GR SUMOylation and formation of an SUMO-SMRT/ NCoR1-HDAC3 repressing complex is mandatory for GC-induced IR nGRE-mediated transrepression

Guoqiang Huaa, Laetitia Paulena, 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 Advanced Study, Illkirch 67404, France; and cCollège de France, Illkirch 67404, France

Contributed by Pierre Chambon, November 19, 2015 (sent for review October 8, 2015; reviewed by Maria G. Belvisi and Andrew Tan Nguan Soon) Unique among the nuclear superfamily, the glucocorticoid effects (1). This led to searches for dissociated ligands that would (GC) receptor (GR) can exert three distinct transcriptional regulatory preferentially induce tethered transrepression and be devoid of functions on binding of a single natural (cortisol in human and transactivation activity. Such a ligand was found to exhibit the ex- corticosterone in mice) and synthetic [e.g., dexamethasone (Dex)] pected dissociation profile in vitro, but its administration in vivo did hormone. The molecular mechanisms underlying GC-induced posi- not confirm this dissociation (1, 5). Our own discovery of a GC- tive GC response element [(+)GRE]-mediated activation of transcrip- induced direct transrepression activity mediated via direct GR tion are partially understood. In contrast, these mechanisms remain binding to evolutionary conserved inverted repeated negative re- elusive for GC-induced evolutionary conserved inverted repeated sponse elements (IR nGREs) indicated that this GC analog failed negative GC response element (IR nGRE)-mediated direct transre- because it had kept the latter activity (6). Thus, a search for im- pression and for tethered indirect transrepression that is mediated proved dissociated anti-inflammatory compounds should aim at by DNA-bound NF-κB/activator 1 (AP1)/STAT3 activators finding GR agonists that would repress expression through and instrumental in GC-induced anti-inflammatory activity. We tethered indirect repression while lacking IR nGRE-mediated demonstrate here that SUMOylation of lysine K293 (mouse K310) transrepression and (+)GRE-mediated transactivation activities. located within an evolutionary conserved sequence in the human However, although the molecular mechanisms involved in (+)GRE- GR N-terminal domain allows the formation of a GR-small ubiqui- mediated transactivation have been deciphered, those underlying tin-related modifiers (SUMOs)-NCoR1/SMRT-HDAC3 repressing tethered indirect transrepression and IR nGRE-mediated trans- complex mandatory for GC-induced IR nGRE-mediated direct re- repression are still poorly understood, thus precluding an educated pression in vitro, but does not affect transactivation. Importantly, design and a differential screening of compounds that would se- these results were validated in vivo: in K310R mutant mice and in lectively exert the anti-inflammatory activities of GCs. We report mice ablated selectively for corepressor 1 (NCoR1)/ here an analysis of the molecular mechanisms involved in direct silencing mediator for retinoid or thyroid-hormone receptors (SMRT) transrepression, which demonstrates at the molecular level the corepressors in skin keratinocytes, Dex-induced direct repression role played by SUMOylation of the GR in this transrepression. and the formation of repressing complexes on IR nGREs were im- paired, whereas transactivation was unaffected. In mice selectively Results ablated for histone deacetylase 3 (HDAC3) in skin keratinocytes, A Conserved Sequence (AA283-295) Within the GR N-Terminal Domain GC-induced direct repression, but not bindings of GR and of co- Is Required for IR nGRE-Mediated Transrepression, Whereas the repressors NCoR1/SMRT, was abolished, indicating that HDAC3 is Ligand Binding Domain Is Dispensable. Human GR cDNAs for instrumental in IR nGRE-mediated repression. Moreover, we dem- full-length (FL) GR, GR isoforms (2), and GR truncated in the onstrate that the binding of HDAC3 to IR nGREs in vivo is mediated through interaction with SMRT/NCoR1. We also show that the GR Significance ligand binding domain (LBD) is not required for SMRT-mediated re- pression, which can be mediated by a LBD-truncated GR, whereas it Glucocorticoids (GCs), acting through binding to the GC re- is mandatory for NCoR1-mediated repression through an interaction ceptor (GR), are peripheral effectors of circadian and stress- with K579 in the LBD. related homeostatic functions fundamental for survival throughout vertebrate life span. They are widely used to combat inflamma- | SUMOylation | GC-induced direct tory and allergic disorders, and their therapeutic effects have transrepression | IR nGRE been mainly ascribed to their capacity to suppress the production of proinflammatory cytokines. The present study unveils, at the lucocorticoids (GCs) hormones, the function of which is trans- molecular level, the mechanisms that underlie the GC-induced Gduced by a single receptor, the GR, have pleiotropic effects on GR direct transrepression function mediated by the evolutionary almost all aspects of physiology. Their anti-inflammatory and im- conserved inverted repeated negative response element. This munosuppressive properties were demonstrated more than 60 years knowledge paves the way to the elucidation of the functions of ago (1, 2). Since then, and despite their multiple side effects, GCs the GR at the submolecular levels and to the future educated have been increasingly used in the treatment of numerous in- design and screening of drugs, which could be devoid of un- flammatory conditions, such as rheumatoid arthritis and allergic desirable debilitating effects on prolonged GC therapy. disorders. The GR regulates the expression of target either by transcriptional transactivation through binding to GC response ele- Author contributions: G.H. and P.C. designed research; G.H. and L.P. performed research; ments (GREs) (3) or by transrepression (1, 2). To initiate “tethered” G.H. and P.C. analyzed data; and G.H. and P.C. wrote the paper. indirect transrepression, the GR is thought to physically interact with Reviewers: M.G.B., Imperial College London; and A.T.N.S., Nanyang Technological University. DNA-bound transcriptional activators (e.g., NF-κB, activator protein The authors declare no conflict of interest. 1 (AP1), STAT3) and to repress their downstream target genes See Commentary on page 1115. (4). Interestingly, it became widely accepted that most GC anti- 1To whom correspondence should be addressed. Email: [email protected]. inflammatory effects can be ascribed to tethered transrepression, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. whereas transactivation was responsible for many undesirable side 1073/pnas.1522821113/-/DCSupplemental.

E626–E634 | PNAS | Published online December 28, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1522821113 Downloaded by guest on October 2, 2021 PNAS PLUS A 1 M 09 K293 633M 420 486 528 NTD DBD 777 GR FL (GRα-A) LBD Regions A/B C D E/F GR 90 (GRα-C2) GR 190 GR 280 GR 336 (GRα-D3) GR 380 GR 420 GR ABCD GR 280-527 GR 336-527 B1,8 Mouse embryonic fibroblasts (MEFs) Mouse GRwt Mouse GRα-D3 SEE COMMENTARY 1,2 * ** ** 0,6 ** ** ** 0 ** ** ** ** Dex Dex Dex Dex Dex Dex Dex Dex Dex Dex Relative RNA level RNA Relative Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Dex+RU Dex+RU Dex+RU Dex+RU Dex+RU Dex+RU Dex+RU Dex+RU Dex+RU Dex+RU IL6 TSLP FGFR3 STRA13 ITPKA PPIL5 JUND PRKCB TNFRSF19 USF1 IR1 nGRE IR1 nGRE IR2 nGRE IR2 nGRE IR0 nGRE IR0 nGRE IR1 nGRE IR1 nGRE IR1 nGRE IR1 nGRE

C Mouse embryonic fibroblasts (MEFs) Vehicle Dex Dex+RU 0,25 0,25 0,3 0,3 0,2 TSLP (IR1 nGRE) 0,2 STRA13 (IR2 nGRE) FGFR3 (IR2 nGRE) IL6 (IR1 nGRE) 0,2 0,15 0,15 0,2 0,1 0,1 0,1 0,1 0,05 0,05 0 0 0 0 GR GR GR GR GR GR IgG IgG IgG IgG IgG IgG GR GR IgG IgG ChIP Assay Input % SMRT SMRT SMRT SMRT SMRT SMRT SMRT SMRT NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 GRwtGRwt GRα-D3 GRwtGRwt GRα-D3 GRwtGRwt GRα-D3GRα-D3 GRwtGRwt GRα-D3GRα-D3 SV40 early promoter D SV40 Enhancer 17mer Gal4 RE pGL3-17mer Gal4 reporter Luciferase pSG5-GR(NTD)-Gal4 pSG5-Gal4 pSG5 GR NTD Gal4(1-147) pSG5 Gal4(1-147) 283 K293 295 E 5000 pGL3-17mer Gal4 reporter F 0,5 pGL3-17mer Gal4 reporter 4000 0,4 GR PHARMACOLOGY SUMO1 3000 0,3 NCoR1 2000 0,2 SMRT IgG 1000 0,1

0 ChIP Assay Input % 0 Vector Gal4 GR(NTD) - GR(NTD GR(NTD

Relative LuciferaseRelative Unit Gal4 GR(NTD)-Gal4 GR(NTD 283- GR(NTD K293R)- Gal4 283-295)-Gal4 K293R)-Gal4 295) -Gal4 Gal4 pSG5 pSG5 G H 1 I 0,1 4000 pGL3-nGRE reporter pGL3-nGRE reporter pGL3-nGRE reporter (+Dex) 0,8 0,08 3000 Vehicle Dex Vehicle NCoR1 0,6 Dex 0,06 SMRT 2000 0,4 0,04 IgG 1000 0,2 0,02 0 ChIP Assay Input % 0 0 VectorVectorGR GR GR GRGR 280- GRGR 336- GRGR GRGR ChIP Assay Input % VectorVector GRGR GR GR GRGR 280- GRGR 336- GRGR GRGR VectorVectorGR GRGR GR GR 280- GRGR 336- GR GRGR Relative LuciferaseRelative Unit ABCD 527 527 ABCD K579A ABCD 280-527 336-527 ABCD K579A ABCD 527 527 K579A ABCD 527 527 ABCD K579A ∆283-295 ABCD 280-527 336-527 ABCDABCD K579A ABCD 280-527 336-527 ABCD K579A 283-295 ∆283-295 ∆283-295 283-295 283-295 J 283 293 295 Homo FIELCTPGV I KQE SUMO consensus : Ψ-K-x-D/E Xenopus YIELCTPGVVKQE : Rattus FIELCTPGV I KQE hGR 275-279: QVKTE (K277) mGR 292-276 QVKTE (K294) : Gallus FIELCTPG - I KQE hGR 291-295: VIKQE (K293) mGR 308-312 VIKQE (K310) Danio I IQLCTPGV I KQE hGR 701-705: IVKRE (K703) mGR 716-720: IVKRE (K718) Salmo FIQLCTPGV I KQE

Fig. 1. GR binding to an IR nGRE in vitro and in vivo requires a discrete sequence located in the NTD. (A) GR NTD and LBD deletion mutants. (B) Quantitative (q)RT-PCR for IR nGRE-containing genes in GRwt and GRα-D3 MEFs. Vehicle, Dex (0.5 μM), and RU (3 μM) treatments were for 6 h. (C) qPCR of ChIP assays performed with GRwt and GRα-D3 MEFs treated with vehicle, Dex (1 μM), and RU (6 μM) for 1 h, showing the association of GR, nuclear receptor corepressor 1, and silencing mediator for retinoid or thyroid- hormone receptors (SMRT) on IR nGREs in the promoter region of genes as indicated. (D) Schematic representation for pGL3-17mer Gal4 reporter, pSG5-Gal4, and pSG5-GR NTD-Gal4 plasmids. (E) Luciferase assays of Cos-1 cells transfected with pGL3-17mer Gal4 reporter and pSG5-Gal4 alone or fused to GR NTD or its mutants as indicated (see D). (F)AsinC, but performed with Cos-1 cells transfected with pGL3-17mer Gal4 reporter and pSG5-Gal4 constructs (see D), showing the association of indicated on the Gal4 RE sequence, using specific antibodies. (G)AsinE, but transfected with pGL3-nGRE reporter and GR mutants. Vehicle or Dex (0.5 μM) treatment was for 6 h. (H and I)As in F but transfected with pGL3-nGRE reporter and GR mutants, showing the binding of GR, NCoR1, and SMRT on an IR nGRE. Cells were treated with vehicle or Dex (1 μM) for 1h.(J) Alignment of the 283–295 AA sequence in human GR with homologous GR sequences in vertebrates (Left) and predicted SUMOylation sites in the human and mouse GR sequence (Right). Values are mean ± SEM. *P < 0.05, **P < 0.01.

Hua et al. PNAS | Published online December 28, 2015 | E627 Downloaded by guest on October 2, 2021 N-terminal domain (NTD) were expressed in the pcDNA3 vector 280-527, and no corepressor was bound to GR 336-527 (Fig. 1I). (Fig. 1A and Fig. S1A). Their transcriptional activities were de- Importantly, analysis of the evolutionary conservation of GR NTD termined in Cos-1 cells (low in GR content) cotransfected with revealed a vertebrate highly conserved sequence: FIELCTPG- pGL3-nGRE or pGL3-(+)GRE luciferase reporters (6) (Fig. S1 B VIKQE (AA283-295) (Fig. 1J, Left), the deletion of which in GR and D–F). From GR FL to GR 280, the pGL3-nGRE activity ABCD totally abolished the IR1 nGRE-mediated transrepression decreased by ∼50% upon dexamethasone (Dex) treatment, (Fig. 1 G and H). Furthermore, this sequence was mandatory for whereas no repression was observed for GR 336 (the GRα-D3 the repressing function of the GR NTD, as its deletion in the GR isoform; Fig. 1A) and beyond (Fig. S1G). Of note, transcripts (NTD)-Gal4(1-147) protein (Fig. 1D) fully reversed the GR NTD- of the IR2 nGRE-containing stimulated by retinoic acid 13 induced repression (Fig. 1E), while SMRT binding to GR NTD (STRA13) gene in Cos-1 cells exhibited similar profiles of Dex- was abolished (ChIP assays; Fig. 1F). induced repression (Fig. S1H), and as expected (6), this repression was relieved by the GC antagonist RU486 (RU) (Fig. S1H). ChIP SUMOylation of Human GR at K293 Is Crucial for IR nGRE-Mediated assays revealed the binding of GR, GR 90, GR 190, and GR 280, Transrepression. The reports of Pascual et al. (10) and Leuenberger but not of GR 336 (the human GRα-D3 isoform), to the IR1 et al. (11) showing that SUMOylation of the LBDs of peroxisome nGRE of the pGL3-nGRE reporter (Fig. S1I). proliferator-activated receptor (PPAR)γ and PPARα were asso- To demonstrate that the mouse GR NTD was similarly man- ciated with transcriptional repression, prompted us to test whether datory for repression in vivo, we removed 352 AAs from the N the SUMOylation site, which was previously identified (12) in the terminus to generate a mutant expressing selectively the mouse NTD of human GR at position K293 within the AA283-295 GRα-D3 (GR 353) isoform similar to the human GR 336 (Fig. S2 conserved sequence (Fig. 1J),couldbeinvolvedinDex-induced and SI Materials and Methods). As 95% of GRα-D3 homozygote IR nGRE-mediated repression. Interestingly, the K293R muta- embryos died before or at birth, mouse embryonic fibroblasts tion, but not those of the additional GR SUMOylation sites at (MEFs) were derived from 13-day embryos. In these MEFs, the position K277 and K703 (12), abolished Dex-induced IR1 nGRE- GRwt, but not GRα-D3, repressed transcription from IR nGRE- mediated repression (Fig. 2A and Fig. S3A, Left). ChIP assays containing genes (Fig. 1B), and interestingly, ChIP assays showed revealed that, upon Dex treatment of transfected Cos-1 cells, GR that GRwt, but not GRα-D3, was associated with SMRT and/or K293R and GR ABCD K293R mutations drastically reduced GR NCoR1 corepressors on IR nGREs of these genes (Fig. 1C; note binding to the IR1 nGRE of the pGL3 reporter, and importantly, the effect of RU486). Together, these in vitro and in vivo results no repressing complex containing SMRT and NCoR1 could be demonstrated that the GR NTD region was instrumental in GR assembled with these mutants (Fig. 2 B and C). Small ubiquitin- binding to an IR nGRE. related modifier (SUMO)1 and SUMO2/3 were associated To determine whether the GR NTD could exert a repressing upon Dex treatment with GR, GR K277R, and GR K703R, function on its own, we used our previous construct pSG5-GR but not with GR K293R, and, as expected, the association of (AB)-GAL (7) [hereafter called pSG5-GR(NTD)-Gal4], and SUMOs with GR ABCD was not ligand dependent (Fig. 2C). The made a luciferase reporter vector by replacing the IR1 nGRE of same SUMO/corepressor repressing complex was associated on the pGL3-nGRE vector with the 17-mer Gal4 DNA response the IR1 nGRE of the Cos-1 cell endogenous thymic stromal element (17mer Gal4 RE), thereby creating the pGL3-17mer lymphopoietin (TSLP)gene(Fig.2D). Importantly, the formation Gal4 luciferase reporter (Fig. 1D). Transactivation by the yeast of this GR complex was prevented by addition of RU486, which is transactivator Gal4 was repressed when the GR NTD was fused known to promote GR translocation to the nucleus (Fig. S1C and to Gal4(1-147) (Fig. 1 D and E). ChIP assays revealed the binding Fig. 2 C and D), indicating that, on its own, SUMOylation of the of GR NTD and SMRT (but not of NCoR1) to 17-mer Gal4 GR is GC dependent. Furthermore, upon topical Dex treatment RE upon transfection of the pSG5-GR(NTD)-Gal4(1-147) vector of the WT epidermis, the same repressing complex was found on (Fig. 1F). These data indicated that the GR NTD on its own can the TSLP IR1 nGRE (Fig. 2E; note the inhibitory effect of exert a repressing function that involves the SMRT corepressor. RU486), thus establishing that SUMOs are associated with Dex- The nuclear ligand binding domain (LBD)-deleted GR (GR induced IR nGRE-mediated transrepression complexes in vivo. ABCD) (Fig. 1A and Fig. S1C) is a constitutive activator of Similar data were obtained for the IR1 nGRE recently found in transcription (8) (Fig. S3 C and D). Similarly, GR ABCD re- exon 6 of the human GR (13). Indeed, upon transfection of WT pressed the expression of the IR1 nGRE reporter in the absence GR, but not of GR K293R, the same Dex-induced SUMO/ of Dex (Fig. 1G), while ChIP assays revealed its IR1 nGRE corepressor repressing complex was associated on the IR1 nGRE binding (Fig. 1H). Interestingly, these assays showed that both present in exon 6 of the Cos-1 cell endogenenous GR gene (Fig. NCoR1 and SMRT could bind together with the GR on an IR1 2F). Interestingly, introduction of the K293R SUMOylation site nGRE, whereas only SMRT was recruited by GR ABCD (Fig. mutation in the GR(NTD)-Gal(1-147) fusion protein (Fig. 1D) 1I). As expected, RU addition did not affect the GR ABCD confirmed that SMRT binding to the GR NTD was dependent on repressing activity and IR nGRE binding (Fig. 2C). Of note, SUMOylation of GR K293 (Fig. 1F) and, consequently, that this the crystal structure of a GR LBD-NCoR1 complex has revealed SUMOylation was instrumental in the repressing function of the a strong hydrogen bond interaction between NCoR1 and the GR GRNTDonitsowninvitro(Fig.1E and F), in keeping with the K579 residue (9). However, mutation of K579 did not affect IR1 data of Holmstrom et al. (14). nGRE-mediated transrepression (Fig. 1G), nor binding of GR to To unequivocally demonstrate that GR SUMOylation is cru- an IR1 nGRE (Fig. 1H), whereas the binding of NCoR1, but not cial for IR nGRE-mediated repression in the mouse in vivo, we of SMRT, was markedly reduced (Fig. 1I), indicating that GR engineered a mouse SUMOylation mutant bearing a K-R mu- K579 is required for NCoR1 binding to the GR. That the in- tation at GR position K310 (homologous to human K293). Ex- teraction between NCoR1 and GR K579 could be instrumental in amining, in the ear skin of this SUMOylation mutant, the effect IR nGRE-mediated transrepression was suggested by transfecting of a topical Dex treatment on the expression of IR nGRE-con- the GR K579A mutant in Cos-1 cells in which SMRT expression taining genes revealed in all cases, including for the GR gene, a was knocked down with siRNA (Fig. S1 J–L, and see below). significant decrease in repression compared with WT (Fig. 2G), Deletions (Fig. 1A) to map the GR sequence involved in the whereas no repression was exerted by GRα-D3 (Fig. 1B). Ac- NTD repressing function showed that GR 280-527 exhibited cordingly, ChIP assays carried out on mouse epidermis showed the same repressing activity and binding to IR1 nGRE as GR that GR, SUMO1, and the corepressor SMRT were associated ABCD, while both were abolished in GR 336-527 (Fig. 1 G with IR nGREs present in genes (including the GR) from WT and H). As expected, SMRT, but not NCoR1, was bound to GR mice, whereas this association was decreased in GR K310R

E628 | www.pnas.org/cgi/doi/10.1073/pnas.1522821113 Hua et al. Downloaded by guest on October 2, 2021 PNAS PLUS A Vehicle Dex Dex+RU C 0,1 pGL3-nGRE reporter 1,5 0,08 NCoR1 STRA13 (IR2 nGRE) 6 SGK1 [(+)GRE] 0,06 SMRT 0,04 1 4 SUMO1 0,02 SUMO2/3 0 0,5 2 GR GR GR GR GR GR GR GR IgG

ChIP Assay Input % K277R K293R K703R ABCD ABCD ABCD Relative RNA level RNA Relative 0 0 K293R GR GR GR GR GR GR GR GR Dex Vehicle Dex+RU RU K277R K293R K703R K277R K293R K703R D0,4 B 1 pGL3-nGRE reporter TSLP (IR1 nGRE) Vehicle Dex Dex+RU SEE COMMENTARY 0,3 0,8 Vehicle 0,2 0,6 Dex 0,1 0,4 0 0,2 GR GR GR GR IgG IgG IgG IgG 0 SMRT SMRT SMRT SMRT NCoR1 NCoR1 NCoR1 NCoR1 SUMO1 SUMO1 SUMO1 GR GR GR GR GR GR SUMO1 ChIP Assay Input % SUMO2/3 SUMO2/3 SUMO2/3 SUMO2/3 K277R K293R K703R ABCD ABCD ChIP Assay Input % GR GR K277R GR K293R GR K703R K293R GR GR K277R GR K293R GR K703R 4 E G * Mouse ear extracts GRwt GR K310R 18 0,2 GR 3 mTSLP (IR1 nGRE) 12 SUMO1 ** 0,16 2 ** * SUMO2/3 * 6 0,12 SMRT 1 0,08 NCoR1 0 0 HDAC2 0,04 Dex Dex Dex Dex Dex Dex Dex Dex HDAC3 Dex IgG

0 Relative RNA level Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle

ChIP Assay Input % Vehicle Dex Dex+RU K5 K14 CYP26A1 N- STRA13 CCND2 GR REDD1 FKBP5 F 0,12 IR nGRE-containing genes (+)GRE-containing genes GR (IR1 nGRE) Vehicle 0,09 H0,3 Dex Mouse epidermis (+Dex) 0,06 0,2 GR SUMO1 0,03 SMRT IgG 0 0,1 GR GR IgG IgG 0 ChIP Assay Input % SMRT SMRT NCoR1 NCoR1 SUMO1 SUMO1 GRwt GR GRwt GR GRwt GR GRwt GR SUMO2/3 SUMO2/3 K310R K310R K310R K310R GR GR K293R K14 K5 GR FKBP5 ChIP Assay Input %

I TSLP (IR1 nGRE) IR nGRE-containing genes (+)GRE-containing genes PHARMACOLOGY 0,350,25 Vehicle J TSLP (IR1 nGRE) 0,2 Dex 10nM 0,40,5 Vehicle 0,15 Dex 100nM 0,3 Dex * Dex+JNK Inhibitor 0,1 ** 0,2 Dex+LiCl (GSK-3 Inhibitor) * 0,1 0,05 * * * ** * * * 0 0 ChIP Assay Input % GR GR GR IgG IgG IgG GR GR GR IgG IgG IgG ChIP Assay Input % SMRT SMRT SMRT SUMO1 SUMO1 SUMO1 NCoR1 NCoR1 NCoR1 SUMO1 SUMO1 SUMO1 SUMO2/3 SUMO2/3 SUMO2/3 SUMO2/3 SUMO2/3 SUMO2/3 Dex 1μM Dex 100nM Dex 10nM GRGR GRGR S226A/S404A S226A/S404AGR GR 5SA 5SA Dex 1μM Dex 100nM Dex 10nM

K 0,4 L M TSLP (IR1 nGRE) SUMO-Interacting Motif (SIM) Input GST GST-SUMO1 0,3 GR ABCD hNCoR1 1623-2440 Human DAXX 733 – IIVLSDSD – 740 GR ABCD S226A/S404A Mouse DAXX 732 – IIVLSDSD – 739 hNCoR1 1623-2434 0,2 GR ABCD 5SA Human NCoR1 2435 – LSDSDD–– 2440 hSMRT 2001-2514 0,1 Mouse NCoR1 2449 – LSDSDD–– 2454 Human SMRT 2510 – LSDSE – 2514 hSMRT 2001-2509 ChIP Assay Input % 0 Mouse SMRT 2464 – LSDSE – 2468

Fig. 2. GR SUMOylation at K293 in the GR NTD is crucial for Dex-induced IR nGRE-mediated direct transrepression through interaction between SUMOs and corepressors NCoR1 and SMRT. (A) qRT-PCR for STRA13 and serum and glucocorticoid-regulated kinase 1 (SGK1) transcripts in Cos-1 cells transfected with GR bearing mutations in SUMOylation sites, treated with vehicle, Dex (0.5 μM), and RU (3 μM) for 6 h. (B and C) qPCR of ChIP assays using Cos-1 cells transfected with pGL3-nGRE reporter and GR expression vectors, treated with vehicle, Dex (1 μM),orRU(1or6μM for the cotreatment) for 1 h, showing the binding of GR, NCoR1, SMRT, SUMO1, and SUMO2/3 to an IR nGRE sequence. (D)AsinB, but showing the association of indicated proteins on the IR1 nGRE of the TSLP gene. (E)AsinD, but using WT mouse dorsal epidermis topically treated with vehicle, Dex (6 nmol/cm2) and RU (36 nmol/cm2)for6h.(F)AsinB, but on the IR1 nGRE present in exon 6 of the GR gene. (G)AsinA, but for IR nGRE and (+)GRE-containing genes from ear extracts of GRwt and GR K310R mutant mice, treated with vehicle or Dex (6 nmol/cm2) for 18 h. (H)AsinE, but with dorsal epidermis from WT and GR K310R mutant mice topically treated with Dex, showing the association of GR, SUMO1, and SMRT on indicated DBSs. (I)AsinD, but using Cos-1 cells transfected with GR, GR S226A/S404A, or GR 5SA, treated as indicated. (J)AsinD, but using A549 cells treated as indicated; 25 μM JNK inhibitor II or/and 20 mM LiCl was added into the medium 30 min before Dex treatment. (K)AsinI, but transfected with GR ABCD or its mutants. (L) Sequence alignment of DAXX SIM with putative human and mouse NCoR1 and SMRT SIM. (M) GST-pull down of 35S-labeled C-terminal moieties of NCoR1 and SMRT proteins by GST or GST-SUMO1 protein. Input, 10% of 35S-labeled proteins used for the binding assay. Values are mean ± SEM. *P < 0.05, **P < 0.01.

Hua et al. PNAS | Published online December 28, 2015 | E629 Downloaded by guest on October 2, 2021 mutants (Fig. 2H), but not abolished as in the GRα-D3 MEFs STRA13), whereas (ii) in another instance, there was no such (Fig. 1C), suggesting that SUMOylation at position K294 (K277 substitution (corepressor-specific binding, as in the case of USF1, in human) could contribute to the GC-induced IR nGRE- PPIL5,andINSR) and no GR binding to IR nGRE elements could mediated repression activity of the GR in vivo, but not in vitro. be detected, and (iii) in a third instance, both corepressors were Interestingly, in all cases the integrity of the SUMOylation site concomitantly found bound in WT MEFs [no preferential binding, (K293 in human, K310 in mice) is mandatory for GR binding to as in the case of TSLP and tumor necrosis factor receptor super- an IR nGRE and assembly of a repressing complex. However, family member 19 (TNFRSF19)] (Fig. 3B and Fig. S4C). However, most of intracellular GR is not SUMOylated (Fig. S3B), which in this latter case, the absence of either one of the two corepressors suggests that the binding of GR to an IR nGRE may precede did not abolish the repression of the TSLP and TNFRSF19 genes its SUMOylation. (Fig. 3A and Fig. S4B), indicating that the simultaneous binding of both NCoR1 and SMRT to their nGREs was not required Phosphorylation of the NTD Facilitates the SUMOylation of the GR, for repression. but Not of GR ABCD. As it has been reported that phosphorylation To extend these observations in vivo, we then generated ker- of the NTD could enhance SUMOylation of the GR (15), we atinocyte-selective KO of NCoR1 and/or SMRT in mouse epi- mutated (serine to alanine) five known phosphorylation sites in dermis, which resulted in decreased expression of NCoR1 (70–80%) the human GR NTD: S134A, S203A, S211A, S226A, and S404A and SMRT (higher than 90%) (Fig. S4D; these single and double − − − − (2). Interestingly, upon GR transfection in Cos-1 cells under mutant mice are hereafter designated as NCoR1ep / , SMRTep / − − limiting Dex concentration (10 nM), the bindings of GR 5SA (all and [SMRT/NCoR1]ep / ). Dex-induced repression of three IR five S mutated), as well as those of SUMOs and SMRT/NCoR1 nGRE-containing genes, STRA13,keratin14(K14), and keratin − − corepressors to the IR1 nGRE of the Cos-1 cell endogenous 5(K5), was abolished in [SMRT/NCoR1]ep / mice, but not in − − − − TSLP gene, were significantly decreased compared with GR WT SMRTep / or NCoR1ep / single mutants (Fig. 3C). ChIP assays (Fig. 2I). Similarly, mutations of the phosphorylation sites for using WT dorsal epidermis showed that upon Dex treatment, GR both the c-Jun N-terminal kinase (JNK for S226) and the gly- was associated on IR nGRE sequences with either both SMRT and cogen synthase kinase-3β (GSK-3β for S404) revealed that, at NCoR1 (for K14 and K5) or NCoR1 alone (for STRA13)(Fig.3D). limiting Dex concentration (10 nM), these sites played a major Neither GR binding nor corepressor recruitment was observed in − − role in the stimulation of GR NTD SUMOylation (Fig. 2I). the [SMRT/NCoR1]ep / mouse epidermis, whereas GR binding Accordingly, when using the JNK inhibitor II and a GSK-3β and corepressor substitution was observed in repressing complexes − − inhibitor (LiCl) to depress GR phosphorylation in A549 cells, for the STRA13 IR1 nGRE in NCoR1ep / cells and for the K14 − − ChIP assays indicated that, at 10 nM Dex, these inhibitors de- IR1 nGRE in SMRTep / cells (note in this case the significant creased the binding of GR and SUMOs to the TSLP IR1 nGRE increase in NCoR1 binding). Thus, depending on the identity of the (relative to no addition of inhibitors; Fig. 2J). Most notably, IR nGRE-containing gene, one of the two corepressors (NCoR1 or nGRE-mediated direct repression was not altered by mutation of SMRT) is indispensable for both GR binding to an IR nGRE se- the NTD phosphorylation sites present in GR ABCD that lacks quence and direct transrepresssion. the LBD (Fig. 2K). Histone Deacetylase 3 Is Essential for Dex-Induced IR nGRE-Mediated NCoR1 and/or SMRT Corepressors Are Mandatory for GR Binding to IR Transrepression in Vivo. The association of histone deacetylase 3 nGREs in Vivo. As Lin et al. (16) reported that the DAXX repressor (HDAC3) with SMRT has been previously demonstrated in vitro possesses a SUMO-interacting motif (SIM), we investigated (17), and we reported that HDAC3 is found together with SMRT/ whether DAXX could be involved in IR nGRE-mediated Dex- NCoR1 in complexes bound on IR nGREs in vivo (6) (Fig. 2E). induced repression. Upon cotransfection of GR and DAXX To examine whether HDAC3 was required for Dex-induced IR into Cos-1 cells, ChIP assays did not provide any evidence sup- nGRE-mediated repression, we selectively ablated HDAC3 in porting a DAXX association with IR nGREs present in TSLP and mouse epidermal keratinocytes (Fig. S4G). No Dex-induced re- − − STRA13 endogenous genes, and their RNA transcripts were not pression was observed in HDAC3ep / mice for K14, K5,and affected in A549 cells on DAXX transfection (Fig. S3 F and G). STRA13 IR nGRE-containing genes (Fig. 3E). ChIP assays using However, alignment of the C-terminal AAs sequences of human mouse epidermis showed that the binding of GR, as well as those of and mouse NCoR1 and SMRT with the DAXX SIM revealed a corepressors SMRT/NcoR1 to IR nGREs, was not affected in − − conserved sequence LSDSD/E (Fig. 2L). Upon its deletion, GST- HDAC3ep / mice (Fig. 3F). Interestingly, selective mutations of pull down assays (SI Materials and Methods) showed that NCoR1 NCoR1 and SMRT abolished HDAC3 binding to IR nGREs, in- and SMRT lost their interaction with SUMO1, demonstrating that dicating that this binding is mediated by SMRT/NCoR1 (Fig. 3D). this sequence was instrumental in these interactions (Fig. 2M). As selective knock down of either SMRT or NCoR1 expression The Repressing Complexes Containing the SUMOylated GR and NCoR1/ with siRNA in A549 cells was not fully effective at abolishing SMRT Corepressors Do Not Repress (+)GRE-Mediated Transactivation Dex-induced IR nGRE-mediated transrepression (6), we pre- and Do Not Bind to a (+)GRE. Dex-induced (+)GRE-mediated − − − − pared NCoR1 / and SMRT / MEFs lacking either NCoR1 or transactivation of 3 genes [regulated in development and DNA SMRT expression (Fig. S4A). Transcript analyses of endogenous damage responses 1 (REDD1), MURF and FK506 binding protein IR nGRE-containing genes showed that, for most of them, Dex- 5(FKBP5)] was not increased in vivo in epidermal keratinocytes of − − − − − − induced repression was not affected in NCoR1 / or SMRT / [SMRT/NCoR1]ep / mice (Fig. S4E), and ChIP assays did not MEFs (Fig. 3A and Fig. S4B), but no Dex-induced repression reveal any SMRT/NCoR1 binding on the [(+)GRE]2× DNA − − was observed in NCoR1 / MEFs for the peptidyl-prolyl isom- binding site (DBS) region of the FKBP5 gene (14) (Fig. S4F). − − erase-like 5 (PPIL5) gene and in SMRT / MEFs for upstream Of note, using the mouse SUMOylation mutant GR K310R in 1 (USF1) and receptor (INSR) genes vivo, we did not find, upon Dex treatment, any increase in the (Fig. 3A and Fig. S4B). ChIP assays showed that, upon Dex expression of the single (+)GRE REDD1 gene, nor of that of the treatment, the binding of GR together with those of NCoR1 and/or [(+)GRE]2× DBS FKBP5 gene, whereas the Dex-induced re- SMRT to IR nGRE elements could be detected in WT MEFs pression was alleviated in IR nGRE-containing genes (Fig. 2G). (Fig. 3B). However, these NCoR1 and SMRT bindings were gene ChIP assays demonstrated that GR from dorsal keratinocytes of dependent as (i) in one instance, one of the two corepressors was both WT and GR K310R mutant mice did bind efficiently to the bound in WT MEFs and, in its absence, was replaced by the other [(+)GRE]2× DBS of the FKBP5 gene, but in both cases, no binding one (corepressor preferential binding, as in the case of IL6 and of SUMO1 and SMRT could be detected (Fig. 2H). Accordingly,

E630 | www.pnas.org/cgi/doi/10.1073/pnas.1522821113 Hua et al. Downloaded by guest on October 2, 2021 PNAS PLUS A Mouse embryonic fibroblasts (MEFs) Vehicle Dex Dex+RU 1,2 1 1,2 0,3 1,5 TSLP IL6 STRA13 PPIL5 USF1 1 1,2 0,75 0,9 0,8 0,2 0,9 0,6 0,5 0,6 0,6 0,4 0,1 0,25 0,3 0,2 0,3 0 0 0 0 0 Relative RNA level RNA Relative WT NCoR1-/- SMRT-/- WT NCoR1 -/- SMRT -/- WT NCoR1 -/- SMRT -/- WT NCoR1 -/- SMRT -/- WT NCoR1 -/- SMRT -/- B 0,3 0,15 TSLP (IR1 nGRE) 0,3 STRA13 (IR2 nGRE) USF1 (IR1 nGRE) 0,2 0,2 0,1 SEE COMMENTARY 0,1 0,1 0,05 0 0 0 GR GR GR GR GR GR GR GR GR IgG IgG IgG IgG IgG IgG IgG IgG IgG SMRT SMRT SMRT SMRT SMRT SMRT SMRT SMRT SMRT NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 ChIP Assay Input % WTWT SMRT-/-SMRT-/-NCoR1-/- NCoR1-/- WTWT SMRT-/-SMRT-/-NCoR1-/- NCoR1-/- WTWT SMRT-/-SMRT-/-NCoR1-/- NCoR1 -/-

Mouse epidermis Vehicle Dex Dex+RU C 2 1,2 1,8 D 0,2 STRA13 1 K14 K5 STRA13 (IR nGRE) 1,5 0,8 1,2 1 0,6 0,1 0,5 0,4 0,6 0,2 0 0 0 0 ChIP Assay Input % GR GR GR GR IgG IgG IgG IgG Relative RNA level RNA Relative SMRT SMRT SMRT SMRT NCoR1 NCoR1 NCoR1 NCoR1 HDAC3 HDAC3 HDAC3 HDAC3 WT SMRT ep -/- NCoR1 ep -/- SMRT/NCoR1 ep -/- 0,2 0,15 K14 (IR nGRE) K5 (IR nGRE) 0,1 0,1 0,05

0 0 GR GR GR GR GR GR GR GR IgG IgG IgG IgG IgG IgG IgG IgG SMRT SMRT SMRT SMRT SMRT SMRT SMRT SMRT NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 NCoR1 HDAC3 HDAC3 HDAC3 HDAC3 HDAC3 HDAC3 HDAC3 HDAC3 ChIP Assay Input %

WT SMRT ep -/- NCoR1 ep -/- SMRT/NCoR1 ep -/- WT SMRT ep -/- NCoR1 ep -/- SMRT/NCoR1 ep -/- PHARMACOLOGY 2 E 0,2 WT HDAC3 ep-/- F K14 (+Dex) 0,2 0,25 1,5 K5 (+Dex) STRA13 (+Dex) 0,15 0,15 0,2 1 0,15 WT 0,1 0,1 HDAC3 ep-/- 0,5 0,1 0,05 0,05 0,05 0 0 0 0 Vehicle Dex Vehicle Dex Vehicle Dex Relative RNA level RNA Relative

K14 K5 STRA13 ChIP Assay Input %

Fig. 3. NCoR1 and/or SMRT corepressors and HDAC3 are required for Dex-induced IR nGRE-mediated transrepression in vivo. (A) qRT-PCR for IR nGRE- − − − − containing genes in MEFs derived from WT, NCoR1 / ,orSMRT / mouse embryos, treated with vehicle, Dex (0.5 μM), and RU (3 μM) for 6 h. (B) qPCR analyses − − − − of ChIP assays performed with WT, SMRT / ,orNCoR1 / MEFs, treated with vehicle, Dex (1 μM), and RU (6 μM) for 1 h, showing GR and corepressors bindings − − − − to the indicated IR nGRE regions. (C)AsinA, but using mouse ear epidermis in WT, SMRTep / and/or NCoR1ep / mutant mice. Mouse ears were treated with − − − − vehicle, Dex (6 nmol/cm2), and RU (36 nmol/cm2) for 18 h. (D)AsinB, but using dorsal epidermis of SMRTep / and/or NCoR1ep / mutant mice, treated as in − − − − C for 6 h. (E)AsinC, but in WT and HDAC3ep / mutant mice. (F)AsinD, but in WT and HDAC3ep / mutant mice. Values are mean ± SEM.

upon GR transfection into Cos-1 cells, ChIP assays revealed that that the repressing SUMOylated GR was bound to the IR1 nGRE, activating complexes [GR, steroid receptor coactivator (SRC)2/ but not to the (+)GRE, whereas the activating non-SUMOylated transcriptional mediator/intermediary factor 2 (TIF2), SRC3] GR was bound only to the (+)GRE. Furthermore, using the same were present on (+)GRE regions of endogenous genes (Fig. vector, but containing a [(+)GRE]3× DBS instead of a single (+)GRE S5A), whereas repressing complexes (GR, SUMOs, NCoR1, DBS, ChIP assays did not reveal any decrease of GR, SUMOs, SMRT) were found only on IR nGRE regions of endogenous and NCoR1/SMRT bound to the IR1 nGRE (Fig. 4B). genes (Fig. S5B). To conclusively demonstrate that the inability of the SUMOylated The GR DNA Binding Domain Is Differentially Involved in (+)GRE- GR repressing complex to inhibit (+)GRE-mediated trans- Mediated Transactivation and IR nGRE-Mediated Transrepression. It activation reflects its inefficiency to bind to a (+)GRE, a vector is known that the GR DNA binding domain (DBD) interacts with containing both a single (+)GREandanIR1nGREDBSsepa- (+)GRE and IR nGRE sequences (18, 19), and that deletion of rated by ∼600 bp was transfected in Cos-1 cells (Fig. 4A). GR, the GR DBD (ΔDBD) or either one of the two zinc fingers (ΔZF1/ SUMOs, and NCoR1/SMRT were bound on the IR1 nGRE, ΔZF2) abolished these interactions (Fig. 5A and Fig. S6 A–C). In- whereas non-SUMOylated GR and the coactivators SRC2(TIF2) terestingly, among the three AA residues (K442, V443, and R447) andSRC3wereassembledonthe(+)GRE (Fig. 4B), thus indicating that were reported to directly interact with (+)GRE and IR nGRE

Hua et al. PNAS | Published online December 28, 2015 | E631 Downloaded by guest on October 2, 2021 A pGL3-(+)GRE-IR1 nGRE reporter pGL3-[(+)GRE]3x-IR1 nGRE reporter ~600bp ~600bp

SV40 early Luc SV40 early Luc (+)GRE IR1 nGRE region promoter [(+)GRE]3x IR1 nGRE region promoter region region B 0,4 0,41,3 1,0 pGL3-(+)GRE-IR1 nGRE reporter pGL3-[(+)GRE]3x-IR1 nGRE reporter 0,90,3 (+)GRE region 1,00,3 (+)GRE region

0,2 0,2

0,1 0,1 Vehicle 0 ChIP Assay Input % 0 Dex Dex+RU 0,6 0,60,3 0,3 pGL3-[(+)GRE]3x-IR1 nGRE reporter 0,5 pGL3-(+)GRE-IR1 nGRE reporter 0,5 IR1 nGRE region IR1 nGRE region 0,2 0,2

0,1 0,1

0 0 ChIP Assay Input %

Fig. 4. SUMOylation of the GR did not affect (+)GRE-mediated transactivation. (A) Schematic representation for pGL3 luciferase reporters containing eitherasingle(+)GRE or a [(+)GRE]3× DBS together with an IR nGRE DBS. (B) qPCR analyses of ChIP assays performed with Cos-1 cells transfected with GR and luciferase reporters (as in A), treated with vehicle, Dex (1 μM), and RU (6 μM) for 1 h, showing the binding of GR, cofactors, and SUMOs on indicated regions.

bases in crystal structures (Fig. 5C) (18, 19), we found that only vitro studies and ChIP assays have revealed that GR SUMOylation K442 was indispensable for GR binding to an IR nGRE, whereas allows the formation of a GC-induced NCoR1/SMRT-con- mutation of all three residues affected GR binding to a (+)GRE taining repressing complex, in which SMRT and NCoR1 are (Fig. 5 A and B and Fig. S6 B and C). Interestingly, a triple AA bound to SUMO through their SUMO-interacting motifs (Fig. 2 L mutation (TM) within the GR first into the corre- and M). Most notably, using GR K310R SUMOylation mutant sponding α (ERα) AAs (G439, S440, and V443 mice, we unequivocally establish that this site is essential for effi- into E, G, and A, respectively) (20) abolished its interaction with cient GC-induced IR nGRE-mediated repression in vivo, whereas (+)GRE, but not with IR nGRE (Fig. 5 A and B and Fig. S6 A–C). SUMOylation at K294 (K277 in human) may also contribute, albeit The double mutation G439E/S440G could only partially affect to a lesser extent (Fig. 2 GandH). In keeping with this conclusion, GC-induced (+)GRE-mediated transactivation, and had no effect MEFs expressing selectively the GRα-D3 isoform, which lacks both on GC-induced IR nGRE-mediated repression (Fig. 5 A and B SUMOylation sites, could not assemble a repressing complex and and Fig. S6 A–C). did not exert any GC-induced IR nGRE-mediated repression (Fig. 1 B and C). In this respect, we note that Gross et al. (22) IR nGREs Are Not STAT3 Binding Elements (SBEs). Langlais et al. (21) found that in U2OS cells expressing the GRα-D isoform only, speculated that IR nGREs could be STAT3 binding elements Dex did not repress the expression of the IR nGRE-containing (SBEs). To investigate this possibility, we analyzed in A549 cells Bcl-xL and Survivin/BIRC5 genes (Fig. S6D) (6). Along the same the binding of GR to the SBE and IR nGRE DBSs present in lines, Paakinaho et al. (23) reported that mutation of all three the regulatory region of the suppressor of cytokine signaling 3 GR SUMOylation sites in HEK293 cells prevents GC-induced (SOCS3) gene promoter. Upon Dex treatment, GR, SMRT, and repression of CCND2 and ZIC2 genes that we found to contain NCoR1 corepressors were associated on the SOCS3 IR0 nGRE IR nGREs (Fig. 2G and Fig. S6D). (Fig. 5D, Left), whereas the association of STAT3 to the SOCS3 Importantly, using either mice in which SMRT and/or NCoR1 SBE was observed upon IL6 treatment on its own, and the teth- were selectively ablated in epidermal keratinocytes (Fig. 3 C and D) − − − − ered association of GR and SMRT with STAT3 bound to the SBE or SMRT / and NCoR1 / MEFs (Fig. 3 A and B), we demonstrate site was detected only after further Dex cotreatment (Fig. 5D, Right). that both SMRT and NCoR1 are instrumental in IR nGRE- Thus, the STAT3 binding element and the IR nGRE are distinct mediated repression in vivo. Intriguingly, depending on the expressed DBSs, which are selectively and independently bound by STAT3 and gene identity, the repressing complex contains both SMRT and the GR, respectively. NCoR1 repressors or only one of them, and in the latter case, its mutation could lead or not to its replacement by the other one (Fig. Discussion 3 B and D). Moreover, the presence of NCoR1 in the repressing SUMOylation of the GR NTD and the Subsequent Formation of an NCoR1/ complex requires an interaction with K579 in the LBD (Fig. 1I). SMRT/HDAC3 Repressing Complex Is Mandatory for GC-Induced IR Whether SMRT may also interact with the LBD is unknown, but it nGRE-Mediated Direct Transrepression, but Does Not Affect (+)GRE- is noteworthy that an active repressing complex containing SMRT, Mediated Transactivation. We demonstrate here that SUMOyla- butnotNCoR1,canbeassembledontheLBD-deletedGRABCD tion of human GR at NTD position K293 (K310 in the mouse) is (Fig. 1 G–I). Structural studies on purified repressing complexes are mandatory for IR nGRE-mediated transrepression (Fig. 2 A–D). In required to establish whether SMRT could similarly bind to the GR

E632 | www.pnas.org/cgi/doi/10.1073/pnas.1522821113 Hua et al. Downloaded by guest on October 2, 2021 PNAS PLUS A SGK1 [(+)GRE] TSLP (IR1 nGRE) 100% C 80% Arg447 Lys442 60% 40% 5’ - CGCCTCCGGGAGAGCT - 3’ 20% transcripts 3’ - GCGGAGGCCCTCTCGA - 5’ Relative RNA RNA Relative 0% GR GR GR TM GR GR GR GR GR DBD (G439E G439E K442A V443A K442A R447A Val443 ΔZF1/ΔZF2 S440G S440G V443A TSLP IR1 nGRE V443A)

B Relative (+)GRE-mediated transactivation of SGK1 gene

Relative IR nGRE-mediated transrepression of STRA13 gene Lys442 Arg447 Val443 SEE COMMENTARY 100% 80% 5’ – NAGAACANNNTGTTCTN - 3’ 60% 3’ – NTCTTGTNNNACAAGAN - 5’ 40% 20% ChIP Assays

Relative binding 0% Val443 Arg447 Lys442 GR GR GR TM GR GR GR GR GR DBD (G439E G439E K442A V443A K442A R447A (+)GRE consensus ΔZF1/ΔZF2 S440G S440G V443A V443A)

D 0,5 0,4 SOCS3 (IR0 nGRE) SOCS3 (SBE) 0,4 0,3 IR0 nGRE: 5’- CTCCGGAG -968 SBE: 5’- TTCCAGGAA -56 Vehicle 0,3 0,2 Dex 0,2 IL6 0,1 0,1 IL6+Dex 0 0 ChIP Assay Input %

Fig. 5. The GR DBD is differently involved in (+)GRE-mediated transactivation and IR nGRE-mediated transrepression. (A) Relative RNA transcripts (nor- malized to GR full length, taken as 100%) for GR mutants as indicated (Fig. S6 A and B). (B) As in A, but for the binding of GR mutants to DBS of indicated genes (Fig. S6 A and C). (C) Representation of interactions between GR DBD AAs and TSLP IR1 nGRE bases and between GR DBD AAs and consensus (+)GRE bases. (D) qPCR analyses of ChIP assays performed with A549 cells, treated with vehicle, IL6 (10 ng/mL), and Dex (1 μM) for 1 h, showing the binding of GR, STAT3, and corepressors to the IR0 nGRE and SBE of the SOCS3 gene. Values are mean ± SEM.

LBD,andalsotoelucidatehowtheassociationofSMRT/NCoR1 assembled on the IR nGRE DBS, whereas only activating com- results in a stable GR binding to IR nGREs, in marked contrast plexes were associated with the (+)GRE DBS (Fig. 4). We con- with the weak in vitro interaction found between the isolated GR clude that the SUMOylated GR cannot assemble a repressing PHARMACOLOGY DBD and an IR nGRE DBS (19). Most notably, the latter study complex on a (+)GRE. revealed an interaction between GR DBD K442 and the TSLP IR1 nGRE (Fig. 5C), which is remarkably in keeping with our own data Two Main GC-Regulated Functions Are Exerted by a Unique GR Under showing that, in transfected cells, mutation of GR K442 prevents the Control of a Single GC Ligand. As the LBD-truncated GR ABCD the GC-induced interaction between the GR and the TSLP IR1 is a constitutive activator of transcription, it was proposed that nGRE (Fig. 5B). Interestingly, the GR DBD is not specifically for GR binding to a (+)GRE, the only requirement is a GC- required for Dex-induced direct repression, because Gal4 trans- dependent conformational modification of the LBD, which on its activation was repressed when the GR NTD was fused to the yeast own unmasks the DBD (8). Similarly, our present results indicated Gal4 DBD (1-147) (Fig. 1 D and E), indicating that the GR NTD that the binding of GR to an IR nGRE requires a GC ligand, on its own could assemble a repressing complex. which, by binding to the LBD, allows the unmasking of not only Of note, and as expected (24), the binding of HDAC3 to IR the DBD, but also of the NTD SUMOylation site and, conse- nGREs in vivo is mediated through interaction with SMRT/NCoR1 quently, the formation of a SMRT/NCoR1-repressing complex on (Fig. 3D). Using mice in which HDAC3 was selectively ablated in the IR nGRE. In this respect, note that the GR ABCD on its own, epidermal keratinocytes, we demonstrated that it is essential for but not its SUMOylation mutant GR ABCD K293R, is readily Dex-induced IR nGRE-mediated repression; however, its ablation SUMOylated to assemble a repressing complex, which efficiently did not affect the binding of GR, nor that of NCoR1/SMRT on binds to an IR nGRE to repress transcription (Fig. 2 B and C). IR nGREs (Fig. 3 E and F). Our results also indicated that al- Note also that under similar conditions, the (+)GRE-mediated though HDAC2 could be found in repressing complexes bound activation of transcription by GR ABCD is unaffected by the on the IR nGRE regions, it was not mandatary for repression K293R mutation (Fig. S3 C–E). (Fig. 3 E and F). Clearly, in addition to its well-established role in the trans- Previous transfection studies have suggested that SUMOylated location of unliganded GR from the cytoplasm to the nucleus, nuclear receptors, including GR, could control negatively the binding of a GC to the GR appears to unveil the DBDs used the expression of luciferase reporter genes containing multiple, for binding to (+)GRE and IR nGRE DBSs. In the case of but not single DNA binding element (14, 25, 26). However, we (+)GREs, this unmasking allows on its own an efficient binding of found that the (+)GRE-mediated transactivation of genes con- the GR DBD to a (+)GRE, which appears to be incompatible taining 1 or 2 (+)GRE DBS was not increased in vivo in epidermal with SUMOylation of the NTD. In contrast, the much weaker − − keratinocytes of [NCoR1/SMRT]ep / mice compared with WT binding of the GR DBD to an IR nGRE (19) requires the sub- mice (Fig. S4 E and F). Similar in vivo results were obtained using sequent SUMOylation of the NTD and the subsequent forma- GR K310R SUMOylation mouse mutants (Fig. 2 G and H). Fur- tion of a repressing SMRT/NCoR1 complex, to stably anchor the thermore, upon transfecting Cos-1 cells with a vector containing GR on the IR nGRE. Interestingly, our results indicate that such both a (+)GRE DBS and an IR1 nGRE DBS separated by 600 bp, unmaskings of the NTD SUMOylation site could involve not ChIP assays showed that repressing complexes were selectively only a ligand-dependent conformational modification of the

Hua et al. PNAS | Published online December 28, 2015 | E633 Downloaded by guest on October 2, 2021 LBD, which is prevented by RU486, but also the phosphoryla- upon weak, but selective, binding of the GR to IR nGREs, which tion of sites located in the NTD. Indeed, upon mutation of five would be in keeping with the observation that only a small fraction such phosphorylation sites, as well as upon addition of selective of the cellular GR is actually SUMOylated (Fig. S3B). inhibitors of two cognate kinases (JNK and GSK-3β), ChIP assays have revealed that, under limiting Dex concentration (10 nM), there Materials and Methods was a decrease in the binding of SUMOylated GR on an IR nGRE Mice. GRα-D3, GR K310R, NCoR1, SMRT,andHDAC3 mutant mice are de- (Fig. 2 I and J). Importantly, the loss of NTD phosphorylation did scribed in SI Materials and Methods. Breeding, maintenance, and experimental not affect the binding of SUMOylated GR ABCD on the IR nGRE manipulation of mice were approved by the Animal Care and Use Committee (Fig. 2K). It appears therefore that SUMOylation of the GR of the Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)/ NTD could be controlled, not only by GCs, but also by site- Institut Clinique de la Souris (ICS). specific phosphorylation mediated by protein kinases, which, The ChIP assay, luciferase assay, siRNA treatment, RNA isolation, and quan- interestingly, have been implicated in inflammation and ho- titative PCR (qPCR) analyses were performed as in ref. 6. Primers used are meostasis (27, 28). available on request. In conclusion, for two of the three GR main functions (trans- activation and direct transrepression), there is an initial common Statistics. Data are represented as mean ± SEM of at least three independent step consisting in the binding of a GC ligand, which in both cases, experiments and were analyzed by Microsoft Excel statistics software using due to a GC-induced conformational modification of the LBD, the Student t test. P < 0.05 was considered significant. leads to the unmasking of the NTD/DBD (ABCD) domains. + ACKNOWLEDGMENTS. We thank the staff of the animal and cell culture Subsequently, the unveiled GR may bind efficiently to a ( )GRE facilities of the Institut de Génétique et de Biologie Moléculaire et Cel- and loosely to an IR nGRE (19). It is then and only then that, in lulaire (IGBMC)/Institut Clinique de la Souris (ICS) for excellent help and the latter case, SUMOylation and assembly of a repressing complex Marie-France Champy (ICS/Institut Clinique de la Souris) for blood anal- may occur, thereby strengthening the binding of the GR to an IR yses. Floxed HDAC3 (HDAC3fl/fl) mice was a gift from Prof. Mitchell A. Lazar. This work was supported by the CNRS, the INSERM, the University nGRE DBS. It is noteworthy that with such operational mecha- of Strasbourg Institute for Advanced Studies, and the Association pour nisms, there might be no need for the existence of a predestined laRechercheàl’IGBMC (ARI). G.H. was supported by a long-term ARI nuclear pool of SUMOylated GR, as SUMOylation may occur only fellowship.

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