DNA binding site sequence directs glucocorticoid receptor structure and activity

Abstract

Genes are not simply turned on or off, but instead their expression is fine-tuned to meet the needs of a cell. How genes are modulated so precisely is not well understood. The glucocorticoid receptor (GR) regulates target genes by associating with specific DNA binding sites, the sequences of which differ between genes. Traditionally, these binding sites have been viewed only as docking sites. Using structural, biochemical, and cell-based assays, we show that GR binding sequences, differing by as little as a single base pair, differentially affect GR conformation and regulatory activity. We therefore propose that DNA is a sequence-specific allosteric ligand of GR that tailors the activity of the receptor toward specific target genes.

Fig. 1

GBSs differentially direct GR activity. (A) GBSs were cloned upstream of a minimal SV40 promoter driving luciferase. Transcriptional activities and binding affinities (humanGR-DBD 380 to 540) for each GBSs ± SEM are shown [number of independent experiments (n) ≥ 3]. K_D, dissociation constant. (B) GBS- specific patterns of domain utilization. GBS reporters respond differentially to mutations in Dim (red, A477T), AF1 (yellow, E219K/F220L/W234R), and AF2 (green, E773R) domains. Fold induction by dex ± SEM (top) and percent induction by mutant GR relative to wild type (bottom) are shown (n ≥ 3). (C) Immunoblots demonstrating short hairpin–mediated RNA (shRNA) knock-down of Brm and CARM1. (D) GBS inductions after CARM1 or Brm knock-down, relative to scrambled shRNA ± SEM, are shown (n = 3).

Fig. 2

DNA sequence-mediated structural differences in GR-DBD. (A) Domain structure of GR. τ₁, tau1. (B) Overlay of chains A and B from GR-DBD:Pal complex shows packed and flipped conformations. (C) Overlay of chain B from GR-DBD complexed with 4-bp spacer (15) (magenta) and 3-bp spacer GBS (green). (D) Composite omit maps of GR-DBD complexed with different GBSs (GilZ, FKBP5, Sgk, and Pal) under the same conditions. Lever arm peptide is shown with 2Fo-Fc (black mesh) and composite omit map (red mesh) overlaid.

Fig. 3

Activities and structure of GRγ. (A) GRγ amino acid sequence, showing Arg insertion in the lever arm. (B) U2OS cells were cotransfected with GRα or GRγ, together with GBS reporters (left) or with an osteocalcin reporter (right). Fold induction (left) and luciferase activity relative to untreated cells (right) ± SEM are shown (n = 3). (C) Regulation of endogenous target genes in U2OS cells stably expressing GRα or GRγ, measured by quantitative real-time fluorescence polymerase chain reaction. (D) Chromatin immunoprecipitation of GR at GBSs of isoform-specific target genes; GR recruitment upon dex treatment ± SEM is shown (n = 3). (E) Overlay of structures for GRα:FKBP5 and GRγ:FKBP5 complexes.

Fig. 4

Receptor activity is modulated by lever arm residues. (A) H472 is critical for tuning activity. Effects of mutating lever arm residues were assayed using GBS reporters; activities are plotted as percentage of wild type ± SEM (n ≥ 3). (B) H472 resides in the DBD pocket formed by the carbonyl adjacent to V468, Y497, and L501. (C) Human DBD sequence alignments reveal variation at V468, Y497, and L501.