed growth at the 6 week stage. Finally, I measured GnRH1 methylation in a non-reproductive context. Since GnRH agonists can interfere with short-term memory in humans, I measured changes in methylation in the GnRH1 gene during memory formation and storage. Animals who successfully learned a memory task showed a correlation with higher methylation at sites in the GnRH1 coding region than non-learners. Learners were successfully able to recall their training after three months; however, the increased methylation in the coding region was no longer present. In summary, DNA methylation is present in A. burtoni and increases on the GnRH1 gene promoter and coding sequence during transitions in development and sexual maturation. As GnRH1 expression levels are known to increase in these cases, increased methylation is not acting canonically as a repressor of expression on GnRH1. During short-term learning, higher methylation is limited to only the coding region of GnRH1, indicating that there may be several different epigenetic regulatory pathways involving this gene. Furthermore, the observation that there are no differences in GnRH1 methylation between stable D and ND males, or, in the long-term, between learners and non-learners, suggests that methylation in this location may be transitory and used as a short-term marker for other regulatory mechanisms.