Genome-Wide Identification of Basic Helix–Loop–Helix and NF-1 Motifs Underlying GR Binding Sites in Male Rat Hippocampus

Abstract

Glucocorticoids regulate hippocampal function in part by modulating gene expression through the glucocorticoid receptor (GR). GR binding is highly cell type specific, directed to accessible chromatin regions established during tissue differentiation. Distinct classes of GR binding sites are dependent on the activity of additional signal-activated transcription factors that prime chromatin toward context-specific organization. We hypothesized a stress context dependency for GR binding in hippocampus as a consequence of rapidly induced stress mediators priming chromatin accessibility. Using chromatin immunoprecipitation sequencing to interrogate GR binding, we found no effect of restraint stress context on GR binding, although analysis of sequences underlying GR binding sites revealed mechanistic detail for hippocampal GR function. We note enrichment of GR binding sites proximal to genes linked to structural and organizational roles, an absence of major tethering partners for GRs, and little or no evidence for binding at negative glucocorticoid response elements. A basic helix–loop–helix motif closely resembling a NeuroD1 or Olig2 binding site was found underlying a subset of GR binding sites and is proposed as a candidate lineage-determining transcription factor directing hippocampal chromatin access for GRs. Of our GR binding sites, 54% additionally contained half-sites for nuclear factor (NF)-1 that we propose as a collaborative or general transcription factor involved in hippocampal GR function. Our findings imply a dose-dependent and context-independent action of GRs in the hippocampus. Alterations in the expression or activity of NF-1/basic helix–loop–helix factors may play an as yet undetermined role in glucocorticoid-related disease susceptibility and outcome by altering GR access to hippocampal binding sites.

MR/GR Signaling in the Brain during the Stress Response

“…In view of their very different effects in the hippocampus, MR and GR should have unique target genes, and this assumption indeed recently has been materialized in three independent studies [31,32,41]. A unique signature of MR binding to DNA loci was discovered and found associated with the NeuroD transcription factor [33].…”

“…Recent studies that evaluated MR and GR binding to the DNA in the hippocampus indicate that the receptors interact with the DNA via their-homologous-DNA-binding domains [31][32][33]. MR and GR share 96% homology in their DNAbinding domain, and both recognize the same "GRE" sequence in the DNA to which they bind as homo-or heterodimers.…”

Thirty years of neuroendocrinology: Technological advances pave the way for molecular discovery

“…The first studies to suggest nuclear hormone receptor interactions with chromatin were reported as early as the 1960s. 28 Additionally, ChIP-Seq has enabled the genome-wide assessment of how binding profiles for GR, ER, PR and AR are highly celltype specific and subject to alteration with changing environmental conditions, [29][30][31][32][33][34][35][36] as well as being dependent upon other regulatory proteins such as AP-1 and FoxA1. Figure 1 shows ChIP-Seq data from rat hippocampal tissue, highlighting a selection of the GR binding events induced by acute stress and exogenous corticosterone treatment.…”

“…Figure 1 shows ChIP-Seq data from rat hippocampal tissue, highlighting a selection of the GR binding events induced by acute stress and exogenous corticosterone treatment. 28 Additionally, ChIP-Seq has enabled the genome-wide assessment of how binding profiles for GR, ER, PR and AR are highly celltype specific and subject to alteration with changing environmental conditions, [29][30][31][32][33][34][35][36] as well as being dependent upon other regulatory proteins such as AP-1 and FoxA1. 30,37 ChIP-Seq has revealed insights into the loss of protein-DNA associations occurring in disease states, such as cancer, increasing our understanding of disease development and potentially leading to the development of more specific therapies.…”

“…60 Similarly, in rat hippocampal tissue, few GR binding sites have been found in promoter regions. 28,61 In MCF7 cells, the majority of ER binding has also been shown to occur at great distances from TSS. 34,57 This has given rise to suggestions of complex chromatin looping to enable interactions between receptors and other transcriptional co-factors to promote transcription.…”