1. Volume 29, Issue 5, Pages 611-624 (March 2008)
Interaction of the Glucocorticoid Receptor with the Chromatin Landscape 
Sam John, Peter J. Sabo, Thomas A. Johnson, Myong-Hee Sung, Simon C. Biddie, Stafford L. Lightman, Ty C. Voss, Sean R. Davis, Paul S. Meltzer, John A. Stamatoyannopoulos, Gordon L. Hager 
Molecular Cell 
Volume 29, Issue 5, Pages (March 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 Functional Effect of dn-Brg1 Expression on Regulation by GR
(A) dnBrg1 effect on GR activated genes. The effect of dnBrg1 expression on inducibility of GR activated genes was examined by microarray analysis. Of the 667 Dex-induced genes in 5555 and 3617 cells, 268 (40%) had a reduced induction level, ranging from 20% to 80% of the WT induction level, in the presence of dnBrg1. The fold change in the Dex induction level due to dnBrg1 is plotted against the WT fold induction by Dex for these 268 genes. The curve represents a lowess regression fit for the data.
(B) Summary of the dnBrg1 effect on induced genes. As shown, 268 of 667 Dex-induced genes show reduced induction after activation of dnBrg1, while 399 are not significantly affected.
(C) Summary of the dnBrg1 effect on repressed genes. For 487 loci shown to be downregulated by GR, only 55 have their functional repression significantly reversed by activation of dnBrg1.
(D) The inhibition of GR activation by dnBrg1 expression was confirmed for six genes by qPCR analysis of RNA transcripts in 3617 and 5555 cells (primer sequences are given in the Supplemental Data). Removal of tet (−tet) induces dnBrg1 in the 5555 cell, and the tTA regulator in 3617 cells. Comparison of RNA levels in the two cell lines in the absence of tet with added ligand (+dex) indicates the effect of dnBrg1 on RNA induction for the target gene. Error bars represent standard deviation.
(E) The effect of dnBrg1 expression on GR negatively regulated genes was examined by qPCR analysis of RNA transcripts in 3617 and 5555 cells (primer sequences are given in the Supplemental Data). Comparison of RNA levels in the two cell lines in the absence of tet with added ligand (+dex) indicates the effect of dnBrg1 on repression of the target gene.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
GR Binding and Nucleoprotein Transitions within the Mt1-Mt2 Chromatin Domain
(A) Site-specific binding of the GR was determined by ChIP-chip analysis with high-density tiling arrays (Experimental Procedures) for chromatin isolated from 3134 cells either treated (+dex; green) or untreated (−dex; gray) with ligand. P values calculated by the sliding window method are presented in Figure S12 for the +dex GR loading data (as negative log values). GR regulation of the Mt2 gene is presented in Figure S11.
(B) Hormone-dependent and -independent DNase I hypersensitive chromatin transitions were determined for the Mt1-Mt2 domain by the method of Dorschner et al. (2004) (Experimental Procedures). qPCR amplification of the individual amplicons was carried out in triplicate for each curve, for a total of six measurements per condition. y axis values represent the recovery of individual amplicons relative to total untreated DNA (see the Supplemental Data). Data are shown for chromatin from hormone-free cells (-♦- and -⋄-) and chromatin from hormone-treated cells (-•- and -○-). The Mt2 and Mt1 data sets were obtained from separate experiments, resulting in a discontinuity in the curves at coordinate Error bars represent standard deviation.
(C) The dnBrg1 effect on hypersensitive sites was determined for the Mt1-Mt2 domain by comparing DNase I-treated chromatin from dnBrg1-free 5555 cells (-♦-) with DNase I-treated chromatin from dnBrg1-induced 5555 cells (-•- and -○-). Cells in both conditions were hormone induced.
(D) The DNase I hypersensitive profile was also determined in 3617 cells, with the tTA regulator either uninduced (+tet; -♦- and -▴-) or induced (−tet; -•- and -○-). No effect of the tTA regulator was observed for hypersensitive sites in any of the gene regions studied.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
GR Binding and Nucleoprotein Transitions within the Glul Chromatin Domain
(A) Site-specific GR binding was determined by ChIP-chip analysis. GR loading ratios are shown for 3134 chromatin from cells either treated (+dex) or untreated (−dex) with hormone. P values are presented for the +dex GR loading data (upper panel).
(B) DNase I hypersensitive chromatin transitions were determined for the Glul domain for chromatin from hormone-free cells (-♦- and -⋄-) and chromatin from hormone-treated cells (-•- and -○-). Error bars represent standard deviation.
(C) The dnBrg1 effect on DHS sites was determined for the Glul domain by comparing chromatin from dnBrg1-free 5555 cells (-♦-) with chromatin from dnBrg1-induced 5555 cells (-•-). Multiple y axis designations relate to the data set indicated in the legend immediately to the right of the axis.
(D) The DNase I DHS profile was determined in 3617 cells, with the tTA regulator either uninduced (+tet; -♦- and -▴-), or induced (−tet; -•- and -○-).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
GR Binding and Nucleoprotein Transitions within the Lcn2 Chromatin Domain
(A) GR loading ratios are shown for 3134 chromatin from cells either treated (+dex) or untreated (−dex) with hormone. P values are for the +dex GR loading data (upper panel).
(B) DNase I hypersensitive chromatin transitions were determined for the Lcn2 domain for chromatin from hormone-free cells (-♦-) and chromatin from hormone-treated cells (-•- and -○-). Error bars represent standard deviation.
(C) The dnBrg1 effect on DHS sites was determined for the Lcn2 domain by comparing chromatin from dnBrg1-free 5555 cells (-♦-) with chromatin from dnBrg1-induced 5555 cells (-•-).
(D) The DNase I DHS profile was determined in 3617 cells, with the tTA regulator either uninduced (+tet; -♦- and -▴-), or induced (- tet; -•- and -○-).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
GR Binding and Nucleoprotein Transitions within the Cxcl5 Chromatin Domain
(A) GR loading ratios are shown for 3134 chromatin from cells either treated (+dex) or untreated (−dex) with hormone. P values are shown for the +dex GR loading data.
(B) DNase I hypersensitive chromatin transitions were determined for the Cxcl5 domain for chromatin from hormone-free cells (-♦- and -⋄-) and chromatin from hormone-treated cells (-•- and -○-). Error bars represent standard deviation.
(C) The dnBrg1 effect on DHS sites was determined for the Cxcl5 domain by comparing chromatin from dnBrg1-free 5555 cells (-♦-) with chromatin from dnBrg1-induced 5555 cells (-•-).
(D) The DNase I DHS profile was determined in 3617 cells, with the tTA regulator either uninduced (+tet; -♦- and -▴-), or induced (−tet; -•- and -○-).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Cell Type Specificity of Gene Activation, GR Binding, and Nucleoprotein Transitions at the Lcn2 Locus
(A) Response of the Lcn2 locus to dexamethasone stimulation was measured in 3134 cells (-□-) and AtT20 cells (-♦-) by qPCR analysis. Error bars represent standard deviation.
(B) Dex-dependent GR loading ratios were determined at the proximal promoter DHS site by ChIP analysis, both in AtT20 cells and 3134 cells.
(C) The DNase I hypersensitive chromatin profile for the Lcn2 locus in 3134 cells is schematically summarized from the data in Figure 4. The dashed line represents the hormone-induced profile.
(D) The DNase I DHS profile was determined for the Lcn2 locus in AtT20 cells, either induced (-♦-), or uninduced (-▪-) with dexamethasone. Each data set represents the average of three biological duplicates, with the qPCR analysis carried out in triplicate for each set of samples. As shown, the dex-dependent DHS site present in 3134 cells is absent in AtT20 cells. Error bars represent standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

8. Figure 7 Dynamic Interactions of the GR with Chromatin
(A) Localization and displacement of H2A.Z at GR response elements. The relative abundance of the H2A.Z histone variant was determined by ChIP analysis at GR interaction sites. These sites include the following: Ampd3 (chr7: coord. 110,564,878 to 5002); Nudt9 (chr5: coord. 104,296,814 to 944); Cxcl5 GRE#1 (chr5: coord. 91,833,422 to 560); and Sgk GRE#1 (chr10: coord. 21,683,042 to 145). Adjacent regions with no evidence of remodeling (non-DHS) were also examined in the Nudt9 locus (see Figure S10; chr5: coord. 104,298,940 to 9065) and the Ampd3 locus (chr7: coord. 110,562,144 to 265). The inset panel shows parallel ChIP analysis for H3 at the same regions. The relative abundance of histone H3 remains unchanged at GR interaction sites and adjacent regions. Error bars represent standard deviation.
(B) Change in histone H2A.Z abundance after GR activation. The equilibrium level of H2A.Z was determined by ChIP analysis at three GR interaction sites during hormone treatment. Solid bars present examples for GR-inducible hypersensitive sites (Sgk, Ampd3, Mt2, and Lcn2), and gray bars show data for a constitutive DHS GR binding site (Cxcl5).
(C) GR interactions with the genome can be classified in four groups. Two types of interaction represent local transitions that are initiated by the receptor, either through the recruitment of the Swi/Snf complex (1) or other uncharacterized remodeling systems (2). The second class of interactions is characterized by pre-existing local perturbations in the chromatin structure, induced either by the Swi/Snf complex (3) or by other remodeling factors (4). These constitutive transitions are likely to be induced by alternate transcription factors (x and z) operating in conjunction with the remodeling systems. As indicated by the octamer details (1–4), nucleosomes at all of the GR interaction sites are enriched in H2A.Z, and this histone variant is subject to exchange at these sites. In addition, there are GR regulatory elements throughout the genome (5) that are unavailable for binding by the receptor due either to a restrictive local chromatin environment, an altered DNA state (such as methylation), or both. Color coding for the transition regions matches that used to highlight the transition domains in Figures 3–6 and Figures S1–S5, S8, and S9.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions