DNA methylation regulates CHRNA7 transcription and can be modulated by valproate.

The CHRNA7 gene encoding the α7 nicotinic acetylcholine receptor (nAChR) has repeatedly been linked with schizophrenia and the P50 sensory gating deficit. The α7 nAChR is considered a promising drug target for treatment of cognitive dysfunction in schizophrenia and improves memory and executive functions in patients and healthy individuals. However, clinical trials with pro-cognitive drugs are challenged by large inter-individual response variations and these have been linked to genotypic variations reducing CHRNA7 expression and α7 nAChR function. Genetic variants as well as environmental conditions may cause epigenetic dysregulation and it has previously been found that DNA methylation of a region surrounding the transcription start site of CHRNA7 is important for tissue specific regulation and gene silencing. In the present study we identify two additional regions involved in epigenetic regulation of the CHRNA7 promoter. In human temporal cortex we find large variations in expression of CHRNA7 and establish evidence for a significant correlation with DNA methylation levels of one region. We then establish evidence that genotypic variations can influence methylation levels of the CHRNA7 promoter. Epigenetic dysregulation can be reversed by pharmacological intervention and in HeLa cells. Valproate, a commonly used mood stabiliser, caused demethylation and increased CHRNA7 expression in HeLA cells. Similar demethylation effect and increased CHRNA7 expression was obtained in SH-SY5Y cells stimulated concomitantly with valproate and nicotine. In summary, both genetic and epigenetic information could be useful to predict treatment outcomes in patients and epigenetic modulation may serve as a mechanism for potentiating the effects of α7 nAChR agonists.

DNA Methylation Analysis of BRD1 Promoter Regions and the Schizophrenia rs138880 Risk Allele.

The bromodomain containing 1 gene, BRD1 is essential for embryogenesis and CNS development. It encodes a protein that participates in histone modifying complexes and thereby regulates the expression of a large number of genes. Genetic variants in the BRD1 locus show association with schizophrenia and bipolar disorder and risk alleles in the promoter region correlate with reduced BRD1 expression. Insights into the transcriptional regulation of BRD1 and the pathogenic mechanisms associated with BRD1 risk variants, however, remain sparse. By studying transcripts in human HeLa and SH-SY5Y cells we provide evidence for differences in relative expression of BRD1 transcripts with three alternative 5' UTRs (exon 1C, 1B, and 1A). We further show that expression of these transcript variants covaries negatively with DNA methylation proportions in their upstream promoter regions suggesting that promoter usage might be regulated by DNA methylation. In line with findings that the risk allele of the rs138880 SNP in the BRD1 promoter region correlates with reduced BRD1 expression, we find that it is also associated with moderate regional BRD1 promoter hypermethylation in both adipose tissue and blood. Importantly, we demonstrate by inspecting available DNA methylation and expression data that these regions undergo changes in methylation during fetal brain development and that differences in their methylation proportions in fetal compared to postnatal frontal cortex correlate significantly with BRD1 expression. These findings suggest that BRD1 may be dysregulated in both the developing and mature brain of risk allele carriers. Finally, we demonstrate that commonly used mood stabilizers Lithium, Valproate, and Carbamazepine affect the expression of BRD1 in SH-SY5Y cells. Altogether this study indicates a link between genetic risk and epigenetic dysregulation of BRD1 which raises interesting perspectives for targeting the mechanisms pharmacologically.

The Schizophrenia-Associated BRD1 Gene Regulates Behavior, Neurotransmission, and Expression of Schizophrenia Risk Enriched Gene Sets in Mice.

BACKGROUND: The schizophrenia-associated BRD1 gene encodes a transcriptional regulator whose comprehensive chromatin interactome is enriched with schizophrenia risk genes. However, the biology underlying the disease association of BRD1 remains speculative. METHODS: This study assessed the transcriptional drive of a schizophrenia-associated BRD1 risk variant in vitro. Accordingly, to examine the effects of reduced Brd1 expression, we generated a genetically modified Brd1+/- mouse and subjected it to behavioral, electrophysiological, molecular, and integrative genomic analyses with focus on schizophrenia-relevant parameters. RESULTS: Brd1+/- mice displayed cerebral histone H3K14 hypoacetylation and a broad range of behavioral changes with translational relevance to schizophrenia. These behaviors were accompanied by striatal dopamine/serotonin abnormalities and cortical excitation-inhibition imbalances involving loss of parvalbumin immunoreactive interneurons. RNA-sequencing analyses of cortical and striatal micropunches from Brd1+/- and wild-type mice revealed differential expression of genes enriched for schizophrenia risk, including several schizophrenia genome-wide association study risk genes (e.g., calcium channel subunits [Cacna1c and Cacnb2], cholinergic muscarinic receptor 4 [Chrm4)], dopamine receptor D2 [Drd2], and transcription factor 4 [Tcf4]). Integrative analyses further found differentially expressed genes to cluster in functional networks and canonical pathways associated with mental illness and molecular signaling processes (e.g., glutamatergic, monoaminergic, calcium, cyclic adenosine monophosphate [cAMP], dopamine- and cAMP-regulated neuronal phosphoprotein 32 kDa [DARPP-32], and cAMP responsive element binding protein signaling [CREB]). CONCLUSIONS: Our study bridges the gap between genetic association and pathogenic effects and yields novel insights into the unfolding molecular changes in the brain of a new schizophrenia model that incorporates genetic risk at three levels: allelic, chromatin interactomic, and brain transcriptomic.

Epigenetic regulation of Dnmt3a and Arc gene expression after electroconvulsive stimulation in the rat.

Electroconvulsive therapy (ECT) remains one of the most effective treatments of major depression. Unfortunately, some patients report side effects, of which the most prominent are memory deficits. The immediate early gene Arc plays a critical role in the maintenance phase of long-term potentiation and consolidation of memory in the rat brain. We recently observed increased methylation of the Arc promoter 24h after acute electroconvulsive stimulation (ECS) in rats, which could cause decreased Arc expression and provide an explanation for the observed memory deficits. In the present study we investigated the methylation and expression changes of Arc at 48h post-ECS and determined the role of de-novo methylation in that process. We initially measured expression of DNA methyltransferases (Dnmt1 and Dnmt3a) and Arc 1, 4, 8, 16, 24, and 48h after a single ECS. Arc expression increased approximately 10-fold at 1 and 4h after ECS, and subsequently decreased below sham levels. Four hours after ECS we also observed a significant increase in Dnmt3a expression, which was attenuated in a second experiment by the use of DNMT inhibitor decitabine (5-aza-2-deoxycytidine). We then investigated Arc gene expression and methylation changes at 48h post-ECS and we found a slightly reduced Arc expression in ECS-treated rats as compared to sham. In animals that received decitabine we observed a significant decrease in Dnmt3a expression and an increase of Arc expression in both ECS and sham groups. The same tendency for reduced Arc expression after ECS, as compared to sham was observed despite the blocking of DNA methylation with decitabine. The DNA methylation as measured by pyrosequencing is decreased 48h post-ECS both in the promoter and intragenic regions as a response to ECS regardless of the treatment with decitabine. Overall the results suggest that DNA methylation is involved in regulating Arc expression but is not the causal mechanism responsible for reducing Arc expression after ECS. We speculate that the decrease is caused by ECS-induced HDAC2 upregulation and decreased H3 acetylation at the Arc promoter.

Temporal gene expression profile after acute electroconvulsive stimulation in the rat.

Electroconvulsive therapy (ECT) remains one of the most effective treatments of major depression. It has been suggested that the mechanisms of action involve gene expression. In recent decades there have been several investigations of gene expression following both acute and chronic electroconvulsive stimulation (ECS). These studies have focused on several distinct gene targets but have generally included only few time points after ECS for measuring gene expression. Here we measured gene expression of three types of genes: Immediate early genes, synaptic proteins, and neuropeptides at six time points following an acute ECS. We find significant increases for c-Fos, Egr1, Neuritin 1 (Nrn 1), Bdnf, Snap29, Synaptotagmin III (Syt 3), Synapsin I (Syn 1), and Psd95 at differing time points after ECS. For some genes these changes are prolonged whereas for others they are transient. Npy expression significantly increases whereas the gene expression of its receptors Npy1r, Npy2r, and Npy5r initially decreases. These decreases are followed by a significant increase for Npy2r, suggesting anticonvulsive adaptations following seizures. In summary, we find distinct changes in mRNA quantities that are characteristic for each gene. Considering the observed transitory and inverse changes in expression patterns, these data underline the importance of conducting measurements at several time points post-ECS.

Low maternal care exacerbates adult stress susceptibility in the chronic mild stress rat model of depression.

In the present study we report the finding that the quality of maternal care, in early life, increased the susceptibility to stress exposure in adulthood, when rats were exposed to the chronic mild stress paradigm. Our results indicate that high, as opposed to low maternal care, predisposed rats to a differential stress-coping ability. Thus rats fostered by low maternal care dams became more prone to adopt a stress-susceptible phenotype developing an anhedonic-like condition. Moreover, low maternal care offspring had lower weight gain and lower locomotion, with no additive effect of stress. Subchronic exposure to chronic mild stress induced an increase in faecal corticosterone metabolites, which was only significant in rats from low maternal care dams. Examination of glucocorticoid receptor exon 17 promoter methylation in unchallenged adult, maternally characterized rats, showed an insignificant tendency towards higher total cytosine methylation in rats from low maternal care dams. Assessment of methylation in the resilient versus anhedonic-like rat phenotypes, revealed only minor differences. Thus, maternal care status seems to be a strong predictor or trait marker for the behavioural phenotype.

Epigenetic regulation of Arc and c-Fos in the hippocampus after acute electroconvulsive stimulation in the rat.

Electroconvulsive stimulation (ECS) remains one of the most effective treatments of major depression. However, the underlying molecular changes still remain to be elucidated. Since ECS causes rapid and significant changes in gene expression we have looked at epigenetic regulation of two important immediate early genes that are both induced after ECS: c-Fos and Arc. We examined Arc and c-Fos protein expression and found Arc present over 4 h, in contrast to c-Fos presence lasting only 1 h. Both genes had returned to baseline expression at 24 h post-ECS. Histone H4 acetylation (H4Ac) is one of the important epigenetic marks associated with gene activation. We show increased H4Ac at the c-Fos promoter at 1 h post-ECS. Surprisingly, we also observed a significant increase in DNA methylation of the Arc gene promoter at 24 h post-ECS. DNA methylation, which is responsible for gene silencing, is a rather stable covalent modification. This suggests that Arc expression has been repressed and may consequently remain inhibited for a prolonged period post-ECS. Arc plays a critical role in the maintenance phase of long-term potentiation (LTP) and consolidation of memory in the rat brain. Thus, this study is one of the first to demonstrate DNA methylation as a regulator of ECS-induced gene expression and it provides a molecular link to the memory deficits observed after ECS.