N6-methyladenine is an epigenetic marker of mammalian early life stress

Abstract

Recent evidence described 6-methyladenine (6 mA) as a novel epigenetic regulator in a variety of multicellular species, including rodents; however, its capacity to influence gene expression in the mammalian brain remains unknown. We examined if 6 mA is present and regulated by early life stress associated with predator odor exposure (POE) within the developing rat amygdala. Our results provide evidence that 6 mA is present in the mammalian brain, is altered within the Htr2a gene promoter by early life stress and biological sex, and increased 6 mA is associated with gene repression. These data suggest that methylation of adenosine within mammalian DNA may be used as an additional epigenetic biomarker for investigating the development of stress-induced neuropathology.

Figure 1

Neonatal predator odor exposure (POE) causes lasting effects on juvenile behavior and gene expression in the amygdala. (A) Mixed-sex litters were acutely exposed to variable predator odor for three consecutive days. Half of the animals were sacrificed for gene expression analysis 30 m post-exposure on P3; the remaining half was raised undisturbed before behavior testing and sacrifice at P33. (B) The elevated plus maze (EPM) shows lasting effect of neonatal, POE stress on anxiety-like behavior. (C) RT-qPCR analysis of the juvenile amygdala reveals significant difference in the mRNA expression of the 6 mA demethylase Alkbh1 and Htr2a. Values shown as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 2

Methylation dependent and sensitive restriction enzyme digests reveal sex and stress effects on 6 mA levels. (A) Schematic of Htr2a gene promoter, showing individual methylation sites analyzed by restriction enzyme digestion of DNA coupled with qPCR. (B) MDRE analysis shows modest but significant sex and treatment effects on global amygdalar 6 mA abundance at Site A within the Htr2a promoter. (C) No significant differences in 5 mC levels were observed via MSRE in this region of the Htr2a promoter. Values shown as mean ± SEM, *p < 0.05.

Figure 3

6 mA and 5mC abundance in the juvenile amygdala of unhandled male and female rats at P35 determined by mass spectrometry. (A) Selected-ion chromatogram (SICs) for monitoring the MRM transitions for unlabeled and labeled 6 mA. (B) Absolute levels of 6 mA isolated from amygdala tissue in female and male rats. (C) SIC for monitoring the transition of 5 mC. (D) Sex differences in the relative levels of 5 mC isolated from amygdala tissue. Values shown as mean ± SEM. *p < 0.05.

Figure 4

Methylated DNA immunoprecipitation (MeDIP) analysis of the Htr2a promoter confirms site-specific sex-differences in relative levels of 6 mA. (A) Schematic of Htr2a gene promoter, showing areas investigated by qPCR after either 6 mA or 5 mC MeDIP. (B) Representative DNA gel for 6 mA MeDIP of Htr2a promoter DNA at Site A; a positive input control and negative IgG control for male and female samples are shown for comparison. (C) Males have higher levels of 6 mA at Site A, but not Site B, in the Htr2a promoter within the P35 amygdala. (D) No significant differences found using 5 mC MeDIP. Values shown as mean ± SEM. *p < 0.05.

Figure 5

Chromatin immunoprecipitation (ChIP) in unhandled P35 male and female amygdalae to examine transcriptional activation of Sites A and B within the Htr2a promoter. (A) Females show significantly greater levels of H3K9 acetylation at Site A, but not at Site B. (B) Females show significantly greater binding of the transcription factor Sp1 at Site A, and trend towards greater binding at Site B. Values shown as mean ± SEM. **p < 0.01, *p < 0.05.

Figure 6

Model showing predicted effect of biological sex and stress on 5-HT signaling in the juvenile amygdala. Htr1a acts as a pre-synaptic autoreceptor, inhibiting 5-HT release into the synaptic terminal. Htr2a is expressed post-synaptically, and initiates signal transduction events in response to 5-HT signaling. Chromatin states for the Htr2a promoter are predicted to relaxed or condensed based on 6 mA status. Clockwise from top left: Control females have low levels of Htr1a and high levels of Htr2a, indicating heightened sensitivity to 5-HT signaling. Low levels of 6 mA allow for a relaxed chromatin state in the Htr2a promoter and greater access by the transcriptional machinery. Control males have both higher levels of 6 mA in the Htr2a promoter, leading to condensed chromatin and diminished transcription of Htr2a, and higher levels of Htr1a presynaptically. This would be predicted to cause blunted activation by 5-HT release. Stress males show no significant differences when compared to control males; stressed females phenocopy control and stressed males.