Although encoded by a single gene GR displays

Although encoded by a single gene, GR displays considerable heterogeneity through the combined effects of alternative mRNA splicing, alternative translation initiation, and complex post-translational modification. Differential expression of the various GR isoforms may contribute to tissue-specific functions 26, 27. GRβ, the best-characterized splice variant, uses an alternate exon 9 that disrupts the structure of helices 11 and 12 in the ligand-binding domain, a region required for co-regulator recruitment [28]. Constitutively localized in the nucleus, GRβ cannot activate gene transcription in response to GC, suggesting that it acts as an endogenous dominant negative of GRα, the classic GR referred to in most studies. Although generally expressed at lower levels than GRα, cellular signals affecting the expression ratio of GRβ to GRα show clinical associations with GC sensitivity, autoimmune disease, and lipid metabolic profiles [26]. Amino-terminal truncations of GR occur through the use of seven alternative translation initiation sites. While none directly prohibit DNA or ligand binding, they can alter GR conformation in a manner that affects its subcellular localization and transcriptional activity [29]. Furthermore, extensive phosphorylation of the N-terminal domain of GR adds to the complexity. Up to six serine residues in human GR are phosphorylated in vitro by mitogen-activated protein kinases (MAPKs), cyclin-dependent kinases (CDKs), or glycogen synthase kinase 3 (GSK3) [26]. Phosphorylation of Ser-211 by p38 is associated with co-activator recruitment and transcriptional activation 30, 31, whereas phosphorylation of Ser-226 by c-Jun N-terminal kinases (JNKs) [32] or CDK5 [33] impairs transcriptional activity 30, 34. Intriguingly, ChIP with phospho-specific GR Chloroquine suggests differential recruitment [34], although the affects of phosphorylation and other post-translational modifications on genome-wide binding for GR remain to be tested. Indeed, a future challenge is the development of molecular tools that can distinguish between the different isoforms to determine their genomic functions.
GR Occupies Open Chromatin through Sequence-Specific Binding Upon binding ligand in the cytoplasm, GR moves to the nucleus to target genes for transcriptional regulation (Figure 2A). It binds as a homodimer, and possibly as a homotetramer [35], to a palindromic DNA sequence approximated as g/aGnACAnnnTGTnCt/c. GR binding in vivo has been dramatically informed by the advent of genome-wide approaches 36, 37. Early chromatin immunoprecipitation with deep sequencing (ChIP-seq) studies set the stage for new and surprising determinants for GR binding in diverse cell types and tissues obtained from mice and humans 10, 38, 39, 40, 41, 42, 43, 44. For a field focused on proximal gene promoters, the discovery that most GR-binding sites (GBSs) reside outside of these regulatory regions was eye opening. Also unforeseen was that GBSs vastly outnumber GC-regulated genes. The identification of thousands to tens of thousands of binding sites in any particular cell type surpasses the number of gene targets by an order of magnitude or more, with the caveat that the number of targets may increase if GR-regulated noncoding RNAs are discovered in large numbers. This difference is partly explained by findings showing that GC-regulated genes are enriched for multiple GBSs. However, an unexpectedly large number of sites do not colocalize with the histone modifications, chromatin remodelers, or transcriptional cofactors associated with active enhancers [45]. It is tempting to consider these as experimental noise or artifact, but they are enriched for the GR motif, suggesting sequence-specific binding. While the size and complexity of the GR cistrome is greater than seemingly needed, the existence of sites without transcriptional characteristics may be understood by principles of constructive neutral evolution [46].