id not affect the expression dynamics of either GR itself or the GR target gene Tsc22d3 with a simple monotonous activation profile. GR is recruited to binding sites associated with Dex-regulated genes The FFL gene regulatory circuitry predicts that the order Pyrroloquinolinequinone disodium salt master TF binds DNA to regulate transcription of both FFL nodes. Using several published mouse ChIP-seq datasets of acute GR recruitment we interrogated Dexinduced genes for the presence of GR binding sites within the gene and 15 Kb upstream and downstream of the gene. With the exception of Sox4 and Klf4, all genes encoding Dex-induced TFs contained at least one peak within the analyzed intervals. To compare these peaks to those in M, we analyzed GR recruitment by ChIP-seq using untreated M as a control and identified 16,657 peaks induced by a 40-min Dex exposure at 2% FDR. Selective comparison of binding site distributions revealed a high level of concordance between Dex-induced peaks in M and those previously described in adipocytes and a partial overlap with a GR cistrome in M polarized with high dose long-term glucocorticoid exposure. By ChIP-qPCR, we detected GR recruitment as early as 40 min post Dex treatment at multiple putative GR binding sites, including those at Per1, Cited2, Klf2, Klf9, Nfil3, Jdp2, Tiparp and Ncoa5. These observations correlate well with the expression data. Although Klf4 was strongly induced by Dex, no glucocorticoid response elements near the gene has been previously reported. We performed a scanning ChIP with evenly spaced primers within the Klf4 gene and several primers flanking potential GR binding sites. Two of the putative GREs located at -3830 bp and +5896 bp relative to the Klf4 transcription start site recruited GR following a 40-min treatment with Dex, consistent with the notion that, similar to genes shown in Chinenov et al. BMC Genomics 2014, 15:656 http://www.biomedcentral.com/1471-2164/15/656 Page 10 of 19 We then used a set of Dex/LPS-regulated genes to analyze the distribution and enrichment of binding sites for TFs that were present in Chip-Enrichment Analysis database. The ChEA database contains curated published genome-wide datasets of TF binding sites in human, mouse and rat. After filtering out TFs that were not expressed in M, we noted that binding sites for several Dex-responsive TFs, such as KLF2, KLF4, ATF3, EGR1, CEBP and IRF1 are enriched among Dex/LPS-regulated genes. Interestingly, binding sites for PPAR, whose expression was inhibited upon prolonged Dex treatment, were found near the majority of Dex-responsive gene expression regulators and highly enriched among Dex/ LPS-regulated genes in general. Discussion Glucocorticoids- and LPS-regulated gene expression programs GR is a ubiquitous ligand-dependent TF capable of eliciting highly divergent transcription programs with up to a third of protein-coding genes differentially expressed following a 24-h glucocorticoid treatment. Establishing the hierarchy of regulatory events upon prolonged hormonal exposure in individual cell types is challenging, which complicates both accurate mechanistic predictions and clinical decisions. Multiple GR ligands have been designed in an attempt to create a highly specific compound that selectively regulates desired subsets of genes. Mechanistic PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19800191 analyses of these ligands usually focus on a specific group of disease-relevant genes and often involve long-term treatments, which obscure primary and transient responses to GR activation by a plethora of