Identification of mineralocorticoid receptor target genes in the mouse hippocampus

Abstract Brain mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) respond to the same glucocorticoid hormones but can have differential effects on cellular function. Several lines of evidence suggest that MR‐specific target genes must exist and might underlie the distinct effects of the receptors. The present study aimed to identify MR‐specific target genes in the hippocampus, a brain region where MR and GR are co‐localised and play a role in the stress response. Using genome‐wide binding of both receptor types, we previously identified MR‐specific, MR‐GR overlapping and GR‐specific putative target genes. We now report altered gene expression levels of such genes in the hippocampus of forebrain MR knockout (fbMRKO) mice, killed at the time of their endogenous corticosterone peak. Of those genes associated with MR‐specific binding, the most robust effect was a 50% reduction in Jun dimerization protein 2 (Jdp2) mRNA levels in fbMRKO mice. Down‐regulation was also observed for the MR‐specific Nitric oxide synthase 1 adaptor protein (Nos1ap) and Suv3 like RNA helicase (Supv3 l1). Interestingly, the classical glucocorticoid target gene FK506 binding protein 5 (Fkbp5), which is associated with MR and GR chromatin binding, was expressed at substantially lower levels in fbMRKO mice. Subsequently, hippocampal Jdp2 was confirmed to be up‐regulated in a restraint stress model, posing Jdp2 as a bona fide MR target that is also responsive in an acute stress condition. Thus, we show that MR‐selective DNA binding can reveal functional regulation of genes and further identify distinct MR‐specific effector pathways.


| INTRODUC TI ON
Endogenous glucocorticoid hormones affect brain function via two closely-related nuclear receptors: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). The ligand concentration in part determines the specific MR/GR responses. High affinity MRs are occupied by endogenous corticosteroids under basal conditions and have been found to be more relevant in the initial phase of a stress response. 1,2 By contrast, the lower affinity GRs are activated only at higher glucocorticoid levels, around the peak of the circadian rhythm and during a stress response. Although GRs are expressed widely throughout the central nervous system, brain glucocorticoid binding MRs are mainly restricted to limbic areas. 3 In the hippocampus, MR and GR are crucial for spatial memory and the modulation of cognition, mood and behaviour. 3 Within the CA1 hippocampal subregion, MR and GR mediate opposite glucocorticoid effects on pyramidal neurone excitability 4 via transcriptional mechanisms. 5 Also, spatial learning in rodents is differentially affected by MR and GR signalling, with MR modulating response selection and GR being essential for memory consolidation. 6,7 Because of intrinsic MR-mediated effects that oppose those of GR, it has long been argued that MR-specific target genes must exist. 8 The existence of MR-specific transcriptional coregulators 9,10 also supports this idea. However, many of the effects that can be attributed specifically to MR function so far comprise rapid non-genomic effects, mediated by the membrane variant of the receptor. 11,12 Several classical genomic MR-targets have been described in various tissues over the past two decades, such as FK506 binding protein 5 (Fkbp5), 13 glucocorticoid-induced leucine zipper (Gilz), 14 period circadian clock 1 (Per1) 15 and serum/glucocorticoid regulated kinase 1 (Sgk1). 16 However, these genes are all known to be GR responsive as well. [17][18][19][20] Of note, not only can the two receptors bind their target DNA as homodimers, but also heterodimerisation of MR/GR has been described. 15 Although MR-selective transrepression and transactivation may occur, 21,22 to date, no hippocampal genomic targets have been reported that are strictly MR-dependent. Transcriptional changes have been attributed to MR function, 23 although they were not formally proven to be direct targets of the receptor and might thus be affected by MR activity in an indirect manner. However, although the presence of glucocorticoid response element (GRE) appears to be crucial for both MR and GR DNA binding in the hippocampus, binding sites for NeuroD transcription factors were found selectively at MR-bound loci. 24 NeuroD factors could coactivate glucocorticoid-induced transactivation and were indeed present near MR-specific binding sites, suggesting that specific GRE-dependent MR target genes do exist.
The present study investigated whether direct MR binding to the hippocampal DNA led to expression regulation of the nearby gene. Based on our recent work that defined MR-specific, MR-GR overlapping and GR-specific chromatin binding sites and the corresponding putative target genes within the rat hippocampus, 24 we examined mRNA levels of several genes in each of these categories.
Forebrain MR knockout (fbMRKO) mice showed altered expression for a subset of genes, including down-regulation of the mixed MR/ GR target Fkbp5, as well as MR-specific Jun dimerization protein 2 (Jdp2), Nitric oxide synthase 1 adaptor protein (Nos1ap) and Suv3 like RNA helicase (Supv3 l1) mRNA levels. Subsequently, corticosterone responsiveness of Jdp2, which is one of the genes with an MR-bound promoter, was validated in mice that were exposed to different durations of restraint stress.

| Animals
Male homozygous fbMRKO and control c57bl/6 mice (n = 7) aged 8-9 weeks were housed under a 12:12 hour light/dark reversed photocycle (lights off 9.00 am). The fbMRKO mice were generated using MR flox mice, with MR exon 3 flanked by loxP sites, and mice expressing Cre recombinase controlled by the CAMKIIα gene. 25 Male MR flox/flox CamKCre Cre/wt mice were crossed with female MR flox/flox mice to generate fbMRKO (MR flox/flox_Cre ) and control (MR flox/flox_wt ) offspring. Because the breeding unexpectedly generated more fb-MRKO than control mice, only part of the control animals were littermates. No differences were found in expression levels between littermate and non-littermate controls for any of the genes measured. Mice were transferred to a novel cage 20 minutes before harvesting the tissue and then killed around the time of their endogenous corticosterone peak, between 9.30 am and 12.00 pm. We assessed the expression of MR, overlapping and GR putative target genes in this condition because both receptor types are activated at peak of the diurnal corticosterone rhythm. The novel cage was included in the protocol to ensure MR and GR binding for chromatin immunoprecipitation (ChIP) analysis in the same animals, under the assumption that mRNA levels will not be affected in this short time span. Trunk blood was collected from all mice and hippocampal hemispheres were freshly dissected and snap-frozen in liquid nitrogen for later analysis.
For validation of Jdp2 down-regulation, male fbMRKO (n = 14) and littermate controls (n = 10) aged 8-12 weeks were housed under a 12:12 hour light/dark photocycle (lights on 7.00 am). Mice were bred as described above. Mice were killed under baseline conditions, between 9.30 am and 10.30 am. Brains were collected and snap-frozen in liquid nitrogen for later analysis.
For MR binding site validation in the mouse brain, male c57bl/6 mice (n = 5) aged 16-19 weeks were housed under a 12:12 hour light/dark photocycle (lights on 8.00 am) and were killed in the afternoon 60 minutes after an i.p. injection of 3.0 mg kg -1 corticosterone Male Balb/c mice (n = 3-6) aged 8-15 weeks were housed under a 12:12 hour light/dark photocycle (lights on 6.00 am) and were exposed to various periods of restraint stress (0, 30, 60, 120 and 240 minutes) and killed directly afterwards, between 9.30 am and 2.00 pm. At this time of the diurnal corticosterone trough, both MR TA B L E 1 Primer sequences used for a quantitative polymerase chain reaction in mouse hippocampal chromatin immunoprecipitation samples (for binding site details, see

| Plasma measurements
Trunk blood was centrifuged for 10 minutes at 7000 g, after which plasma was transferred to new tubes. Corticosterone levels of the fbMRKO experiment were determined using an enzyme immunoassay (Immunodiagnostic Systems, East Boldon, UK) and adrenocorticotrophic hormone (ACTH) and corticosterone levels of the restraint stress mice were determined using an enzyme-linked immunosorbent assay (IBL International, Hamburg, Germany) in accordance with the manufacturers' instructions.

| Target gene selection
MR-specific, MR-GR overlapping and GR-specific binding sites were annotated to the nearest gene. 24 To increase the chances of correct annotation and identifying functional target genes, we focused on binding sites located intragenic or in the proximal promoter (up to-5 kb). Furthermore, hippocampal expression 26 of the putative target genes, the degree of coexpression with NeuroD factors (Neurod1/2/6) and face validity of ChiP-sequencing (ChIP-seq) peaks were assessed. The total numbers of putative target genes measured for MR-specific, overlapping (including classical targets) and GR-specific subset were 12, 10 and 9, respectively.

TA B L E 3 Selected putative target genes to validate
Additional binding site information can be found in the supplemental data of van Weert et al. 24 Yellow: MR specific loci; Green: Mixed MR/GR loci; Blue: GR specific loci. Sequences represent the rat glucocorticoid response element (GRE) (upper) and mouse GRE (lower) with mismatches to the rat sequence indicated in red. ABA, Allen Brain Atlas (http://mouse.brain-map.org/); TSS, transcription start site.

| Real-time quantitative PCR
Mouse hippocampal hemispheres were homogenised in TriPure (Roche, Basel, Switzerland) by shaking the tissue with 1.0-mm diameter glass beads for 20 seconds at 6.5 m s -1 in a FastPrep-24 5G instrument (MP Biomedicals, Santa Ana, CA, USA). Total RNA was isolated, cDNA was generated and quantitative real-time PCR was performed as described previously. 24 Because Actb (β-actin) expression was regulated between fbMRKO and control mice, genes of interest were normalised against the in both experiments stably expressed housekeeping gene Rplp0, encoding a ribosomal protein. Primer sequences are listed in Table 2.

| In situ hybridisation
Frozen brains were sectioned at 18 μm in a cryostat microtome, collected on SuperFrost Plus slides (Thermo Scientific, Walthaqm, MA, USA) and stored at −80°C until further use. In situ hybridisation using 35 S UTP-labelled ribonucleotide probes for Jdp2 was performed as described previously. 28

| Statistical analysis
In the fbMRKO experiment, independent t-tests were used and P < 0.01 was considered as a statistically significance cut-off to correct for multiple gene testing. For the ChIP-quantitative PCR validation, we performed one-tailed paired t tests. The predictable directionality (ie, the MR signal is higher than the background IgG signal) justifies the use of a one-tailed test. Because a decrease in signal might also be relevant, we note that all statisitically significant results were at P < 0.025 and therefore would also be significant using a two-tailed test. We considered a paired test appropriate because MR and IgG are measured on the same chromatin sample and this allows correction for the corresponding background levels.
Again, one-tailed unpaired t tests gave essentially the same results.
For the gene Nos1ap, one of the samples was excluded from analysis because of a missing value as a result of non-detectable IgG levels.

| RE SULTS
To explore the functional effects of previously detected MR/GR DNA binding (ie, transcription regulation), binding sites were associated with their nearest gene. This resulted in lists of MR-specific, MR-GR overlapping and GR-specific putative target genes. 24 Gene expression levels, for a subset of each category (Table 3) Figure 1C). Only for Supv3 l1 (P = 0.1784) were we unable to detect the expected MR binding. Also, for classical target genes F I G U R E 2 Hippocampal mRNA levels of glucocorticoid target genes assessed in wild-type (WT) and forebrain mineralocorticoid receptor (MR) knockout (fbMRKO) mice. Gene expression of (A) MR-specific, (B) overlapping and (C) glucocorticoid receptor (GR)-specific targets and (D) classical glucocorticoid targets in fbMRKO versus WT mice, as assessed by independent t-tests with P < 0.01 as the statistically significant cut-off. Other genes measured but not differentially expressed between WT and fbMRKO mice were: Adam23, Arl8b, Dgkb, Els1, Myo16 and Nob1 as MR-specific targets; Grb2, Luzp1 and Map1lc3b as overlapping targets; Arntl, B3galt1, Map2k5, Pglyrp1 and Slc3a2 as GRspecific targets. a.u., arbitrary unit. #P < 0.05 (considered a trend), **P < 0.01, ***P < 0.001, ****P < 0.0001  Several MR-specific putative targets showed lower expression levels in the fbMRKO compared to WT mice (Figure 2A). The most robust effect was found in the Jdp2 mRNA levels, which were reduced by 50% (P < 0.0001). Other differentially expressed genes were MR-specific Nos1ap (P = 0.0005) and Supv3 l1 (P = 0.0061), as well as MR-GR overlapping Camk1d (P = 0.0016) and Kif1c (P = 0.0022), which were also all down-regulated in the fbMRKO compared to WT mice (Figure 2A,B). Moreover, two of the GR-specific genes, Acsl6 (P = 0.0002) and Mrpl48 (P = 0.0065), were expressed at lower levels, and C4ST1 showed a trend for lowered expression (P = 0.0138) ( Figure 2C).
Besides the brain-related putative MR/GR target genes, we measured the expression of the classical target genes Fkbp5, Gilz, Per1 and Sgk1 ( Figure 2D). These genes are all known to be bound and/ or regulated by both MR and GR, comprising our identified MR-GR overlapping target subset contained Fkbp5 and Per1 (Table 3) that was mostly affected in fbMRKO mice showed an increase in response to stress ( Figure 3C) in those animals exposed to restraint for 60-240 minutes. Other genes associated with MR and/or GR binding loci that we had selected for validation did not show transcriptional effects upon restraint stress ( Figure 3C).
Finally, we confirmed Jdp2 down-regulation measured by in situ hybridisation in an independent experiment in fbMRKO (Figure 4). In the absence of MR, Jdp2 mRNA levels were decreased in the principal neurones of the dorsal hippocampus, as was apparent from a significantly lower expression in the CA2 (P = 0.0001), CA3 (P = 0.0357) and dentate gyrus (P = 0.0005) subregions. For the CA1, this occurred at the trend level (P = 0.0901).

| D ISCUSS I ON
Based on non-overlapping MR-GR binding sites, we defined putative MR-specific and GR-specific hippocampal target genes. We identified tends to be modest in response to steroids. 30 Despite possible false negative results, we were able to find robust changes in several MRspecific and classical glucocorticoid target genes.
It is of note that gene regulation by MR knockout and restraint stress was validated in a mouse model, whereas the MR/GR binding loci were obtained from experiments in rats. We were able to show MR binding in the mouse hippocampus at five out of six MR targets originally detected in the rat brain. Evolutionary conservation can increase the predictive value of functional GREs. 31,32 Moreover, because brain MR/GR-mediated regulation is considered to be part of a general adaptive response, the genes regulated in the rat would also be expected to be affected in mice. However, the species difference is an additional potential cause for absence of mRNA regulation. The fbMRKO animals become MR deficient during embryonic development and loss of MR protein is completed after birth. 25 In the present study, down-regulated MR expression was validated and slight up-regulation of GR expression in the hippocampus was observed as described previously. 25 It is possible that MR-dependent gene expression is normalised as a result of compensation by GR or other factors. We cannot exclude that such compensatory mechanisms might also affect expression of Jdp2, Nos1ap and Supv3 l1 in fbMRKO mice. Also, redundancy in gene regulation is not uncommon and, although complete dependence of target genes to a single transcription factor can occur, 33  of an ultra-short feedback loop in which it is induced by glucocorticoids, whereas, in turn, Fkbp5 prevents GR activation. 39 Besides the observed up-regulation of GR expression itself, the lowered Fkbp5 levels could contribute to a compensatory mechanism by relieving the repression of GR function to overcome the lack of MR signalling.
Overall, the Jdp2 gene was the most robust MR target identified in the present study. Initially, Jdp2 was discovered as a negative regulator of activator protein-1 (AP-1) function, by dimerising to c-Jun and preventing transcriptional effects. 40 Later, it was found that Jdp2 can also act in a stimulating fashion as a coactivator for the progesterone receptor. 41 In this latter study, Jdp2 was also shown to have a coactivating effect on transactivation by GR, as confirmed by Garza et al. 42 We found Jdp2 to be a bona fide MR target. A feedforward mechanism could be speculated in which MR can increase Jdp2 levels, which, in turn, could enhance GR activity. A recent ChIP-seq study in mouse neuroblastoma cells found the Jdp2 binding motif near both MR-and GR-bound sites. 43 Besides the differential affinity of MR and GR for their hormone, temporal responses to glucocorticoids could be accounted for by such a feedforward loop.
Feedforward models have been described previously for GR 44 and other nuclear receptors. 45,46 It is worth noting that Jdp2 has been implicated in AP-1 modulation during fear extinction, 47 and polymorphisms in the Nos1ap gene have been linked to post-traumatic stress disorder and depression, 48 also demonstrating a functional role of these genes in the stress system.
In conclusion, we found three novel hippocampal MR-specific target genes, comprising Jdp2, Nos1ap and Supv3 l1, of which Jdp2 is also responsive in an acute stress situation. Dissecting the glucocorticoid response in MR-specific, common and GR-specific pathways will enable us to better understand the stress physiology and pathophysiology of stress-related disorders.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflicts of interest.