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. 2010 Mar;24(3):511-25.
doi: 10.1210/me.2009-0443. Epub 2010 Jan 21.

Glucocorticoid receptor activates poised FKBP51 locus through long-distance interactions

Ville Paakinaho  1 , Harri Makkonen, Tiina Jääskeläinen, Jorma J Palvimo
Affiliations

Glucocorticoid receptor activates poised FKBP51 locus through long-distance interactions

Ville Paakinaho et al. Mol Endocrinol. 2010 Mar.

Abstract

Recent studies have identified FKBP51 (FK506-binding protein 51) as a sensitive biomarker of corticosteroid responsiveness in vivo. In this work, we have elucidated the molecular mechanisms underlying the induction of FKBP51 by the glucocorticoid receptor (GR) in human A549 lung cancer cells showing robust accumulation of FKBP51 mRNA in response to dexamethasone exposure. Our quantitative chromatin immunoprecipitation scans and enhancer activity analyses indicate that activation of the FKBP51 locus by glucocorticoids in vivo is triggered by the loading of GR to enhancers at about 34 kb 5' and about 87 kb 3' of the transcription start site. Interestingly, the region encompassing these enhancers is bordered by CCCTC-binding factor- and cohesin-binding sites. Dexamethasone treatment also decreased the histone density at several regions of the gene, which was paralleled with the occupancy of SWI/SNF chromatin remodeling complexes within the locus. Moreover, silencing of BRM subunit of the SWI/SNF complex blunted the glucocorticoid induction of the locus. The proximal promoter region along with the major intronic enhancer at approximately 87 kb, at which the GR binding peaked, had elevated levels of histone 3 acetylation and H3K4 trimethylation, whereas H3K36 trimethylation more generally marked the gene body and reflected the occupancy of RNA polymerase II. The occurrence of these active chromatin marks within the FKBP51 locus before glucocorticoid exposure suggests that it is poised for transcription in A549 cells. Taken together, these results indicate that the holo-GR is capable of activating transcription and evoking changes in chromatin structure through distant-acting enhancers.

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Figures

Fig. 1.
Fig. 1.
Rapid and robust activation of FKBP51 expression by dex in A549 cells. A, Cells were treated with indicated concentrations of synthetic glucocorticoid (dex) for 12 h, and the amounts of FKBP51 variant 1 (var. 1) and variant 2 (var. 2), S100P, and GILZ mRNAs were analyzed by RT-QPCR and normalized as described in Materials and Methods. B, A549 cells were treated with 100 nm dex for indicated times, and the analysis of mRNAs was performed as above. Columns represent the mean ± sd of three independent experiments.
Fig. 2.
Fig. 2.
Function of the putative FKBP51 GRE-containing regions as enhancers. A, Schematic locations of the GREs and regions analyzed in reporter and ChIP assays. Bars depict the positions of exons, and arrows indicate the TSSs of the different FKBP51 transcript variants, with the bolded arrow indicating the major transcript start site. Roman numerals depict the in silico-identified GREs, and Arabic numerals indicate the regions amplified for enhancer analyses and ChIP assays. B, Sequences of the potential GREs in FKBP51 gene. Top, Position weight matrix used for identification of putative GREs. The frequency of nucleotides at each of the 15 positions in the GRE in the matrix is shown. Lower, List of the in silico-identified GREs, their localization in the FKBP51 gene, and their sequences. C and D, Transcriptional activity of the GRE-containing 300-bp FKBP51 fragments was assessed by reporter gene assays. A549 (C) and COS-1 (D) cells were transiently transfected with TATA-LUC constructs driven by indicated regions of FKBP51 or empty pTATA-LUC (TATA) as described in Materials and Methods. For COS-1 (D) analyses, pSG5-hGR (0.2 μg/well) was cotransfected with the reporter constructs. Cotransfection of pCMVβ and β-galactosidase activity was used for normalization of transfection efficiency. The cells were treated with vehicle (ethanol, EtOH) or 100 nm dex for 16 h before the cells were harvested for reporter analyses. Results are shown as relative LUC activities and fold inductions of glucocorticoid-treated samples in relation to the activity of ethanol-treated samples are shown above the columns. Columns represent the mean ± sd of three independent experiments.
Fig. 3.
Fig. 3.
Temporo-spatial loading of GR to the FKBP51 locus in response to glucocorticoid exposure in A549 cells. A, Cells were treated with 100 nm dex for indicated times, and ChIPs with anti-GR antibody followed by real-time PCR were carried out as described in Materials and Methods to investigate the occupancy of GR within 17 different regions spanning approximately 160 kb of chromosome 6 that covers the FKBP51 locus plus its surroundings (cf. Fig. 2A). A region in the middle of DSCAM in chromosome 22 served as a nontarget control. ChIP assays with normal IgG monitored nonspecific binding. Results are shown as percentages of the input samples. Columns represent the mean ± sd of three experiments. B, Binding of GR to the FKBP51 regions −3, 8, 9, and 10 plotted against time after addition of the hormone. Ctrl., Control.
Fig. 4.
Fig. 4.
Effect of RU486 on the FKBP51 expression and the GR loading to the locus chromatin. A, Accumulation of FKBP51 mRNA in response to exposure of A549 cells to dex (100 nm) or RU486 (1000 nm) alone or in combination relative to the mRNA level with vehicle (EtOH) for 12 h. RNAs were analyzed by RT-QPCR analysis as described in Fig. 1. B, Loading of GR to the FKBP51 locus in response to agonist or antagonist exposure alone or in combination for 2 h in A549 cells. ChIP scans were performed using anti-GR antibody followed by quantitative PCR analysis. Results are shown as percentage of the input samples and represent the mean ± sd of three experiments.
Fig. 5.
Fig. 5.
Occupancy of RNA PolII and occurrence of H3 modifications within the FKBP51 locus in A549 cells in the presence and absence of glucocorticoid. Cells were treated with vehicle (EtOH) or 100 nm dex for 2 h, and ChIP assays were performed using antibodies against GR (A), PolII (B), H3 (C), H3K9,14ac (D), H3K4me3 (E), or H3K36me3 (F). ChIP samples were used as templates in qPCR. Results are shown as fold over the normal rabbit IgG-precipitated samples and represent the mean ± sd of three experiments.
Fig. 6.
Fig. 6.
Evidence for the involvement of SWI/SNF type chromatin-remodeling complexes in the regulation of the FKBP51 locus. The effect of glucocorticoid on the occupancy of BRM- or BRG1-containing complexes within the FKBP51 locus was analyzed in A549 cells treated with vehicle or dex as in Fig. 4 by ChIP assays using antibodies against BRM (A) or BRG1 (B) followed by QPCR analysis. Results are shown as fold over the normal rabbit IgG-precipitated samples and represent the mean ± sd of three experiments. C, Depletion of BRM from A549 cells transfected with BRM-specific siRNA as assessed by immunoblotting. The level of glyceraldehyde-3-phosphate dehydrogenase monitored the loading of protein in the samples. D, The effect of BRM siRNA on the glucocorticoid induction of FKBP51 mRNA as analyzed by RT-QPCR analysis. ***, P < 0.001 compared with the cells transfected with unspecific target siRNA (siSCR) and treated with dex. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.
Fig. 7.
Fig. 7.
Differential activation of C6orf81 in A549 and VCaP cells by glucocorticoid and androgen, respectively. A, Schematic map of the FKBP51 and its neighboring genes C6orf81 and TULP1 in chromosome 6. Angled arrows indicate the TSSs of different genes, bolded regions show the schematic locations of the genes, and dashed line depicts extra 5′-region of the FKBP51 transcript variant 2. A549 cells and VCaP cells were treated with increasing concentrations of dex (B) and R1881 (D), respectively, for 12 h or with 100 nm dex (C) or 10 nm R1881 (E) for indicated times, and mRNAs were analyzed by RT-QPCR analysis as described in Fig. 1. Columns represent the mean ± sd of three independent experiments. F, Loading of AR to the FKBP51 locus in VCaP cells. Cells were treated with vehicle (EtOH) or 10 nm R1881 for 2 h. ChIP assays were performed using anti-AR antibody, and the precipitated DNAs were used as templates in QPCR. Chr., Chromosome.
Fig. 8.
Fig. 8.
Occupancy of the CTCF within the FKBP51 locus and its surroundings in A549 and VCaP cells. A, Schematic map of the FKBP51 and its neighboring genes C6orf81 and TULP1 in chromosome 6 with the circles showing the locations of the major GR-binding sites (identified in the preceding experiments). The locations of putative CTCF-binding regions Ins1-Ins9 (depicted as vertical lines) screened by ChIP assays are based on the information in the CTCF-binding site database (41 42 43 ). The other screened regions served as controls. A549 cells and VCaP cells treated with and without cognate agonist for 2 h were analyzed by ChIP with anti-CTCF antibody (B and D) or with anti-RAD21 antibody (C) followed by qPCR analysis. Results are shown as fold over the normal rabbit IgG-precipitated samples and represent the mean ± sd of three experiments. var., Variant; Ins, insulator.
Fig. 9.
Fig. 9.
A hypothetical model of how CTCF/cohesin complexes that border the region encompassing the major GR-binding sites in the FKBP51 locus might contribute to the long-range interactions and loop formation before (on the left) and after glucocorticoid exposure (on the right). CoA, Coactivator.
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