Abstract
Glucocorticoid hormones (GCs) — acting through hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) — are critical to physiological regulation and behavioural adaptation. We conducted genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus to elucidate MR- and GR-regulated genes under circadian variation or acute stress. In a subset of genes, these physiological conditions resulted in enhanced MR and/or GR binding to DNA sequences and associated transcriptional changes. Binding of MR at a substantial number of sites however remained unchanged. MR and GR binding occur at overlapping as well as distinct loci. Moreover, although the GC response element (GRE) was the predominant motif, the transcription factor recognition site composition within MR and GR binding peaks show marked differences. Pathway analysis uncovered that MR and GR regulate a substantial number of genes involved in synaptic/neuro-plasticity, cell morphology and development, behavior, and neuropsychiatric disorders. We find that MR, not GR, is the predominant receptor binding to >50 ciliary genes; and that MR function is linked to neuronal differentiation and ciliogenesis in human fetal neuronal progenitor cells. These results show that hippocampal MRs and GRs constitutively and dynamically regulate genomic activities underpinning neuronal plasticity and behavioral adaptation to changing environments.
Introduction
Glucocorticoid hormones (GCs) are of critical importance for central nervous system functioning both during daily activities and after a stressful challenge. They play a pivotal role in stress resilience and behavioral adaptation1,2,3,4,5. Impaired GC secretion and function has been associated with stress-related mental disorders like major depression and post-traumatic stress disorder3,5,6.
GC secretion from the adrenal glands varies over the circadian cycle, peaking at the onset of the active phase. Exposure to a stressful event results in a transient surge of GC secretion that is superimposed on the circadian variation. In the brain, GCs bind to two types of receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR)7, which show striking differences in localization and function in the brain. Whilst expression of MR is predominantly in neurons within specific limbic structures such as the hippocampus and lateral septum, GR is widely distributed throughout the brain, including the hippocampus8. MRs and GRs co-localize in hippocampal neurons8,9. Once bound and activated by GCs, MRs and GRs translocate to the cell nucleus to act as ligand-dependent transcription factors. It is thought that genomic actions in the hippocampus underlie the distinct roles of MR and GR in the control of circadian and stress-related physiology, cognition and behaviour, however, the molecular underpinnings of these receptor-mediated actions are still largely unresolved10,11.
Comprehensive knowledge about genome-wide MR and GR interactions within the hippocampus under physiological conditions is currently lacking. Specifically, which genes are regulated by MR and/or GR after stress or across the circadian cycle is still unknown. Previously, we conducted chromatin immunoprecipitation (ChIP) analyses to assess the binding of MR and GR to well-known GC-target genes in the hippocampus of rats killed under baseline and stress conditions12. This study showed for the first time that MRs and GRs bind to GC-response elements (GREs) within the GC-target genes Fkbp5 (FK506 binding protein 5), Per1 (Period circadian regulator 1) and Sgk1 (serum/glucocorticoid regulated kinase 1) as homo- or heterodimers in a gene- and GRE-dependent manner in vivo12. Despite providing novel insight into the complexity of MR/GR interaction, the conclusions of this study were limited to those genes. Therefore, a comprehensive genome-wide study into GC receptor interaction with the DNA was required to identify MR- and GR-regulated genes and clarify the possible existence of selectively MR- or GR-regulated genes. Moreover, as long as the identity of all MR- and GR-targeted genes is undetermined, the full implications of GC signaling in the hippocampus will be unknown.
Therefore, in this study, we investigated the interaction of MR and GR with the entire genome of the rat hippocampus after an acute stress exposure (forced swimming (FS)) as well as under baseline early morning (BLAM) and baseline late afternoon (BLPM) conditions. Moreover, we integrated these data with genome-wide gene transcriptional responses occurring in the rat hippocampus under the same conditions. This approach revealed a comprehensive map of the genomic loci bound by MR and/or GR, the transcription factor binding motifs involved, as well as the associated gene transcriptional responses providing novel insights into the function of MR and GR in the hippocampus….
