2013). is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions. Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB. Glucocorticoids play a crucial role in normal physiology and are 9-Aminoacridine highly effective anti-inflammatory drugs with diverse clinical indications including asthma, rheumatoid arthritis, lupus, and inflammatory bowel disease, among many others (Morand 2000; Barnes 2006; Gerber 2015; Kim et al. 2017). Glucocorticoids exert their potent effects through binding to the glucocorticoid receptor (NR3C1, also known as the GR), which causes the GR to translocate 9-Aminoacridine to the nucleus and regulate gene expression through directly interacting with specific DNA sequences (Meijsing 2015; Sacta et al. 2016). Expression changes caused by glucocorticoids include gene induction and repression, with repression encompassing negative FGFA regulation of responses to inflammatory signals such as tumor necrosis factor (TNF) and lipopolysaccharide (LPS), including robust repression of cytokine expression (Rao et al. 2011; Uhlenhaut et al. 2013). Consequently, transcriptional repression is central to glucocorticoid-mediated anti-inflammatory effects (Clark and Belvisi 2012; Chinenov et al. 2013). Pregenomics and deep sequencing-based approaches have established that inductive gene regulation by the GR is typically nucleated through protein-DNA interactions between homodimeric GR and high-affinity palindromic or semi-palindromic consensus GR binding sequences, 9-Aminoacridine which are found in regulatory regions 9-Aminoacridine of glucocorticoid-induced genes (La Baer and Yamamoto 1994; So et al. 2007; John et al. 2008). Mechanisms underpinning GR-mediated gene repression are less well understood. Although protein products resulting from GR-induced gene expression, such as TSC22D3 and DUSP1, are known to indirectly contribute to glucocorticoid-mediated transcriptional repression (Auphan et al. 1995; Ronchetti et al. 2015; Newton et al. 2017), direct repressive effects of the GR on inflammatory transcription factors, such as NF-kB, have long been viewed as principally responsible for the potent repressive effects of glucocorticoids on cytokine expression (Cruz-Topete and Cidlowski 2015; Vandewalle et al. 2018). Such primary repressive effects have been variably attributed to proteinCprotein tethering of the monomeric GR to DNA-associated inflammatory transcription factors, commonly referred to as transrepression (Ratman et al. 2013; De Bosscher et al. 2014), and also to protein-DNA interactions between the GR and so-called negative glucocorticoid response elements (nGREs) found within regulatory regions for inflammatory genes (King et al. 2013). Both mechanisms are purported to ultimately result in GR-centered recruitment of repressive complexes and down-regulation of specific inflammatory genes. Controversy has emerged regarding putative repressive mechanisms. Enrichment for nGRE sequences within GR-occupied regions has not been evident on a genome-wide basis (Rao et al. 2011; Kadiyala et al. 2016; Oh et al. 2017). Similarly, repressive tethering interactions between the GR and NF-kB have not been uniformly observed in ChIP-seq studies (Uhlenhaut et al. 2013; Oh et al. 2017). Accordingly, the notion that GR-mediated repression is largely secondary, that is, a result of GR-induced targets exerting repressive effects, has recently been suggested (Cohen and Steger 2017; Oh et al. 2017). However, experiments with cycloheximide have indicated that protein synthesis is not 9-Aminoacridine required for at least partial glucocorticoid-based transcriptional repression (King et al. 2013). The structure of GR-nGRE complexes.