The brain is a major target of glucocorticoids (GCs) that are secreted by the hypothalamus-pituitary-adrenal axis in response to stress. In the brain there are two receptors for glucocorticoids, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), that differ in their expression pattern and affinity for GCs. GR is abundantly expressed throughout the brain both in neurons and non-neuronal cells such as microglia and astrocytes [1
]. GR has a relatively low affinity for its ligand, cortisol in humans and corticosterone in rodents (both abbreviated as CORT), and is activated when CORT levels rise, for example during stress. Upon CORT binding, GR migrates from the cytoplasm to the nucleus where it is involved in the regulation of gene transcription.
Transcriptional regulation by GR is complex and several molecular mechanisms have been described involving both homodimers and monomers of GR. Direct binding of GR dimers to Glucocorticoid Response Elements (GREs) in the vicinity of target genes, a process known as transactivation, is the classical mode of action which generally results in a potentiation of transcription [5
]. However, GR also exhibits extensive crosstalk with other transcription factors (TFs), and besides simple GREs composite sites exist that contain a binding site for another TF in close proximity to the GRE, resulting in either a synergistic activation or a repression of transcription [6
]. Furthermore, GR monomers can also exert effects on gene transcription by indirectly binding to the DNA via an intermediate DNA-bound TF in so called tethering response elements [8
], mostly resulting in a repression of transcription of the associated gene, a process referred to as transrepression. This extensive crosstalk of GR with other TFs not only vastly expands the range of GR-control on physiological processes compared to the classical GRE-driven transcriptional control in simple GREs, but it also underlies the highly context-dependent action of GCs.
Several TFs have been described that participate in this crosstalk with GR, including Oct1, Ets1, AP-1 and CREB at composite GREs and NF-κB, AP-1, CREB, Oct-1/2, STAT6, SMAD3,4 and PU.1/Spi-1 at tethering sites [6
]. However, most of these crosstalk partners of GR have been identified in studies on the immunosuppressive and the tumor suppressor properties of GR [17
], while very little is known about crosstalk partners in a neuronal context.
In neuronal cells GR regulates the expression of a wide diversity of genes involved in general cellular processes such as energy metabolism, cell cycle and response to oxidative stress, but also clearly is involved in regulating a wide variety of genes important for neuronal structure and plasticity [20
]. Despite the fact that many neuronal GC-responsive genes have been identified [21
], it remains unclear whether these genes are primary or downstream targets of GR. The onset of high-throughput sequencing combined with chromatin immunoprecipitation (ChIP-Seq) has made it possible to characterize genome-wide binding sites of TFs and today several studies have used this approach to identify global primary GR-targets in a variety of cell types, including human lung carcinoma cells (A549), mouse adipocytes (3T3-L1), premalignant breast epithelial cells (MCF10A-Myc), murine mammary epithelial cells (3134) and pituitary (AtT-20) cells [24
]. These studies have yielded an unprecedented insight into genome wide GR targets as well as molecular mechanisms of GR-signaling, but perhaps one of the most striking findings is the low degree of overlap in GR binding sites when comparing different cell types, indicating that GR occupancy is highly cell type specific [25
]. Therefore, in order to gain insight into global GR primary target in neurons, it is essential to characterize GR binding in a neuronal context. So far no studies have taken a ChiP-Seq approach to characterize GR-binding in a neuronal context.
The aim of the current study was to analyze genome-wide GR-binding sites (GBS) in rat neuronal PC12 cells using ChIP-Seq. The PC12 cell line is derived from a pheochromocytoma of the rat adrenal medulla and can be differentiated into a neuronal phenotype by stimulation with nerve growth factor [28
]. NGF- treated PC12 cells stop dividing, develop neurites, display electrical activity and develop many other properties similar to those of sympathetic neurons [28
]. They are considered a useful model system for neurosecretion and neuronal differentiation [30
] and have been extensively used to study neuronal function in relation to GCs [31
]. In this study, besides identifying the binding sites of GR in neuronal PC12 cells, we analysed which genes were located in the vicinity of the binding sites, which gene ontology classes were overrepresented, whether GR-binding resulted in regulation of gene expression of nearby genes and the motif composition of the binding sites.