RESUMEN
In the limbic brain, mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) both function as receptors for the naturally occurring glucocorticoids (corticosterone/cortisol) but mediate distinct effects on cellular physiology via transcriptional mechanisms. The transcriptional basis for specificity of these MR- vs GR-mediated effects is unknown. To address this conundrum, we have identified the extent of MR/GR DNA-binding selectivity in the rat hippocampus using chromatin immunoprecipitation followed by sequencing. We found 918 and 1450 nonoverlapping binding sites for MR and GR, respectively. Furthermore, 475 loci were co-occupied by MR and GR. De novo motif analysis resulted in a similar binding motif for both receptors at 100% of the target loci, which matched the known glucocorticoid response element (GRE). In addition, the Atoh/NeuroD consensus sequence was found in co-occurrence with all MR-specific binding sites but was absent for GR-specific or MR-GR overlapping sites. Basic helix-loop-helix family members Neurod1, Neurod2, and Neurod6 showed hippocampal expression and were hypothesized to bind the Atoh motif. Neurod2 was detected at rat hippocampal MR binding sites but not at GR-exclusive sites. All three NeuroD transcription factors acted as DNA-binding-dependent coactivators for both MR and GR in reporter assays in heterologous HEK293 cells, likely via indirect interactions with the receptors. In conclusion, a NeuroD family member binding to an additional motif near the GRE seems to drive specificity for MR over GR binding at hippocampal binding sites.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Encéfalo/metabolismo , Neuropéptidos/fisiología , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Elementos de Respuesta/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , ADN/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Neuropéptidos/metabolismo , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
In the present study, genomic binding sites of glucocorticoid receptors (GR) were identified in vivo in the rat hippocampus applying chromatin immunoprecipitation followed by next-generation sequencing. We identified 2470 significant GR-binding sites (GBS) and were able to confirm GR binding to a random selection of these GBS covering a wide range of P values. Analysis of the genomic distribution of the significant GBS revealed a high prevalence of intragenic GBS. Gene ontology clusters involved in neuronal plasticity and other essential neuronal processes were overrepresented among the genes harboring a GBS or located in the vicinity of a GBS. Male adrenalectomized rats were challenged with increasing doses of the GR agonist corticosterone (CORT) ranging from 3 to 3000 µg/kg, resulting in clear differences in the GR-binding profile to individual GBS. Two groups of GBS could be distinguished: a low-CORT group that displayed GR binding across the full range of CORT concentrations, and a second high-CORT group that displayed significant GR binding only after administering the highest concentration of CORT. All validated GBS, in both the low-CORT and high-CORT groups, displayed mineralocorticoid receptor binding, which remained relatively constant from 30 µg/kg CORT upward. Motif analysis revealed that almost all GBS contained a glucocorticoid response element resembling the consensus motif in literature. In addition, motifs corresponding with new potential GR-interacting proteins were identified, such as zinc finger and BTB domain containing 3 (Zbtb3) and CUP (CG11181 gene product from transcript CG11181-RB), which may be involved in GR-dependent transactivation and transrepression, respectively. In conclusion, our results highlight the existence of 2 populations of GBS in the rat hippocampal genome.
Asunto(s)
Genoma/genética , Hipocampo/metabolismo , Receptores de Glucocorticoides/metabolismo , Adrenalectomía , Animales , Sitios de Unión/genética , Inmunoprecipitación de Cromatina , Mapeo Cromosómico/métodos , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Estudios de Validación como AsuntoRESUMEN
BACKGROUND: Glucocorticoids, secreted by the adrenals in response to stress, profoundly affect structure and plasticity of neurons. Glucocorticoid action in neurons is mediated by glucocorticoid receptors (GR) that operate as transcription factors in the regulation of gene expression and either bind directly to genomic glucocorticoid response elements (GREs) or indirectly to the genome via interactions with bound transcription factors. These two modes of action, respectively called transactivation and transrepression, result in the regulation of a wide variety of genes important for neuronal function. The objective of the present study was to identify genome-wide glucocorticoid receptor binding sites in neuronal PC12 cells using Chromatin ImmunoPrecipitation combined with next generation sequencing (ChIP-Seq). RESULTS: In total we identified 1183 genomic binding sites of GR, the majority of which were novel and not identified in other ChIP-Seq studies on GR binding. More than half (58%) of the binding sites contained a GRE. The remaining 42% of the GBS did not harbour a GRE and therefore likely bind GR via an intermediate transcription factor tethering GR to the DNA. While the GRE-containing binding sites were more often located nearby genes involved in general cell functions and processes such as apoptosis, cell motion, protein dimerization activity and vasculature development, the binding sites without a GRE were located nearby genes with a clear role in neuronal processes such as neuron projection morphogenesis, neuron projection regeneration, synaptic transmission and catecholamine biosynthetic process. A closer look at the sequence of the GR binding sites revealed the presence of several motifs for transcription factors that are highly divergent from those previously linked to GR-signaling, including Gabpa, Prrx2, Zfp281, Gata1 and Zbtb3. These transcription factors may represent novel crosstalk partners of GR in a neuronal context. CONCLUSIONS: Here we present the first genome-wide inventory of GR-binding sites in a neuronal context. These results provide an exciting first global view into neuronal GR targets and the neuron-specific modes of GR action and potentially contributes to our understanding of glucocorticoid action in the brain.
Asunto(s)
Sitios de Unión/genética , Genómica , Unión Proteica/genética , Receptores de Glucocorticoides/metabolismo , Animales , Inmunoprecipitación de Cromatina , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Células PC12 , Regiones Promotoras Genéticas , Ratas , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Reproducibilidad de los Resultados , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Glucocorticoid (GC) hormones, released by the adrenals in response to stress, are key regulators of neuronal plasticity. In the brain, the hippocampus is a major target of GC, with abundant expression of the GC receptor. GC differentially affect the hippocampal transcriptome and consequently neuronal plasticity in a subregion-specific manner, with consequences for hippocampal information flow and memory formation. Here, we show that GC directly affect the mammalian target of rapamycin (mTOR) signaling pathway, which plays a central role in translational control and has long-lasting effects on the plasticity of specific brain circuits. We demonstrate that regulators of the mTOR pathway, DNA damage-induced transcript (DDIT)4 and FK506-binding protein 51 are transcriptionally up-regulated by an acute GC challenge in the dentate gyrus (DG) subregion of the rat hippocampus, most likely via a GC-response element-driven mechanism. Furthermore, two other mTOR pathway members, the mTOR regulator DDIT4-like and the mTOR target DDIT3, are down-regulated by GC in the rat DG. Interestingly, the GC responsiveness of DDIT4 and DDIT3 was lost in animals with a recent history of chronic stress. Basal hippocampal mTOR protein levels were higher in animals exposed to chronic stress than in controls. Moreover, an acute GC challenge significantly reduced mTOR protein levels in the hippocampus of animals with a chronic stress history but not in unstressed controls. Based on these findings, we propose that direct regulation of the mTOR pathway by GC represents an important mechanism regulating neuronal plasticity in the rat DG, which changes after exposure to chronic stress.
Asunto(s)
Glucocorticoides/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Masculino , Unión Proteica , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Glucocorticoids act in part via glucocorticoid receptor binding to hormone response elements (HREs), but their direct target genes in vivo are still largely unknown. We developed the criterion that genomic occurrence of paired HREs at an inter-HRE distance less than 200 bp predicts hormone responsiveness, based on synergy of multiple HREs, and HRE information from known target genes. This criterion predicts a substantial number of novel responsive genes, when applied to genomic regions 10 kb upstream of genes. Multiple-tissue in situ hybridization showed that mRNA expression of 6 out of 10 selected genes was induced in a tissue-specific manner in mice treated with a single dose of corticosterone, with the spleen being the most responsive organ. Caveolin-1 was strongly responsive in several organs, and the HRE pair in its upstream region showed increased occupancy by glucocorticoid receptor in response to corticosterone. Our approach allowed for discovery of novel tissue specific glucocorticoid target genes, which may exemplify responses underlying the permissive actions of glucocorticoids.