Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat

Abstract

The synthesis of arginine vasopressin (AVP) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus is sensitive to increased plasma osmolality and a decreased blood volume, and thus is robustly increased by both dehydration (increased plasma osmolality and decreased blood volume) and salt loading (increased plasma osmolality). Both stimuli result in functional remodelling of the SON and PVN, a process referred to as functional-related plasticity. Such plastic changes in the brain have recently been associated with altered patterns of DNA methylation at CpG (cytosine-phosphate-guanine) residues, a process considered to be important for the regulation of gene transcription. In this regard, the proximal Avp promoter contains a number of CpG sites and is recognised as one of four CpG islands for the Avp gene, suggesting that methylation may be regulating Avp transcription. In the present study, we show that, in an immortalised hypothalamic cell line 4B, the proximal Avp promoter is highly methylated, and treatment of these cells with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine to demethylate DNA dramatically increases basal and stimulated Avp biosynthesis. We report no changes in the expression of DNA methyltransferases, Dnmt1 and Dnmt3a, whereas there is decreased expression of the demethylating enzyme ten-eleven-translocation 2, Tet2, in the SON by dehydration and salt loading. We found higher methylation of the SON Avp promoter in dehydrated but not salt-loaded rats. By analysis of individual CpG sites, we observed hypomethylation, hypermethylation and no change in methylation of specific CpGs in the SON Avp promoter of the dehydrated rat. Using reporter gene assays, we show that mutation of individual CpGs can result in altered Avp promoter activity. We propose that methylation of the SON Avp promoter is necessary to co-ordinate the duel inputs of increased plasma osmolality and decreased blood volume on Avp transcription in the chronically dehydrated rat.

Keywords: arginine vasopressin; hyperosmotic; hypovolaemia; methylation; supraoptic nucleus.

Figure 1

Schematic diagram of the Avp gene and the presence of CpG (cytosine‐phosphate‐guanine) sites in the proximal promoter region. (a) Avp gene contains three exons indicated by open boxes. The primers used to amplify the 302‐bp Avp promoter are indicated. (b) Transcription factor binding sites (highlighted) and CpG sites (red) investigated within the 350 bp of Avp promoter are shown. The location of forward and reverse primers for amplification of bisulphite converted DNA are underlined. Lower: location of CpG sites in the Avp promoter. TSS, transcription start site; CAAT box; CRE, cAMP response element; AP, activator protein.

Figure 2

Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. (a) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. (b) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μm) increases Avp synthesis. (c) Forskolin (10 μm) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P < 0.001 (b, one‐way anova with Tukey's post‐hoc test; c, two‐way anova with a Bonferonni post‐hoc test). DMSO, dimethyl sulphoxide.

Figure 3

mRNA expression of genes involved in hyperosmotic stress and methylation in the supraoptic nucleus (SON) and cortex of dehydrated and salt‐loaded rats. (a) Brain section stained with 1% (w/v) toludine blue/70% (v/v) ethanol shows the punched area of the SON. The high magnification image shows that the punch samples were confined within the area of magnocellular neurones of the SON. cDNA synthesis and subsequent quantitative polymerase chain reaction analysis was performed using RNA extracted from these punch samples. mRNA expression of hnAvp, c‐Fos, Creb3l1, Dnmt1, Dnmt3a, Tet1, Tet2 and Tet3 was examined in SON (a) and cortex (b). Error bars indicate the mean ± SEM (n = 4–5 per group). *P < 0.05, **P < 0.01, ***P < 0.001 (one‐way anova with Tukey's post‐hoc test). OC, optic chiasm; MCN, magnocellular neurone; DH, dehydration; SL, salt loading; hnAvp, heteronuclear RNA of Avp. Scale bar = 200 μm.

Figure 4

Methylation status of the Avp promoter in response to dehydration and salt loading in the supraoptic nucleus (SON). Genomic DNA was extracted from the SON and cortex of control, 3 days dehydrated and 7 days salt‐loaded rats (n = 4–5). The DNA was bisulphite converted, Avp promoter region amplified and cloned into TA vector for sequencing (n = 20 for each sample). (a) Tile diagrams showing the methylation status of seven CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter extracted from the SON. (b) Percentage of global methylation on the Avp promoter in the SON and cortex of dehydrated and salt‐loaded rats is shown. (c, d) Change in methylation status of CpGs in (c) SON and (d) cortex Avp promoters in response to dehydration and salt loading. (e) Correlation analysis of methylation level with the expression of hnAvp in control SON samples. (f) Methylation level of CpG 1–7 on the Avp promoter of dehydrated and salt‐loaded rats compared to control. Black, hypermethylation; white, hypomethylation; grey, no change in methylation level compared to control. Error bars indicate the mean ± SEM (n = 4–5 per group). *P < 0.05, **P < 0.01 (one‐way anova with Tukey's post‐hoc test); ^#P < 0.05 (unpaired t‐test); DH, dehydration; SL, salt loading.

Figure 5

Methylation of CpG (cytosine‐phosphate‐guanine) sites on the Avp promoter in vitro. The substitution (C‐A) at CpG sites in the Avp promoter by overlap extension polymerase chain reaction was used to prevent methylation at specific CpG sites. The mutation sites are shown in (a). The mutated plasmids were subsequently methylated by methyltransferase enzyme. (b) Successful methylation was determined using methylation sensitive restriction enzyme PmlI. (c, d) Luciferase assays were performed by co‐transfection of plasmid expressing Creb3l1 and 350 bp Avp promoter contructs with (c) unmethylated and (d) methylated plasmid. Error bars indicate the mean ± SEM (n = 4 per group). *P < 0.05; **P < 0.01; ***P < 0.001 (one‐way anova with Tukey's post‐hoc test). 5‐Aza‐dc, 5‐Aza‐2′‐deoxycytidine.