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Abstract Background High throughput microarray analyses result in many differentially expressed genes that are potentially responsible for the biological process of interest. In order to identify biological similarities between genes, publications from MEDLINE were identified in which pairs of gene names and combinations of gene name with specific keywords were co-mentioned. Results MEDLINE search strings for 15,621 known genes and 3,731 keywords were generated and validated. PubMed IDs were retrieved from MEDLINE and relative probability of co-occurrences of all gene-gene and gene-keyword pairs determined. To assess gene clustering according to literature co-publication, 150 genes consisting of 8 sets with known connections (same pathway, same protein complex, or same cellular localization, etc.) were run through the program. Receiver operator characteristics (ROC) analyses showed that most gene sets were clustered much better than expected by random chance. To test grouping of genes from real microarray data, 221 differentially expressed genes from a microarray experiment were analyzed with CoPub Mapper, which resulted in several relevant clusters of genes with biological process and disease keywords. In addition, all genes versus keywords were hierarchical clustered to reveal a complete grouping of published genes based on co-occurrence. Conclusion The CoPub Mapper program allows for quick and versatile querying of co-published genes and keywords and can be successfully used to cluster predefined groups of genes and microarray data.

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.

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.

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.

The glucocorticoid receptor (GR) is an ubiquitously expressed ligand-activated transcription factor that mediates effects of cortisol in relation to adaptation to stress. In the brain, GR affects the hippocampus to modulate memory processes through direct binding to glucocorticoid response elements (GREs) in the DNA. However, its effects are to a high degree cell specific, and its target genes in different cell types as well as the mechanisms conferring this specificity are largely unknown. To gain insight in hippocampal GR signaling, we characterized to which GRE GR binds in the rat hippocampus. Using a position-specific scoring matrix, we identified evolutionary-conserved putative GREs from a microarray based set of hippocampal target genes. Using chromatin immunoprecipitation, we were able to confirm GR binding to 15 out of a selection of 32 predicted sites (47%). The majority of these 15 GREs are previously undescribed and thus represent novel GREs that bind GR and therefore may be functional in the rat hippocampus. GRE nucleotide composition was not predictive for binding of GR to a GRE. A search for conserved flanking sequences that may predict GR-GRE interaction resulted in the identification of GC-box associated motifs, such as Myc-associated zinc finger protein 1, within 2 kb of GREs with GR binding in the hippocampus. This enrichment was not present around nonbinding GRE sequences nor around proven GR-binding sites from a mesenchymal stem-like cell dataset that we analyzed. GC-binding transcription factors therefore may be unique partners for DNA-bound GR and may in part explain cell-specific transcriptional regulation by glucocorticoids in the context of the hippocampus.

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.

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(xxx)b, generated by distal splicing in exon 8 (exon 8b). VEGF(165)b, the first described exon 8b-containing isoform, antagonises VEGF(165) and is anti-angiogenic in vivo., Methods: Using VEGF(xxx)b-specific antibodies, we investigated its expression quantitatively and qualitatively in developing kidney, and measured the effect of VEGF(165)b on renal endothelial and epithelial cells., Results: VEGF(xxx)b formed 45% of total VEGF protein in adult renal cortex, and VEGF(165)b does not increase glomerular endothelial cell permeability, it inhibits migration, and is cytoprotective for podocytes. During renal development, VEGF(xxx)b 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(xxx)b isoforms are highly expressed in adult and developing renal cortex, and suggest that the VEGF(xxx)b family plays a role in glomerular maturation and podocyte protection by regulating the pro-angiogenic pro-permeability properties of VEGF(xxx) isoforms., (Copyright 2008 S. Karger AG, Basel.)

Objective: Nuchal edema (NE) is a clinical indicator for aneuploidy, cardiovascular anomalies, and several genetic syndromes. Its etiology, however, is unknown. In the nuchal area, the endothelium of the jugular lymphatic sacs (JLS) develops by budding from the blood vascular endothelium of the cardinal veins. Abnormal distension of the jugular sacs is associated with NE. We hypothesize that a disturbed lymphatic endothelial differentiation and sac formation causes NE. We investigated endothelial differentiation of the jugular lymphatic system in human and mouse species with NE., Methods: Aneuploid human fetuses (trisomy 21; trisomy 18) were compared with euploid controls (gestational age 12 to 18 weeks). Trisomy 16 mouse embryos were compared with wild type controls (embryonic day 10 to 18). Trisomy 16 mice are considered an animal model for human trisomy 21. Endothelial differentiation was investigated by immunohistochemistry using lymphatic markers (prox-1, podoplanin, lymphatic vessel endothelial hyaluronan receptor [LYVE]-1) and en blood vessel markers (neuropilin [NP]-1 and ligand vascular endothelial growth factor [VEGF]-A). Smooth muscle actin (SMA) was included as a smooth muscle cell marker., Results: We report a disturbed venous-lymphatic phenotype in aneuploid human fetuses and mouse embryos with enlarged jugular sacs and NE. Our results show absent or diminished expression of the lymphatic markers Prox-1 and podoplanin in the enlarged jugular sac, while LYVE-1 expression was normal. Additionally, the enlarged JLS showed blood vessel characteristics, including increased NP-1 and VEGF-A expression. The lumen contained blood cells and smooth muscle cells lined the wall., Conclusion: A loss of lymphatic identity seems to be the underlying cause for clinical NE. Also, abnormal endothelial differentiation provides a link to the cardiovascular anomalies associated with NE.