Identifying signaling pathways and functionally coordinated regions
2016 Study Abstract
Steroid receptors are pleiotropic transcription factors that coordinate adaptation to different physiological states. An important target organ is the brain, but even though their effects are well studied in specific regions, brain-wide steroid receptor targets and mediators remain largely unknown due to the complexity of the brain.
Here, we tested the idea that novel aspects of steroid action can be identified through spatial correlation of steroid receptors with genome-wide mRNA expression across different regions in the mouse brain.
- First, we observed significant coexpression of six nuclear receptors (NRs) [androgen receptor (Ar), estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), glucocorticoid receptor (Gr), mineralocorticoid receptor (Mr), and progesterone receptor (Pgr)] with sets of steroid target genes that were identified in single brain regions. These coexpression relationships were also present in distinct other brain regions, suggestive of as yet unidentified coordinate regulation of brain regions by, for example, glucocorticoids and estrogens.
- Second, coexpression of a set of 62 known NR coregulators and the six steroid receptors in 12 nonoverlapping mouse brain regions revealed selective downstream pathways, such as Pak6 as a mediator for the effects of Ar and Gr on dopaminergic transmission.
- Third, Magel2 and Irs4 were identified and validated as strongly responsive targets to the estrogen diethylstilbestrol in the mouse hypothalamus. The brain- and genome-wide correlations of mRNA expression levels of six steroid receptors that we provide constitute a rich resource for further predictions and understanding of brain modulation by steroid hormones.
… We measured the response of genes that are highly coexpressed with Esr1 in the HY to estrogen diethylstilbestrol (DES) in castrated male mice using quantitative PCR (qPCR). In the male brain, testosterone can be metabolized to estrogen or act directly via the AR. To avoid interpretation difficulties, we decided to activate brain estrogen receptors directly with the selective ligand DES. We selected the top 10 most strongly coexpressed genes with Esr1 in the HY. As a negative control, we used the set of genes that are not coexpressed with Esr1 in the HY. Fig. 5A shows examples, from the ISH experiments of the ABA, of Irs4 and Magel2, two of the strongly coexpressed genes selected for validation. Because Esr1 is not homogeneously expressed across the HY, we analyzed the responsiveness of the set of top 10 genes to DES in the anterior (MPO) and posterior (ARH) parts of the HY separately. Fold-change up-regulation was modest, which may be due to nonresponsiveness, a modest transcriptional response of brain targets, or dilution of the signal in the hypothalamic homogenates. To confirm colocalization further, we performed quantitative double ISH (dISH) for Esr1 and the six mRNAs (Irs4, Magel2, Adck4, Unc5, Ngb, and Gdpd2) that showed more than 1.3-fold enrichment in qPCR. Esr1 mRNA was consistently down-regulated more than twofold upon DES treatment, validating the treatment. Irs4 and Magel2 mRNA were both significantly up-regulated by DES treatment in MPO (1.9-fold and 2.4-fold, respectively), whereas only Magel2 was up-regulated in ARH (2.6-fold). A 1.3-fold induction of Ngb mRNA in ARH did not reach statistical significance, whereas Gdpd2, Unc5d, and Adck4 mRNA levels showed no trend of regulation after DES treatment. Using gene expression measurements (qPCR and dISH), we validated the responsiveness of Irs4 and Magel2 as predicted ESR1 targets to DES treatment. …
Sources and more information
- Full text (free access) : Genome-wide coexpression of steroid receptors in the mouse brain: Identifying signaling pathways and functionally coordinated regions, Proceedings of the National Academy of Sciences, NCBI PubMed PMC4791033, 2016 Mar 8.
- Response of Irs4 and Magel2 to DES treatment in castrated mice in the MPO and ARH using dISH featured image credit PMC4791033/figure/fig05.