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.