Glucocorticoid receptor control of transcription: precision and plasticity via allostery

Abstract

The glucocorticoid receptor (GR) is a constitutively expressed transcriptional regulatory factor (TRF) that controls many distinct gene networks, each uniquely determined by particular cellular and physiological contexts. The precision of GR-mediated responses seems to depend on combinatorial, context-specific assembly of GR-nucleated transcription regulatory complexes at genomic response elements. In turn, evidence suggests that context-driven plasticity is conferred by the integration of multiple signals, each serving as an allosteric effector of GR conformation, a key determinant of regulatory complex composition and activity. This structural and mechanistic perspective on GR regulatory specificity is likely to extend to other eukaryotic TRFs.

Figure 1 |. GR signalling and DNA binding.

a | Linear domain structure of glucocorticoid receptor (GR). GR comprises: the amino-terminal domain (NTD), DNA-binding domain (DBD), hinge region and ligand-binding domain (LBD). Embedded in these domains are segments that participate, context-specifically, in transcription regulation: activation function domain 1 (AFl,τ1),tau2 (τ2) and AF2. Insets: Crystal structures are shown for a single DBD (green; adapted from RCSB Protein Data Bank identifier (PDB ID): 1R4R), with coordinated zinc ions shown as grey spheres, and for a LBD (purple) liganded with cortisol (yellow) and complexed with steroid receptor co-regulator 2 (SRC-2) peptide (not shown) (PDB ID: 4P6X). b | Overview of signalling mediated by the natural GR ligand, cortisol. Activating ligand interacts with monomeric GR associated with molecular chaperone-containing complexes in the cytosol. This induces local and remote allosteric changes that potentiate nuclear transport and other activities. Within the nucleus, GR nucleates multi-component transcription regulatory complexes containing various other transcriptional regulatory factors (TRFs) and transcriptional co-regulators at different glucocorticoid response elements (GREs) to activate or repress transcript ion of particular target genes. GRE1 and GRE2 represent distinct GREs within the genome, Gene X and Gene Y represent the genes under the control of GRE1 and GRE2, respectively.

Figure 2 |. Modes of site-specific GR-genome interactions.

A | Glucocorticoid receptor (GR) associates with specific genomic sites in multiple ways. Aa | Two GR monomers bind a canonical GR-binding seguence (GBS) present in a glucocorticoid response element (GRE) in a head-to-head fashion; dimerization is achieved through interactions in the sister DNA-binding domains (DBDs). Ab | GR bindsto inverted-repeat GBSs (IR-GBSs).The crystal structure of this interaction (see also part Ac) shows two GR monomers bound to opposite sides of the DNA in a head-to-tail fashion; however, negative cooperativity argues that GR may bind as a monomer to IR-GBSs in vivo (thus, the second GR monomer is shown faded). Ac | GR can interact with GBS half sites; these elements typically contain a single hexamer related to the consensus seguence that is palindromic in the full GBS and may operate in conjunction with proximal non-GR transcriptional regulatory factors (TRFs), although there is no evidence for enrichment of particular TRF binding site (TRF-BS) motifs contiguous with the GBSs. Ad | GR can interact at specific genomic sites without directly binding to DNA. Here, GR is tethered to a non-GR TRF through protein-protein interactions. The faded GR monomers depict uncertainty of whether monomeric or dimeric GR binds at tethering elements. B| Overall crystal structure of the DBD of GR with the canonical GBS(RCSB Protein Data Bank identifier (PDB ID: 3FYL).C| Overall crystal structure of the complex between the DBD of GR and the IR-GBS (PDB ID: 4HN5 (REF. 25)). D | Zoom of structure shown in B.The GR DBD makesthree base-specific contacts within the major groove of the DNA at the GBS.This interaction is mediated by hydrogen bonds (red dashed lines) and van der Waals interactions (black dashed lines). E | Zoom of structure shown in C. The GR DBD makescontactsto an IR-GBSthat are similartothose made in the case of the canonical GBS, with the exception of Arg447, which does not make any contacts with the DNA. F | Superposition of all deposited structures of the interactions between DBD and DNA reveals especially striking conformational differences within the GR lever arm (residues 469–474, a loop connecting the ‘DΝΑ-reading helix’with the dimerization region), demonstrating allosteric modulation of GR by DNA seguence. DNA seguences from each structure are listed and colour coded, the gene the seguence is derived from is listed in parenthesis (PDB IDs listed in the top-down order of the seguences given: 3FYL, 3G99 (REF. 18), 3G6U, 1GLU, 1R4O, 1R4R, 3G9P, 3G9O, 3G9M, 3G9J and 4HN5 (REF. 25)). CGT, ceramide UDP-galactosyltransferase (also known as UGT8); FKBP5, FK506-binding protein 5 (also known as FKBP1B); LBD, ligand-binding domain; PAL, synthetic palindromic consensus site with AAA spacer; SGK, serum/glucocorticoid-regulated kinase 1; TSLP, thymic stromal lymphopoietin protein receptor (also known as CRLF2).

Figure 3 |. Context-specific GR occupancy and gene regulation.

a | Glucocorticoid receptor (GR)-regulated genes are commonly linked to multiple GR-occupied regions (GORs), usually >10 kb from the transcription start site of the regulated gene, one or more of which may be a functional glucocorticoid response element (GRE) for that gene. As shown here, GREs may be near (<100 bp) to or far (>100 kb) from their target genes. b | Illumina Human Ref8 beadchip analysis of glucocorticoid-regulated genes. Genes regulated by 4-hour, 100-nM dexamethasone treatment in lung carcinoma (A549) (K.R.Y., S. Cooper, S.-H. Chen and B. Schiller, unpublished observations) and osteosarcoma (U20S) cells. Differentially responsive genes are upregulated in one cell line and downregulated in the other. Common genes are similarly regulated in both cell lines. Unigue genes are regulated in one cell line and not regulated in the other. The abundance of genes within the unigue and differentially regulated classes demonstrates cell-context-specific regulation by GR. Differentially responsive genes demonstrate distinct mechanisms of regulation of target genes in the two cell types. AF1, activation function domain 1; DBD, DNA-binding domain; GTFs, general transcription factors; LBD, ligand-binding domain; Pol II, RNA polymerase II.

Figure 4 |. GR-ligand interactions.

a | Cortisol, dexamethasone and RU-486 are three glucocorticoid receptor (GR) ligands; RU-486 also binds to progesterone receptor (PR); oestradiol, which binds to oestrogen receptor (ER) but not GR, is shown for comparison; cholesterol is shown to provide the sterol carbon numbering convention. b | Ligand-binding pocket (purple) of the ligand-binding domain (LBD) of GR bound to its endogenous ligand, cortisol (yellow). Residues within 4.2 Å are shown, and hydrogen bonds are depicted by dashed red lines. This structure highlights the intricate network of interactions between GR and its ligand, as well as the amount of unoccupied space within the ligand-binding pocket with the ligand bound. This is a zoom of the ligand binding pocket from structure shown in panel c. c | Overall structure of the GR LBD (purple) bound to cortisol (yellow) and the steroid receptor co-regulator 2 (SRC-2) peptide (green) (RCSB Protein Data Bank identifier (PDB ID): 4P6X).The inset cartoon represents how the LXXLL motif (the so called nuclear receptor box) of the co-regulator peptide interacts with GR. The peptide is held in place by a conserved charge clamp interaction; the positively charged Lys579 residue (+) and negatively charged Glu755 residue (−) on helix 3 (H3) and helix 12 (H12), respectively, mediate this interaction. There is an additional charge clamp that occurs through the residues Arg585 and Asp590. This is referred to as the active conformation, and is associated with specific co-regulator binding. d | Overall structure of the GR LBD bound to RU-486 (yellow) and the nuclear receptor co-repressor (NCoR) co-regulator peptide (pink) (PDB ID: 3H52 (REF. 164)). The inset cartoon represents how the extended co-repressor nuclear receptor (CoRNR) boxes of the co-regulator peptide interact with GR. The extended peptide makes only one of the conserved interactions, through Lys579, and H12 is displaced. This is considered an inactive form of the LBD. Differences in GR conformations presented in parts c and d indicate that different ligands can promote the formation of alternative protein surfaces on GR that in turn differentially affect co-regulator binding.

Figure 5 |. Sites of glucocorticoid receptor post-translational modifications.

Major reported modifications, including phosphorylation (P), sumoylation (S), ubiguitylation (U), acetylation (A) and nitrosylation (N), are mapped onto the glucocorticoid receptor domain schematic. AF, activation function domain; DBD, DNA-binding domain; LBD, ligand-binding domain; NTD, amino-terminal domain.

Figure 6 |. A model for transcription regulation: precision and plasticity of TRF function achieved via allostery.

In our model for metazoan transcription regulation, transcriptional regulatory factors (TRFs), such as glucocorticoid receptor (GR), may lack intrinsic regulatory activities, serving instead as molecular scaffolds that can assume different conformations in response to modification by different combinations of specific signalling inputs. Four classes of signalling input are described: ligands and post-translational modifications (PTMs) provide physiological context, DNA-binding seguences provide gene context, and non-GRTRFs provide cell context. Each class of signal confers distinct allosteric effects, and their integrated actions can produce a vast range of GR conformations, which induce, expose or stabilize context-specific proteinsurfacesthat are read by co-regulators. These co-regulators, which are generally large multi-component enzyme-containing complexes, interact in distinct combinations with patterns of cognate GR surfaces and enzymatically modify the general transcriptional machinery and/or surrounding chromatin in and around glucocorticoid response elements (GREs) and target promoters and genes, resulting in the activation or repression of gene expression. Upper panel: lines around GR domains depict direct and allosteric conformational alterations imposed by the signalling inputs. Note that a lack of net regulation (that is, neither activation nor repression) could reflect balanced actions of one or more GREs acting on a single gene (not shown). AF, activation function domain; DBD, DNA-binding domain; LBD, ligand-binding domain. Circles with letters A, P,S indicate different PTMs.