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Glucocorticoid-Induced Tethered Transrepression Requires Sumoylation of GR and Formation of a SUMO-SMRT/Ncor1-HDAC3 Repressing C

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Glucocorticoid-Induced Tethered Transrepression Requires Sumoylation of GR and Formation of a SUMO-SMRT/Ncor1-HDAC3 Repressing C Glucocorticoid-induced tethered transrepression PNAS PLUS requires SUMOylation of GR and formation of a SUMO-SMRT/NCoR1-HDAC3 repressing complex Guoqiang Huaa, Krishna Priya Gantia, and Pierre Chambona,b,c,1 aInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR7104, INSERM U964, Illkirch 67404, France; bUniversity of Strasbourg Institute for SEE COMMENTARY Advanced Study, Illkirch 67404, France; and cCollège de France, Illkirch 67404, France Contributed by Pierre Chambon, December 2, 2015 (sent for review October 19, 2015; reviewed by Maria G. Belvisi and Andrew Tan Nguan Soon) Upon binding of a glucocorticoid (GC), the GC receptor (GR) can dissected the molecular mechanisms involved in GR-mediated exert one of three transcriptional regulatory functions. We GC-induced IR nGRE-mediated direct transrepression, which recently reported that SUMOylation of the GR at position K293 revealed that GR SUMOylation in its N-terminal domain in humans (K310 in mice) within the N-terminal domain is indis- (NTD) at K293 (K310 in mice), as well as the subsequent for- pensable for GC-induced evolutionary conserved inverted re- mation of a small ubiquitin-related modifier (SUMO)-associated peated negative GC response element (IR nGRE)-mediated direct SMRT/NCoR1-HDAC3 repressing complex, are instrumental in transrepression. We now demonstrate that the integrity of this GR direct transrepression (11). This led us to investigate whether the SUMOylation site is mandatory for the formation of a GR-small same GR SUMOylation could also be implicated in GC-induced ubiquitin-related modifiers (SUMOs)-SMRT/NCoR1-HDAC3 repres- tethered transrepression. We report here the molecular mechanism sing complex, which is indispensable for NF-κB/AP1-mediated GC- underlying GC-induced tethered indirect transrepression and induced tethered indirect transrepression in vitro. Using GR K310R demonstrate the important role played by SUMOylation of the mutant mice or mice containing the N-terminal truncated GR iso- GR in this transrepression. We also report preliminary data il- form GRα-D3 lacking the K310 SUMOylation site, revealed a more lustrating how the elucidation of the molecular mechanisms severe skin inflammation than in WT mice. Importantly, cotreat- underlying the three main functions of the GR could be used in ment with dexamethasone (Dex) could not efficiently suppress a searches for selective GR agonists (SEGRAs), which would 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced skin inflam- selectively exert the beneficial antiinflammatory activities of mation in these mutant mice, whereas it was clearly decreased in glucocorticoids while being devoid of their (+)GRE and IR WT mice. In addition, in mice selectively ablated in skin keratino- nGRE-mediated debilitating activities. cytes for either nuclear receptor corepressor 1 (NCoR1)/silencing mediator for retinoid or thyroid-hormone receptors (SMRT) core- Results pressors or histone deacetylase 3 (HDAC3), Dex-induced tethered SUMOylation of the GR Is Not Required for Binding to DNA-Bound transrepression and the formation of a repressing complex on NF-κB, AP1, and STAT3, but Is Mandatory for Tethered Transrepression PHARMACOLOGY DNA-bound NF-κB/AP1 were impaired. We previously suggested and Is Involved in GC Antiinflammatory Effects. We previously that GR ligands that would lack both (+)GRE-mediated transacti- demonstrated that the SUMOylation site located at K293 in the vation and IR nGRE-mediated direct transrepression activities of GR NTD is mandatory for dexamethasone (Dex)-induced IR GCs may preferentially exert the therapeutically beneficial GC anti- nGRE-mediated direct transrepression (11). The report of Gross inflammatory properties. Interestingly, we now identified a non- et al. (12), showing that the GRα-D3 isoform (Fig. 1A), which steroidal antiinflammatory selective GR agonist (SEGRA) that selectively lacks both Dex-induced (+)GRE-mediated transactiva- Significance tion and IR nGRE-mediated direct transrepression functions, while still exerting a tethered indirect transrepression activity and could therefore be clinically lesser debilitating on long-term GC therapy. The antiinflammatory property of natural glucocorticoids (GCs) was demonstrated more than 60 years ago. Since then, syn- glucocorticoid receptor | SUMOylation | thetic GCs have been widely used to combat inflammatory and NF-κB/AP1-mediated GC-induced tethered repression allergic disorders. However, multiple severe undesirable side effects associated with long-term GC treatments, as well as induction of glucocorticoid resistance associated with such lucocorticoids (GC) are widely used in clinical treatments to treatments, limit their therapeutic usefulness. In the present Gsuppress inflammatory and allergic disorders. However, study, we unveiled the molecular mechanism underlying the various associated undesirable side effects limit their therapeutic GC-induced GC receptor (GR)-mediated tethered indirect trans- usefulness (1). Upon GC binding, the GC receptor (GR) regulates repression. This knowledge paves the way to the future edu- the expression of target genes either by transcriptional activation cated design and screening of drugs, collectively named selective through direct binding to (+)GRE DNA binding sites (DBS) (2), GR agonists, which would exhibit the major therapeutically direct transrepression through binding to evolutionary conserved beneficial properties of GCs, but would be devoid of undesir- inverted repeated negative response element (IR nGRE DBSs) able debilitating effects upon prolonged GC therapy. (3), or tethered indirect transrepression mediated through in- teraction with transactivactors such as NF-κB/activator protein 1 Author contributions: G.H. and P.C. designed research; G.H. and K.P.G. performed re- (AP1)/STAT3 bound to their cognate DBSs (4, 5). The beneficial search; G.H. and P.C. analyzed data; and G.H. and P.C. wrote the paper. antiinflammatory effects are generally ascribed to tethered trans- Reviewers: M.G.B., Imperial College London; and A.T.N.S., Nanyang Technological repression, whereas many of the undesirable side effects appear to University. be related to transactivation (1) and direct transrepression (3). The authors declare no conflict of interest. Tethered transrepression of NF-κB and AP1 by the GR has been See Commentary on page 1115. proposed to result from alteration in the assembly of coactivator 1To whom correspondence should be addressed. Email: [email protected]. – (6 8) or from interference with serine-2 phophorylation at the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. C-terminal domain of RNA polymerase II (9, 10). We recently 1073/pnas.1522826113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1522826113 PNAS | Published online December 28, 2015 | E635–E643 Downloaded by guest on September 29, 2021 A 1 M 09 K293 633M 420 486 528 NTD DBD GR FL (GRα-A) LBD Regions A/B C D E/F GR 336 (GRα-D3) GR ABCD B Mouse embryonic fibroblasts (MEFs) 36 9 24 6 12 3 0 0 GRwt MEFs β IL1β IL1 IL1β IL1β IL1β IL1β IL1β IL1β α +Dex +Dex GR -D3 MEFs β+Dex β+Dex β Relative RNA level RNA Relative Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle IL1β+Dex IL1β IL1β+Dex IL1β+Dex IL1 IL1β+Dex IL1 IL1 IL6IL6MMP13 MMP13RANTES RANTES COX2COX2TSLP CCL4 CCL4 IL1α IL1β C β β 0,25 Vehicle 0,2 IL1 IL1 +Dex 0,25 0,2 TSLP (NFκB) 0,16 IL6 (NFκB) 0,2 MMP13 (AP1) 0,15 0,12 0,15 0,1 0,08 0,1 0,05 0,04 0,05 0 0 0 GR GR GR GR GR GR IgG IgG p65 p65 IgG IgG IgG IgG p65 p65 p65 p65 c-jun c-jun c-jun c-jun c-jun c-jun SMRT SMRT SMRT SMRT SMRT SMRT NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 SUMO1 SUMO1 SUMO1 SUMO1 SUMO1 SUMO1 ChIP Assay Input % SUMO2/3 SUMO2/3 SUMO2/3 SUMO2/3 SUMO2/3 SUMO2/3 GRwtGRwt MEFs MEFs GRα-D3GRα-D3 MEFsMEFs GRwtGRwt MEFs GRα-D3GRα-D3 MEFs MEFs GRwtGRwt MEFs GRα-D3GRα-D3 MEFsMEFs D Mouse ear extracts 6 5 * 5 E GRwt GRα-D3 4 GRwt GR K310R 4 3 3 2 2 * 1 1 0 0 Relative RNA level RNA Relative Relative RNA level RNA Relative TPA TPA TPA TPA TPA TPA TPA TPA TPA TPA TPA TPA Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle TPA+Dex TPA+Dex TPA+Dex TPA+Dex TPA+Dex TPA+Dex TPA+Dex TPA+Dex TPA+Dex TPA+Dex TPA+Dex TPA+Dex MMP13MMP13COX2 COX2TSLP TSLP TNFαTNFα IL6IL6 IL1βIL1β MMP13MMP13COX2 COX2 TNFαTNFα IL6IL6IL17f IL17fRANTES RANTES F Mouse epidermis treated with TPA+Dex 0,2 0,15 0,15 MMP13 (AP1) COX2 (NFκB) TNFα (NFκB) 0,15 0,12 0,09 0,1 0,1 0,06 0,05 0,05 0,03 0 0 0 GR GR GR GR GR GR IgG IgG IgG IgG IgG IgG p65 p65 p65 p65 p65 p65 ChIP Assay Input % c-jun c-jun c-jun c-jun c-jun c-jun SMRT SMRT SMRT SMRT SMRT SMRT NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 SUMO1 SUMO1 SUMO1 SUMO1 SUMO1 SUMO1 SUMO2/3 SUMO2/3 SUMO2/3 SUMO2/3 SUMO2/3 SUMO2/3 WTWT GRGR K310R K310R WT GRGR K310RK310R WT GRGR K310R Fig. 1. SUMOylation of the GR is mandatory for tethered transrepression and involved in GC antiinflammatory effects. (A) Schematic presentation for GR FL, GR K293R, GR 336, and GR ABCD.(B) Quantitative (q)RT-PCR for transcripts of endogenous NF-κB/AP1 DBS-containing genes in GRwt and GRα-D3 MEFs, treated with vehicle, IL1β (5 ng/mL), and Dex (0.5 μM) for 6 h. (C) ChIP assays performed with GRwt and GRα-D3 MEFs treated with vehicle, IL1β (5 ng/mL), and Dex (0.5 μM) for 1 h. qPCR analyses were performed on NF-κB/AP1 DBS regions, as indicated. (D)AsinB, but using ear extracts from GRwt or GRα-D3 mice topically treated with vehicle, TPA (1 nmol/cm2), and Dex (6 nmol/cm2)for3d.(E)AsinD, but for GRwt or GR K310R mice topically treated with TPA and Dex for 5 d.
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