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.

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.

The tetrapod limb provides several examples of heterochrony-changes in the timing of developmental events. These include species differences in the sequence of skeletal chondrogenesis, in gene transcription in the developing limbs, and in the relative time at which forelimb and hind limb buds develop. Here, we examine (i) phylogenetic trends in limb heterochrony; (ii) changes in developmental mechanisms that may lead to heterochrony; and (iii) the possible role that heterochrony plays in generating adaptive traits. We analyze the published literature and present preliminary data on turtle (Emys orbicularis) and bat (Rousettus amplexicaudatus) limb development. Teleosts, marsupials, and some urodeles show extreme timing differences between forelimb (or pectoral fin) and hind limb (or pelvic fin) development; this heterochrony may, in some cases, be adaptive. Published data on limb chondrogenesis reveal sequence elements that are strongly conserved (possibly owing to constraints); and others that vary between higher taxa (for unknown reasons). We find little evidence that chondrogenic sequences are modified by selection for limb functional traits. There are a few examples of developmental mechanisms that may be modified under heterochrony to produce adaptive changes in the limb (e.g. some cases of hyperphalangy or limb reduction). In conclusion, numerous examples of limb heterochrony have been recorded. However, few cases are obviously adaptive. Indeed, current data and methodologies make it difficult to identify the developmental changes, or selective pressures, that may underlie limb heterochrony. More integrative studies, including studies of heterochrony within populations, are needed to assess the role of timing shifts in limb evolution.

Background/Aim: Vascular endothelial growth factor (VEGF), required for renal development, is generated by alternative splicing of 8 exons to produce two families, pro-angiogenic VEGF_xxx, formed by proximal splicing in exon 8 (exon 8a), and anti-angiogenic VEGF_xxxb, generated by distal splicing in exon 8 (exon 8b). VEGF₁₆₅b, the first described exon 8b-containing isoform, antagonises VEGF₁₆₅ and is anti-angiogenic in vivo. Methods: Using VEGF_xxxb-specific antibodies, we investigated its expression quantitatively and qualitatively in developing kidney, and measured the effect of VEGF₁₆₅b on renal endothelial and epithelial cells. Results: VEGF_xxxb formed 45% of total VEGF protein in adult renal cortex, and VEGF₁₆₅b does not increase glomerular endothelial cell permeability, it inhibits migration, and is cytoprotective for podocytes. During renal development, VEGF_xxxb was expressed in the condensed vesicles of the metanephros, epithelial cells of the comma-shaped bodies, invading endothelial cells and epithelial cells of the S-shaped body, and in the immature podocytes. Expression reduced as the glomerulus matured. Conclusion: These results show that the anti-angiogenic VEGF_xxxb isoforms are highly expressed in adult and developing renal cortex, and suggest that the VEGF_xxxb family plays a role in glomerular maturation and podocyte protection by regulating the pro-angiogenic pro-permeability properties of VEGF_xxx isoforms.

BackgroundGlucocorticoids, 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).ResultsIn 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.ConclusionsHere 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.