Two Populations of Glucocorticoid Receptor-Binding Sites in the Male Rat Hippocampal Genome

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. - See more at: http://press.endocrine.org/doi/10.1210/en.2012-2187?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed#sthash.LqK088DP.dpuf本研究通过染色质免疫共沉淀（chromatin immunoprecipitation）结合下一代测序（next-generation sequencing）技术，于大鼠海马体内鉴定了糖皮质激素受体（glucocorticoid receptors, GR）的基因组结合位点。本研究共鉴定出2470个显著性GR结合位点（GR-binding sites, GBS），并随机选取覆盖广泛P值范围的部分结合位点，验证了GR与其的结合活性。对显著性GBS的基因组分布分析显示，基因内结合位点占比极高。携带GBS或紧邻GBS的基因中，显著富集了参与神经元可塑性及其他关键神经元活动的基因本体（gene ontology, GO）聚类。对雄性去肾上腺大鼠给予梯度剂量的GR激动剂皮质酮（corticosterone, CORT），剂量范围为3至3000 μg/kg，结果导致不同GBS的GR结合谱出现显著差异。可将GBS分为两类：一类为低剂量响应组，在所有浓度的CORT处理下均能检测到GR结合；另一类为高剂量响应组，仅在给予最高剂量CORT时才呈现显著的GR结合。低、高剂量响应组中所有验证过的GBS均能结合盐皮质激素受体（mineralocorticoid receptor），且在CORT剂量为30 μg/kg及以上时，结合水平保持相对稳定。基序（motif）分析显示，几乎所有GBS均包含与文献中共识基序相似的糖皮质激素反应元件（glucocorticoid response element）。此外，本研究还鉴定出与新型潜在GR互作蛋白对应的基序，例如含锌指和BTB结构域蛋白3（zinc finger and BTB domain containing 3, Zbtb3）以及CUP（CG11181基因转录本CG11181-RB的编码产物），二者可能分别参与GR介导的转录激活（transactivation）与转录抑制（transrepression）过程。综上，本研究结果揭示了大鼠海马基因组中存在两类GBS群体。- 详情请见：http://press.endocrine.org/doi/10.1210/en.2012-2187?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed#sthash.LqK088DP.dpuf