3), nor was there any support for the existence of negative GR binding elements (nGRE) (30, 31) in either mouse or human GR ChIP-seq datasets

3), nor was there any support for the existence of negative GR binding elements (nGRE) (30, 31) in either mouse or human GR ChIP-seq datasets. of conserved sites. We conclude that enhancer divergence underlies the difference in transcriptional activation after GC treatment between mouse and human macrophages. Only the shared inducible loci show evidence of selection and therefore these loci may be important for the subset of responses to GC that is shared between species. Keywords: enhancer, evolution, glucocorticoid, macrophage == Introduction == The gene complements of different mammals are remarkably similar (1), which implies that phenotypic variation is driven mainly by differences in transcriptional regulation (24). Expression of orthologous genes can differ between closely related primates (5) and even between individuals within a species (6), with genes involved in extracellular processes and the immune response being the most divergent (7). The most highly-conserved, and highly-inducible promoters are, paradoxically, the most sensitive to variation in expression across species (8). Much of this divergence is driven by the evolution of cis-regulatory elements (9), such as enhancers (10). Deep evolutionary conservation has been used to identify candidate enhancers (11, 12). Nevertheless, complete gain and loss of enhancers between species is also common (1315). In mammals, MDL 105519 at least, turnover of cis-regulatory elements can occur rapidly enough to be identified between strains of a single species (16). Most evolutionarily labile enhancers can be aligned to the genomes of distantly-related species (17), suggesting that the acquisition of novel enhancers can occur through mobilization of existing sequences, including transposable elements (18). Species-specific regulatory elements display a level of nucleotide diversity consistent with a relaxation in evolutionary constraint (14). Since not all transcription factor-binding sites have a direct effect on gene expression (1820), these sites may represent functionally neutral sequence. The profound differences in the immune systems of mouse and man have long been recognised (8, 2123), and are accompanied by both high levels of gene expression divergence and cis-regulatory element turnover (7). This divergence is likely driven by the evolutionary pressure of host-pathogen interactions, alongside changes in the expressed protein-coding sequences driven by positive natural selection (24). Glucocorticoids (GC) are powerful metabolic hormones that are released in response to stress (25) and that provide natural feedback regulation of immune function. Exogenous GC are widely-used as anti-inflammatory therapy (26). Accordingly, their actions on immune cells are likely to be affected by evolutionary selection. GC act by binding to an intracellular nuclear hormone receptor – the glucocorticoid receptor (GR). Nuclear GR may bind directly to DNA, classically as a homo-dimer (27), to a canonical glucocorticoid response element (GRE), or may act indirectly by binding other transcription factors such as NFB and AP-1 (28), as well as by recruiting coregulators, for example GRIP1 (29). Gene repression by GC in inflammation has been linked to binding of negative GR binding elements (nGRE) (30, 31), that are distinct from the consensus GRE. Aside from their ability to repress the actions of proinflammatory stimuli, GC alone act directly on macrophages, producing changes in cell survival, proliferation, morphology and phagocytosis (3235). In other cellular systems, GR binds DNA mainly at cis-regulatory elements (36) and alters MDL 105519 chromatin organization (3739). We sought evidence of the evolutionary pressure on GC actions by comparing the responses of human and mouse macrophages. Only limited expression data for macrophages responding to GC has been generated previously (4042). We confirmed that the majority of genes with a significant shift in expression in response to GC are upregulated (36), but few target genes were shared between the two species. GR binding sites were enriched near to inducible genes in MDL 105519 both species and species-specific binding was associated with species-specific upregulation of genes in the same genomic region. However , these species-specific sites do not appear to be experiencing a selective pressure and the only selection we could detect was for the preservation of the small set of GR-binding sites that were shared between human and mouse. == Methods == == Ethics == Procedures involving human volunteers were approved by the South East Itga3 Scotland National Health Service Research Ethics Committee. All volunteers gave informed consent. Animals were cared for and managed within the Roslin Institutes guidelines for animal safety and welfare. == Cell.