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• W2008654807 abstract "The glucocorticoid (GC) receptor (GR), when liganded to GC, activates transcription through direct binding to simple (+)GRE DNA binding sequences (DBS). GC-induced direct repression via GR binding to complex “negative” GREs (nGREs) has been reported. However, GR-mediated transrepression was generally ascribed to indirect “tethered” interaction with other DNA-bound factors. We report that GC-induces direct transrepression via the binding of GR to simple DBS (IR nGREs) unrelated to (+)GRE. These DBS act on agonist-liganded GR, promoting the assembly of cis-acting GR-SMRT/NCoR repressing complexes. IR nGREs are present in over 1000 mouse/human ortholog genes, which are repressed by GC in vivo. Thus variations in the levels of a single ligand can coordinately turn genes on or off depending in their response element DBS, allowing an additional level of regulation in GR signaling. This mechanism suits GR signaling remarkably well, given that adrenal secretion of GC fluctuates in a circadian and stress-related fashion. The glucocorticoid (GC) receptor (GR), when liganded to GC, activates transcription through direct binding to simple (+)GRE DNA binding sequences (DBS). GC-induced direct repression via GR binding to complex “negative” GREs (nGREs) has been reported. However, GR-mediated transrepression was generally ascribed to indirect “tethered” interaction with other DNA-bound factors. We report that GC-induces direct transrepression via the binding of GR to simple DBS (IR nGREs) unrelated to (+)GRE. These DBS act on agonist-liganded GR, promoting the assembly of cis-acting GR-SMRT/NCoR repressing complexes. IR nGREs are present in over 1000 mouse/human ortholog genes, which are repressed by GC in vivo. Thus variations in the levels of a single ligand can coordinately turn genes on or off depending in their response element DBS, allowing an additional level of regulation in GR signaling. This mechanism suits GR signaling remarkably well, given that adrenal secretion of GC fluctuates in a circadian and stress-related fashion. Distinct response elements mediate direct transrepression by glucocorticoid receptor This direct repression, distinct from “tethered” transrepression, controls many genes Direct repression is important for homeostasis, circadian and stress functions Our discovery paves the way to improved anti-inflammatory glucocorticoids Glucocorticoids (GCs) are peripheral effectors of circadian and stress-related homeostatic functions fundamental for survival throughout vertebrate life span (Chrousos, 2009Chrousos G.P. Stress and disorders of the stress system.Nat. Rev. Endocrinol. 2009; 5: 374-381Crossref PubMed Scopus (1523) Google Scholar, Nader et al., 2010Nader N. Chrousos G.P. Kino T. Interactions of the circadian CLOCK system and the HPA axis.Trends Endocrinol. Metab. 2010; 21: 277-286Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). They are widely used to combat inflammatory and allergic disorders and their therapeutic effects have been mainly ascribed to their capacity to suppress the production of proinflammatory cytokines (Rhen and Cidlowski, 2005Rhen T. Cidlowski J.A. Antiinflammatory action of glucocorticoids–new mechanisms for old drugs.N. Engl. J. Med. 2005; 353: 1711-1723Crossref PubMed Scopus (1970) Google Scholar). GCs act by binding to the GC receptor (GR), a member of the nuclear receptor (NR) superfamily. In absence of GCs, GR is maintained in the cytoplasm by molecular chaperones. Binding of GCs generates a conformational switch in the GR ligand binding domain (LBD) which affects GR interactions with chaperones and facilitates nuclear translocation (Ricketson et al., 2007Ricketson D. Hostick U. Fang L. Yamamoto K.R. Darimont B.D. A conformational switch in the ligand-binding domain regulates the dependence of the glucocorticoid receptor on Hsp90.J. Mol. Biol. 2007; 368: 729-741Crossref PubMed Scopus (48) Google Scholar). Once in the nucleus, GR binds to GC response elements (GREs) and regulates transcription of target genes. “Simple” GREs belong to a family of imperfect palindromes consisting of two inverted hexameric half-site motifs separated by three base pairs (bp) (Meijsing et al., 2009Meijsing S.H. Puffal M.A. So A.Y. Bates D.L. Chen L. Yamamoto K.R. DNA binding site sequence directs glucocorticoid receptor structure and activity.Science. 2009; 324: 407-410Crossref PubMed Scopus (489) Google Scholar). Such “simple” (+)GRE confer transcriptional transactivation to agonist-liganded GR through association with coactivators (e.g., SRC1, TIF2/SRC2 and SRC3) (Lonard and O'Malley, 2007Lonard D.M. O'Malley B.W. Nuclear receptor coregulators: judges, juries and executioners of cellular regulation.Mol. Cell. 2007; 27: 691-700Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). “Composite” GREs consist of DNA binding sites (DBS) for GR which, in association with binding sites for other factors, can act synergistically to mediate transactivation or transrepression. In a few of cases, binding of GR to promoter regions has been implicated in GC-induced transrepression, but no consensus sequence for “repressing” negative GREs (nGREs) has emerged (Dostert and Heinzel, 2004Dostert A. Heinzel T. Negative glucocorticoid receptor response elements and their role in glucocorticoid action.Curr. Pharm. Des. 2004; 10: 2807-2816Crossref PubMed Scopus (171) Google Scholar). Remarkably, “tethering” GREs do not contain DBS for GR per se, but instead contain binding sites for other DNA-bound regulators, such as NFκB and AP1, that recruit GR (Karin, 1998Karin M. New Twists in Gene Regulation by Glucocorticoid Receptor: Is DNA Binding Dispensable?.Cell. 1998; 93: 487-490Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, Kassel and Herrlich, 2007Kassel O. Herrlich P. Crosstalk between the glucocorticoid receptor and other transcription factors: Molecular aspects.Mol. Cell. Endocrinol. 2007; 275: 13-29Crossref PubMed Scopus (212) Google Scholar). Thus, tethering GREs confer “indirect” transrepression to agonist liganded GR. Atopic dermatitis (AD) is an inflammatory skin disease that exhibits a high prevalence (Bieber, 2008Bieber T. Atopic dermatitis.N. Engl. J. Med. 2008; 358: 1483-1494Crossref PubMed Scopus (1419) Google Scholar). We recently developed mouse models which closely mimic human AD (Li et al., 2005Li M. Messadeq N. Teletin M. Pasquali J.L. Metzger D. Chambon P. Retinoid X receptor ablation in adult mouse keratinocytes generates an atopic dermatitis triggered by thymic stromal lymphopoietin.Proc. Natl. Acad. Sci. USA. 2005; 102: 14795-14800Crossref PubMed Scopus (165) Google Scholar, Li et al., 2006Li M. Hener P. Zhang Z. Kato S. Metzger D. Chambon P. Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.Proc. Natl. Acad. Sci. USA. 2006; 103: 11736-11741Crossref PubMed Scopus (310) Google Scholar), and revealed that induction of the Thymic Stromal Lymphopoietin (TSLP) cytokine in epidermal keratinocytes is necessary and sufficient to trigger a human AD-like syndrome. As topical GCs are important tools for AD treatment, we wondered whether their effect could result from TSLP repression. We report that GCs transcriptionally repress TSLP expression in AD mouse models, and demonstrate that this repression is mediated through direct binding of GR to a “simple” nGRE, which belongs to a novel family of evolutionary-conserved cis-acting negative response elements (IR nGREs) found in numerous GC-repressed genes. The GC agonist fluocinolone acetonide (FA) was applied to ears of mice concomitantly treated with the “low-calcemic” Vitamin D3 (VitD3) analog MC903 (Calcipotriol; hereafter called MC) to trigger TSLP expression (Li et al., 2006Li M. Hener P. Zhang Z. Kato S. Metzger D. Chambon P. Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.Proc. Natl. Acad. Sci. USA. 2006; 103: 11736-11741Crossref PubMed Scopus (310) Google Scholar). In wild-type (WT) mice, FA application inhibited basal TSLP RNA level by ∼50%, which interestingly could be relieved by coapplication of the GC antagonist RU486 (mifepristone, hereafter named RU), while MC-induced increase of TSLP RNA, which was fully blocked by FA, was also restored by RU cotreatment (Figure 1A ). Similarly, the retinoic acid (RA)-induced increase of TSLP transcripts (Li et al., 2006Li M. Hener P. Zhang Z. Kato S. Metzger D. Chambon P. Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.Proc. Natl. Acad. Sci. USA. 2006; 103: 11736-11741Crossref PubMed Scopus (310) Google Scholar) was blocked by FA and restored by RU (Figure S1A available online). As expected, the expression of the GC-inducible GPX3 (glutathione peroxidase 3) gene which harbours a (+)GRE (Tuckermann et al., 1999Tuckermann J.P. Reichardt H.M. Arribas R. Richter K.H. Schutz G. Angel P. The DNA binding-independent function of the glucocorticoid receptor mediates repression of AP-1-dependent genes in skin.J. Cell Biol. 1999; 147: 1365-1370Crossref PubMed Scopus (162) Google Scholar) was enhanced by FA (Figure 1A) and inhibited by RU cotreatment, while FA or RU had no effect on MC-dependent expression of the CYP24A1 gene (a VitD3 target) (Figure 1A). Inhibition of TSLP expression by FA was not dependent on its induction by MC or RA, as a 3-day FA application to ears of RXRαβep−/− or VDR/RARαγep−/− mice (selectively lacking in epidermal keratinocytes both RXRα and β, or VDR and both RARα and RARγ, respectively) which express high levels of TSLP in epidermal keratinocytes in the absence of MC or RA treatment (Li et al., 2005Li M. Messadeq N. Teletin M. Pasquali J.L. Metzger D. Chambon P. Retinoid X receptor ablation in adult mouse keratinocytes generates an atopic dermatitis triggered by thymic stromal lymphopoietin.Proc. Natl. Acad. Sci. USA. 2005; 102: 14795-14800Crossref PubMed Scopus (165) Google Scholar and unpublished data) reduced TSLP RNA by ∼70% (Figure 1B, left panel, and data not shown).Figure S1Glucocorticoid-Induced Recruitment of GR and Corepressors to the TSLP IR1 nGRE Prevent RA and Active VD3-Induced Recruitment of RAR, VDR, Coactivators, and pol II to the TSLP DR2b RARE, DR3d VDRE and Proximal Promoter Regions in Epidermis and Intestinal Epithelium of Wild-Type Mouse and in Human A549 Cells, Related to Figure 1Show full caption(A) Q-RT-PCR of TSLP RNA from skin of WT mice topically-treated for 6 hr with the indicated compounds (mean ± SEM). FA, fluocinolone acetonide; RA, all-trans retinoic acid; VD3, active Vitamin D3; RU, glucocorticoid antagonist RU486. All values were normalized with respect to those obtained for HPRT.(B) Q-RT-PCR of TSLP, GPX3 and Cyp24A1 RNA in epithelial cells of WT mouse ileum (mean ± SEM). Mice were intraperitoneally injected with vehicle, FA, RU 486 (RU) and/or VD3, as indicated. All values were normalized with respect to those obtained for HPRT.(C) Q-RT-PCR of TSLP, GILZ and Cyp24A1 RNA in A549 cells, treated for 6 hr as indicated. Values were normalized with respect to those obtained for GAPDH (mean ± SEM).(D) Mouse TSLP promoter region showing the position of DR2b RARE with respect to other sequence elements. Related to Figure 1F.(E) FA-treated mouse epidermis was processed for ChIP using antibodies and primers 1 to 7 to amplify amplicons 1 to 7, as indicated. Amplicon 5 encompasses the nGRE.(F) ChIP assays for (+) GRE (located 1.65kb from the transcription start site) of GPX3 gene using aliquots of samples used in Figure 1G.(G) ChIP assays as in Figure 1G with indicated HDAC antibodies.(H) Glucocorticoid-induced repression of the TSLP promoter is initiated in vivo through binding of the GR to the nGRE, along with corepressors. ChIP assays showing the FA-induced binding of GR and corepressors to the TSLP nGRE region and its reversion by RU 486 (RU). Dorsal skin from WT mice was topically treated with vehicle, RA, FA and RU for 6 hr prior to epidermis isolation. ChIP-immunoprecipitated DNA from epidermal whole cell extracts was PCR-amplified using primers flanking the TSLP nGRE, TSLP RARE (DR2b) and TSLP PP (Proximal Promoter) regions (see panel D). IP antibody indicates the antibodies used for immunoprecipitation. Control antibody corresponds to rabbit IgG. 10% input indicates the signal obtained after PCR amplification of a given DNA region contained in 10% of the chromatin used for immunoprecipitation of each sample with a given antibody.(I) ChIP assays of intestine epithelium of WT mouse, intraperitoneally-injected as indicated with vehicle, VD3, FA and RU, for 6 hr before sacrifice. Epithelial cells were isolated and processed through ChIP assays using indicated antibodies. Immunoprecipitated DNA was PCR- amplified using primers flanking the TSLP nGRE, DR3d and PP regions, as well as the GPX3 (+) GRE region.(J) ChIP assays of A549 whole cell extracts showing FA-induced binding of GR and corepressors to the human TSLP nGRE (hTSLP nGRE) region and its reversion by RU.(K) ChIP assays of A549 whole cell extracts showing FA-induced binding of GR and coactivators to the hGILZ (+) GRE region, and its reversion by RU. Aliquots of the immunoprecipitated DNA described in panel (G) were PCR-amplified using primers flanking the (+) GRE region of the human GILZ gene.(L) Schematic representation of the Alu I sites flanking the PP, nGRE and DR3d VDRE regions of the TSLP promoter.(M) Chromosome conformation capture (3C) assay using MC 903-treated WT mouse dorsal epidermis shows that topical treatment with FA prevents the interaction of the Alu I region A that contains the VDRE (DR3d) with the Alu I region E that contains the proximal promoter (PP) region (see panel I). Dorsal skin was topically treated with vehicle, MC, FA and/or RU for 6 hr, prior to isolation of epidermis. Non-cross linked and cross-linked chromatin were digested with Alu I enzyme, diluted and incubated with T4 DNA ligase. Ligated DNA was PCR-amplified using primers flanking the junction between either VDRE (Alu I fragment A) or nGRE (Alu I fragment C) regions with the PP (Alu I fragment E) region. PCR product was separated in a 2% agarose gel and Southern-hybridized to a [32P]-5′ labeled probe corresponding to 20 nucleotides upstream and 20 nucleotides downstream from the junction between the Alu I regions A and E and C and E. Upper and lower panels reveal interaction between region C containing the nGRE and region E containing the PP region, and between region A containing the VDRE and region E. A BAC containing a 50kb genomic DNA sequence encompassing the TSLP coding and flanking sequences was processed along as a positive control for efficiency of ligation (TSLP BAC).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Q-RT-PCR of TSLP RNA from skin of WT mice topically-treated for 6 hr with the indicated compounds (mean ± SEM). FA, fluocinolone acetonide; RA, all-trans retinoic acid; VD3, active Vitamin D3; RU, glucocorticoid antagonist RU486. All values were normalized with respect to those obtained for HPRT. (B) Q-RT-PCR of TSLP, GPX3 and Cyp24A1 RNA in epithelial cells of WT mouse ileum (mean ± SEM). Mice were intraperitoneally injected with vehicle, FA, RU 486 (RU) and/or VD3, as indicated. All values were normalized with respect to those obtained for HPRT. (C) Q-RT-PCR of TSLP, GILZ and Cyp24A1 RNA in A549 cells, treated for 6 hr as indicated. Values were normalized with respect to those obtained for GAPDH (mean ± SEM). (D) Mouse TSLP promoter region showing the position of DR2b RARE with respect to other sequence elements. Related to Figure 1F. (E) FA-treated mouse epidermis was processed for ChIP using antibodies and primers 1 to 7 to amplify amplicons 1 to 7, as indicated. Amplicon 5 encompasses the nGRE. (F) ChIP assays for (+) GRE (located 1.65kb from the transcription start site) of GPX3 gene using aliquots of samples used in Figure 1G. (G) ChIP assays as in Figure 1G with indicated HDAC antibodies. (H) Glucocorticoid-induced repression of the TSLP promoter is initiated in vivo through binding of the GR to the nGRE, along with corepressors. ChIP assays showing the FA-induced binding of GR and corepressors to the TSLP nGRE region and its reversion by RU 486 (RU). Dorsal skin from WT mice was topically treated with vehicle, RA, FA and RU for 6 hr prior to epidermis isolation. ChIP-immunoprecipitated DNA from epidermal whole cell extracts was PCR-amplified using primers flanking the TSLP nGRE, TSLP RARE (DR2b) and TSLP PP (Proximal Promoter) regions (see panel D). IP antibody indicates the antibodies used for immunoprecipitation. Control antibody corresponds to rabbit IgG. 10% input indicates the signal obtained after PCR amplification of a given DNA region contained in 10% of the chromatin used for immunoprecipitation of each sample with a given antibody. (I) ChIP assays of intestine epithelium of WT mouse, intraperitoneally-injected as indicated with vehicle, VD3, FA and RU, for 6 hr before sacrifice. Epithelial cells were isolated and processed through ChIP assays using indicated antibodies. Immunoprecipitated DNA was PCR- amplified using primers flanking the TSLP nGRE, DR3d and PP regions, as well as the GPX3 (+) GRE region. (J) ChIP assays of A549 whole cell extracts showing FA-induced binding of GR and corepressors to the human TSLP nGRE (hTSLP nGRE) region and its reversion by RU. (K) ChIP assays of A549 whole cell extracts showing FA-induced binding of GR and coactivators to the hGILZ (+) GRE region, and its reversion by RU. Aliquots of the immunoprecipitated DNA described in panel (G) were PCR-amplified using primers flanking the (+) GRE region of the human GILZ gene. (L) Schematic representation of the Alu I sites flanking the PP, nGRE and DR3d VDRE regions of the TSLP promoter. (M) Chromosome conformation capture (3C) assay using MC 903-treated WT mouse dorsal epidermis shows that topical treatment with FA prevents the interaction of the Alu I region A that contains the VDRE (DR3d) with the Alu I region E that contains the proximal promoter (PP) region (see panel I). Dorsal skin was topically treated with vehicle, MC, FA and/or RU for 6 hr, prior to isolation of epidermis. Non-cross linked and cross-linked chromatin were digested with Alu I enzyme, diluted and incubated with T4 DNA ligase. Ligated DNA was PCR-amplified using primers flanking the junction between either VDRE (Alu I fragment A) or nGRE (Alu I fragment C) regions with the PP (Alu I fragment E) region. PCR product was separated in a 2% agarose gel and Southern-hybridized to a [32P]-5′ labeled probe corresponding to 20 nucleotides upstream and 20 nucleotides downstream from the junction between the Alu I regions A and E and C and E. Upper and lower panels reveal interaction between region C containing the nGRE and region E containing the PP region, and between region A containing the VDRE and region E. A BAC containing a 50kb genomic DNA sequence encompassing the TSLP coding and flanking sequences was processed along as a positive control for efficiency of ligation (TSLP BAC). The GR involvement in FA-induced inhibition of MC-induced TSLP expression in keratinocytes was demonstrated using adult mice in which GR was selectively ablated in keratinocytes (GRep−/− mice). Although the basal TSLP level was similar in vehicle-treated WT and GRep−/− mice, FA blocked MC-induced TSLP expression in WT, but not in GRep−/− mice (Figure 1B, right panel). MC treatment was more efficient in GRep−/− than in WT mice, indicating that endogenous GCs may partially inhibit MC-induced TSLP expression in WT epidermis. TSLP expression was similarly repressed by FA and restored by RU in mouse intestinal epithelium (Figure S1B) and in human lung epithelial cells A549 (Figure S1C), whereas expression of the (+)GRE-containing mouse GPX3 and human GILZ (Wang et al., 2004Wang J.C. Derynck M.K. Nonaka D.F. Khodabakhsh D.B. Haqq C. Yamamoto K.R. Chromatin immunoprécipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes.Proc. Natl. Acad. Sci. USA. 2004; 101: 15603-15608Crossref PubMed Scopus (247) Google Scholar) GC-induced genes was enhanced by FA and inhibited by RU. Nuclear run-on assays demonstrated that GR-mediated FA inhibition of TSLP expression was transcriptional (Figure 1C). As neither NFκB nor AP1 are involved in TSLP induction by MC in epidermis (unpublished data), its repression was unlikely to be mediated by a tethering GRE. A bioinformatics analysis of 20 kb of DNA located upstream and downstream from the mouse and human TSLP translation startsite (+1) did not reveal any (+)GRE or known “composite” activating or repressing GRE, but unveiled the presence of a palindromic sequence consisting of two inverted repeated (IR) motifs separated by one bp (called hereafter IR1 nGRE), in the upstream promoter region of both mouse (m) and human (h) TSLP genes (Figure 1E). Recombinant human GR protein in electrophoretic mobility shift (EMSA) and supershift assays with GR antibody showed that this putative mTSLP IR1 nGRE and its human counterpart, as well as the TAT (+)GRE (Meijsing et al., 2009Meijsing S.H. Puffal M.A. So A.Y. Bates D.L. Chen L. Yamamoto K.R. DNA binding site sequence directs glucocorticoid receptor structure and activity.Science. 2009; 324: 407-410Crossref PubMed Scopus (489) Google Scholar), bound to the GR protein (Figure 1D, left panel). These bindings were specific, as shown by lack of GR binding to a mutant (+)GRE and to three mTSLP IR1 nGRE mutants (Mut1, 2, and 3) (Figures 1E and 1D, middle panel). Complexes formed between the recombinant GR and either putative IR1 nGREs or (+)GRE similarly migrated. As GR binds (+)GREs as a dimer (Wrange et al., 1989Wrange O. Eriksson P. Perlmann T. The purified activated glucocorticoid receptor is a homodimer.J. Biol. Chem. 1989; 264: 5253-5259Abstract Full Text PDF PubMed Google Scholar), two GR monomers may bind these putative nGREs. Competition bindings between [32P]-labeled mTSLP IR1 nGRE probe and excess cold (+)GRE probe, and vice-versa, indicated that GR has a higher affinity for (+)GRE than for TSLP IR1 nGRE (Figure 1D, right panel). GC-induced binding of GR to TSLP IR1 nGRE, the generation of a repressing complex, and its effect on the organization of the TSLP promoter regions, were investigated in vivo by chromatin immunoprecipitation (ChIP) with WT epidermis and intestinal epithelium, as well as in vitro with cultured A549 cells. Four regions of the TSLP promoter were analyzed: the proximal promoter region (PP), the region containing the IR1 nGRE, and those containing the DR3d VitD3 (VDRE) and the DR2b Retinoic Acid (RARE) response elements (unpublished data) (Figure 1F and Figure S1D). ChIP assays of epidermis revealed weak bindings of GR, as well as of SMRT and NCoR corepressors (Lonard and O'Malley, 2007Lonard D.M. O'Malley B.W. Nuclear receptor coregulators: judges, juries and executioners of cellular regulation.Mol. Cell. 2007; 27: 691-700Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar) to the nGRE region, which were strongly increased upon a 6hr topical FA treatment (Figure 1G). The concomitant disappearance of both GR and SMRT/NCoR bindings to the nGRE region in GRep−/− mutant mice (Figure 1G) indicated that corepressor bindings were associated with that of GR, which was confirmed by colocalization of GR and corepressors, when shorter segments of the nGRE region were explored (Figure S1E). Upon FA treatment, in the same cells, GR as well as SRC2, SRC3 and Pol II but not SMRT and NCoR, were recruited to the GPX3 (+)GRE region (Figure S1F). Moreover, binding of GR to (+)GRE, was antagonized by RU, which on its own, did not allow the binding of GR to GPX3 (+)GRE (Figure S1F). SMRT and NCoR are known to recruit histone deacetylase (HDACs) to repressing complexes. As for GR and corepressors, HDAC2 and HDAC3 were weakly bound to the nGRE in vehicle-treated epidermis, and FA strongly enhanced this recruitment (Figure S1G), further supporting that GC-induced binding of GR to TSLP IR1 nGRE generates a repressing complex. RU topical treatment precluded FA-induced generation of this repressing complex on nGRE, whereas application of MC (Figure 1G and Figure S1G) or retinoic acid (RA) (Figure S1H, upper panels) had no effect. We also used mouse intestinal epithelium, which revealed, upon FA intraperitoneal injection, a strong binding of GR together with SMRT and NCoR corepressors to the TSLP IR1 nGRE region. RU addition precluded the generation of this FA-induced repressing complex, whereas VitD3 had no effect (Figure S1I, upper panel). Similarly, FA addition to A549 cells resulted in a stronger binding of GR together with SMRT and NCoR to the nGRE region (which was suppressed by RU, Figure S1J), whereas it induced binding of an activating complex to the GILZ gene (+)GRE (Figure S1K). To demonstrate that SMRT and NCoR are instrumental in GC-induced IR1 nGRE-mediated TSLP repression, we knocked-down their expression in A549 cells by 60% and 80%, respectively, with selective siRNA (Figure 1H). Upon single siRNA treatment, ChIP assays showed a marked decrease in SMRT binding and complete disappearance of NCoR binding to TSLP nGRE, while no SMRT and NCoR binding could be detected upon siSMRT and siNCoR RNAs cotreatment (Figure 1I). Dex-induced TSLP repression was prevented by concomitant knockdowns of SMRT and NCoR, but not significantly affected by their single knockdown, thus demonstrating that SMRT and NCoR are instrumental in GC-induced IR1 nGRE-mediated repression, and that these two corepressors can be functionally redundant. Interestingly, the ChIP data suggest that GR is less efficiently bound to the IR1 nGRE in the absence of the two corepressors (Figure 1I). Note that SMRT and NCoR knockdowns (on their own or together) did not affect GR expression and Dex-induced transactivation of the (+)GRE GILZ gene (Figure 1H). In the absence of an agonist ligand, a repressing complex containing VDR and SMRT was bound to TSLP DR3d VDRE in epidermal keratinocytes, whereas it was replaced by a VDR-SRC2/SRC3-Pol II activating complex upon MC topical treatment (Figure 1G and unpublished results). Upon MC and FA cotreatment, VDR association with the VDRE was not inhibited, whereas those of SRC2, SRC3, and Pol II were drastically reduced, and an association of SMRT was observed (Figure 1G). No association of GR to DR3d VDRE was detected, but these latter changes are clearly related to binding of FA to GR, as RU cotreatment (MC+FA+RU) restored the activation binding pattern observed upon treatment with MC alone (Figure 1G). No GR binding to the PP region could be detected upon FA treatment. However, this treatment precluded VDR, SRC2, SRC3 and Pol II bindings induced by MC treatment, and a RU cotreatment (MC+FA+RU) reversed the effect of FA, indicating an involvement of FA-liganded GR in preventing the association of VDR, SRC2/SRC3 and Pol II with the PP region (Figure 1G, lower panels). Similarly, the generation of a repressing complex on IR1 nGRE precluded the formation of an activating complex on DR2b RARE and the PP regions (Figure S1H). Note that, in contrast to the DR3d VDRE complex, the DR2b RARE complex contains SRC2 only. In keeping with the above data, in intestinal epithelium, the generation of a repressing complex on TSLP IR1 nGRE also precluded formation of a VitD3-induced activating complex on DR3d VDRE and the PP regions (Figure S1I, middle panels). That, in presence of MC, the same activating complexes (VDR, SRC2/SRC3 and Pol II) were associated with DR3d VDRE and PP regions indicated that these two regions could be in close apposition through chromatin looping. We therefore performed Chromosome Conformation Capture (3C) assays on epidermal chromatin of mice topically treated with vehicle, FA, MC, MC+FA, RU, and MC+FA+RU. Cross-linked chromatin was digested with Nla III restriction enzyme to separate DR3d, nGRE and PP regions (Figure 1F), which were then ligated to reveal possible interactions between PP and DR3d VDRE or nGRE regions (Figure 1J). No interaction between nGRE and PP region, nor between nGRE and VDRE, could be detected upon FA or RU treatment, whereas an interaction was observed upon MC treatment between DR3d VDRE and PP regions, which was precluded by FA cotreatment (MC+FA), and restored upon RU addition (MC+FA+RU) (Figure 1J). Interactions between DR3d VDRE and PP regions were similarly revealed upon Alu I digestion (Figures S1L and S1M). Thus, the TSLP IR1 nGRE which is located ∼1.3 kb upstream from the PP region could act as a silencer element precluding the formation of a chromatin loop between the PP and the VDRE enhancer region located ∼7.3 kb upstream. To investigate whether additional DNA elements could be required to generate a repressing activity, we inserted the TSLP IR1 nGRE upstream of an enhancerless SV40 early promoter located 5′ to the luciferase coding sequence of pGL3 vector (Figure 2A ). A VDRE separated from the IR1 nGRE by a 314bp-long DNA segment devoid of any known transregulator binding site (not shown) was inserted to generate a luciferase-expressing reporter plasmid (pGL3 vector 1), which was transfected into A549 cells, followed by addition of VitD3 and/or FA. FA addition did not affect luciferase expression in absence of IR1 nGRE, whereas its presence resulted in decreased expression (which could be prevented by RU addition) of basal and VDRE-mediated VitD3-induced transcription (Figures 2A and 2B). As expected, FA-induced increase in luciferase expression was observed when IR1 nGRE was replaced b" @default.
• W2008654807 created "2016-06-24" @default.
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• W2008654807 creator A5024429879 @default.
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• W2008654807 date "2011-04-01" @default.
• W2008654807 modified "2023-10-18" @default.
• W2008654807 title "Widespread Negative Response Elements Mediate Direct Repression by Agonist- Liganded Glucocorticoid Receptor" @default.
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• W2008654807 doi "https://doi.org/10.1016/j.cell.2011.03.027" @default.
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