Cross-talk of genetically and environmentally modulated epigenetic factors in the development of anxiety-related behavior

Here, I studied the role of two candidate genes i.e. neuropeptide S receptor 1 (Npsr1) and transmembrane protein 132D (Tmem132d), in two psychopathological animal models of anxiety-related behavior because of recent studies showing importance of these candidates in limbic areas and the frontal cortex of panic disorder patients, respectively. The two animal models are rat (r) and mouse (m), high anxiety-related behavior (rHAB/mHAB) and low anxiety-related behavior(rLAB/mLAB). To understand the anxiolytic role of neuropeptide S (NPS), basal Npsr1 mRNA expression was studied in limbic brain regions of HAB vs. LAB rodents, i.e. the paraventricular nucleus of hypothalamus (PVN) and amygdala, because these regions have been implicated in anxiety and fear attenuating responses of NPS and also due to differences in long-term activity based on cytochrome c oxidase activity in mHAB vs. mLAB. There was significantly lower basal Npsr1 mRNA expression in the basolateral amygdala of mHAB and also in the PVN of rHAB compared to corresponding LABs. To study the genetic underpinnings underlying this differential expression, Npsr1 DNA sequencing was carried out, which revealed several polymorphisms including single-nucleotide polymorphisms (SNP), insertions and deletions. By using dual reporter (luciferase) assays, I could show that the SNPs in the whole HAB promoter construct cause a significant decrease in promoter activity, thus confirming our in vivo findings in both rats and mice. Interestingly, however, when the promoter constructs were shortened to 500 bp relative to translational (ATG) start site, there was a two-fold higher HAB promoter activity, which could be attributed to the introduction of a polymorphism with putative binding site for the glucocorticoid receptor (GR) transcription factor. The higher HAB promoter activity was suppressed by dexamethasone (a GR activator), thus suggesting the presence of a polymorphism that favors GR binding. These findings are analogous to the higher HAB specific allele expression in cross-mated F1 offspring, which allows us to study the HAB vs. LAB alleles in the same cellular environment, irrespective of any epigenetic or other environmentally mediated factors that might modulate or interact with cis-acting factors. In addition, there was no difference in Npsr1 mRNA expression in the basolateral amygdala of mHAB and mLAB subjected to environmental enrichment (EE) and unpredictable chronic mild stress (UCMS), respectively. Thus it is a non-plastic gene as it does not respond to environmental challenges faced by the susceptible animal models. Similarly, for Tmem132d, using dual luciferase assays, two SNPs in the mHAB promoter region were shown to cause an increase in its corresponding promoter activity, and there was no difference in DNA methylation in the mHAB vs. mLAB Tmem132d promoter region, which explains the observed higher Tmem132d mRNA expression in the anterior cingulate cortex of mHAB. However, mHABs subjected to EE had higher Tmem132d mRNA expression, while mLAB undergoing UCMS had corresponding lower gene expression. To study the cis-trans interaction, we also subjected cross-mated F1 offspring to EE or UCMS and found that both groups have higher mLAB allelic expression, which could be attributed to differences in DNA methylation. Finally, I could show that there was no difference in DNA methylation in the basal mHAB vs. mLAB Tmem132d promoter and that two SNPs in the mHAB promoter were sufficient to cause a higher corresponding promoter activity, which explains the in vivo findings observed in the anterior cingulate cortex. Furthermore, F1 offspring subjected to EE or UCMS had a significantly lower mHAB-specific allele expression which was negatively correlated with DNA methylation, in the Tmem132d promoter region, thus this suggests cross-talk between genetic and environmentally mediated epigenetic factors. In summary, the data suggests a strong evolutionary conserved role of the NPS system considering the similar findings in rats and mice. However, Npsr1 is a nonplastic gene as it is not amenable to the different environmental manipulations applied to the animals. On the other hand, the plastic gene Tmem132d, is differentially expressed, thus making the animals more susceptible to environmental influences. Here, it could be revealed, that SNPs in the mHAB Tmem132d promoter cause higher promoter activity and that environmental manipulation can modulate the gene’s corresponding expression through DNA methylation.