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

Abstract

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.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 0 -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.Key words: methylation, arginine vasopressin, supraoptic nucleus, hyperosmotic, hypovolaemia doi: 10.1111/jne.12371The neuropeptide hormone arginine vasopressin (AVP) is synthesised in magnocellular neurones of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. Increases in plasma osmolality are detected by osmosensitive neurones in circumventricular organs that provide direct excitatory inputs leading to increased Avp synthesis by magnocellular neurones of the SON and PVN, as well as AVP secretion from the posterior pituitary (1). An increase in plasma osmolality of only 1% is sufficient to drive increased AVP synthesis and secretion (2).Vasopressin synthesis and secretion is also sensitive to nonosmotic cues, including changes in blood volume and pressure (3-7). A decrease in blood ...

Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1) Expression by Orphan Nuclear Receptor Nr4a1

“…In addition, our findings from in vitro cell cultures hinted that methylation could be important in regulating Creb3l1 expression in different cell-types of the PVN, namely AVP and OT neurones, particularly as Creb3l1 expression increases exclusively in AVP neurones by DH (Greenwood et al, 2014 ). We recently described altered methylation patterns of the Avp promoter with DH in the SON and in this study we observed decreased expression of the demethylating enzyme ten-eleven translocation 2, Tet2, by DH and SL in support of methylation changes in the hypothalamus (Greenwood et al, 2016a ). Interestingly there was an absence of methylation marks on this region of the Creb3l1 promoter in vitro in the PVN.…”

“…Treatment with Aza alone increased Avp expression, and when combined with FSK this increase in expression was markedly enhanced. To demonstrate that increased Avp expression was not the result of demethylation of the Avp promoter as we have previously reported (Greenwood et al, 2016a ), we overexpressed Creb3l1 in N2a cells and were able to successfully reproduce this response. Therefore, methylation strongly influences the actions of Nr4a1 as a transcriptional regulator of the Creb3l1 gene in vitro .…”

“…The punches were collected bilaterally from three brain slices and dispensed into 0.5 ml tubes (six punches per tube) placed on dry ice within the cryostat. Total RNA and genomic DNA were extracted from each sample as previously described (Greenwood et al, 2016a ).…”

“…Genomic DNA from AVP and OT PVN punches (whole sample) and cultured cells (200 ng) was bisulfite converted using Zymo EZ DNA Methylation-Gold kit (Cambridge Bioscience) as previously described (Greenwood et al, 2016a ). Primers for amplification of bisulfite converted DNA were designed using MethPrimer software; (mouse 5′-TTAGAGAGTTGAGTTAGTTAAGGAAA-3′ and 5′-CAAAAATCTCTCTAAATCTCTTCC-3′; rat 5′-AGGAAGTTTATAGTTTTTAGGATAG-3′ and 5′-AAAAATCTCTCTAAATCTCTTCCTC-3′; human 5′-TTGGAAGGATGGAAATAGTTTT-3′ and 5′-AAACCCCTAACTAACTAACCCAACTA-3′).…”

The effects of aging on biosynthetic processes in the rat hypothalamic osmoregulatory neuroendocrine system

“…This has led some to suggest that deficits in mechanisms controlling transcription, mRNA stability, or translation in the aging SON magnocellular neurons may be responsible ( Lucassen et al., 1997 ). Moreover, we recently showed that dehydration initiates the formation of new methylation marks on the rat Avp promoter ( Greenwood et al., 2016a ), suggesting that altered methylation patterns could lie beneath these transcriptional changes in aging AVP neurons.…”