Perturbations of Neuron-Restrictive Silencing Factor Modulate Corticotropin-Releasing Hormone Gene Expression in the Human Cell Line BeWo

Abstract

Stress exacerbates disease, and understanding its molecular mechanisms is crucial to the development of novel therapeutic interventions to combat stress-related disorders. The driver of the stress response in the hypothalamic-pituitary-adrenal axis (HPA) is corticotropin-releasing hormone (CRH), a neuropeptide synthesized in the paraventricular nucleus of the hypothalamus. Evidence supports that CRH expression is epigenetically modified at the molecular level by environmental stimuli, causing changes in the stress response. This effect is mediated by a concert of factors that translate environmental change into alterations in gene expression. An important regulator and epigenetic modulator of CRH expression is neuron-restrictive silencing factor (NRSF). Previously, our lab identified numerous splice variants of NRSF that are specific to humans and predictive of differential regulatory effects of NRSF variants on targeted gene expression. The human cell line BeWo has endogenous CRH and NRSF expression providing an in vitro model system. Here, we show that manipulation of NRSF expression through siRNA technology, overexpression by plasmid vectors, and direct cAMP induction that CRH expression is linked to changes in NRSF expression. Accordingly, this epigenetic regulatory pathway in humans might be a critical mechanism involved in the regulation of the stress response.

Keywords: Corticotropin-releasing hormone; Neuron-restrictive silencing factor; Stress regulation.

Fig. 1

a NRSF baseline expression and splicing pattern of variants driven by three alternative promoters (Pa, Pb, Pc) and splicing in NRSF Pa producing variants with partial and complete exon 2 exclusion. b Exponential curve of fluorescence accumulation upon qPCR with CRH-specific primers targeting exon 2, along with no template control, along with single expected amplicon observation by gel electrophoresis of the PCR product, also observed by melting curve analysis.

Fig. 2

a NRSF's coding DNA sequence (CDS) and the respective binding of the siRNA. b Decreased expression of NRSF upon treatment with 10 μM siRNA, no significant difference in total expression decrease by the siRNAs (n = 4). c Differential increase in CRH expression by siRNA downregulation of NRSF, suggesting binding site but not NRSF quantitative decrease is responsible (n = 4). ANOVA with repeated measures was performed to assess significance: ns, p > 0.05; ** p < 0.01; *** p < 0.001.

Fig. 3

Transfection with 2 μg pcDNA3.0 (CTL), NRSF insert (NRSF), and NRSF4 insert (NRSF4). Cells were transfected in 2 independent experiments and selected over a period of 2 weeks for generation of a clonal population. a The endogenous mechanism of NRSF4 splicing. b NRSF expression induction upon plasmid transfection. c CRH expression changes due to overexpression of NRSF isoform constructs.

Fig. 4

Differential changes in NRSF differential promoter (a, b, c) variant expression (b), and increasing trend in CRH expression (a) upon time course treatment with 250 μM 8-Br-cAMP. Independent experiments (n = 4) where analyzed with paired two-tailed t test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 5

a Scatter of NRSF and CRH RPKMs matched to patients across different brain regions; line corresponds to linear regression output. b Distribution of CRH and NRSF RPKMs. A binomial distribution is observed due to the threshold observed in NRSF RPKM (approximately 1.5).