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Science 324 (5925): 407-410

Copyright © 2009 by the American Association for the Advancement of Science

DNA Binding Site Sequence Directs Glucocorticoid Receptor Structure and Activity

Sebastiaan H. Meijsing1*, Miles A. Pufall1*, Alex Y. So1,2, Darren L. Bates3, Lin Chen3, and Keith R. Yamamoto1,2{dagger}

1 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA.
2 Chemistry and Chemical Biology Program, University of California, San Francisco, CA 94107, USA.
3 Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA.

Figure 1 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]. KD, 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). [View Larger Version of this Image (47K GIF file)]

Figure 2 Fig. 2.. DNA sequence-mediated structural differences in GR-DBD. (A) Domain structure of GR. {tau}1, 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. [View Larger Version of this Image (56K GIF file)]

Figure 3 Fig. 3.. Activities and structure of GR{gamma}. (A) GR{gamma} amino acid sequence, showing Arg insertion in the lever arm. (B) U2OS cells were cotransfected with GR{alpha} or GR{gamma}, 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 GR{alpha} or GR{gamma}, 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 GR{alpha}:FKBP5 and GR{gamma}:FKBP5 complexes. [View Larger Version of this Image (35K GIF file)]

Figure 4 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. [View Larger Version of this Image (50K GIF file)]

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