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FEBS Letters 588 (2014) 4513–4519

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The GFI1 negatively regulates NLRP3 inflammasome activation in macrophages ⇑ Liuluan Zhu a,b,1, Qingcai Meng a,b,1, Shuntao Liang a,b, Yaluan Ma c, Rui Li a,b, Guoli Li a,b, Hui Zeng a,b,

a Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China b Beijing Key Laboratory of Emerging Infectious Diseases, Beijing, China c Institute of Basic Medical Theory of Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, China

article info abstract

Article history: Interleukin-1b (IL-1b) secretion downstream of Toll-like (TLR) activation is tightly con- Received 5 August 2014 trolled at the transcriptional and post-translational levels. NLRP3 inflammasome is involved in Revised 13 October 2014 the maturation of pro-IL-1b, with NLRP3 expression identified as the limiting factor for inflamma- Accepted 22 October 2014 some activation. Previously, we had demonstrated that the zinc-finger protein GFI1 inhibits Available online 4 November 2014 pro-IL-1b transcription. Here, we show that GFI1 inhibits NLRP3 inflammasome activation and Edited by Michael R. Bubb IL-1b secretion in macrophages. GFI1 suppressed Nlrp3 transcription via two mechanisms: (1) by binding to the Gli-responsive element 1 (GRE1) in the Nlrp3 promoter; and (2) by antagonizing the nuclear factor- B (NF- B) transcriptional activity. Thus, GFI1 negatively regulates TLR-mediated Keywords: j j Gfi1 IL-1b production at both transcriptional and post-translational levels. NLRP3 Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Inflammasome IL-1b Macrophage

1. Introduction which, in turn, proteolytically cleaves pro-IL-1b to release mature IL-1b [11–14]. Innate immune cells initiate an immediate response to Among the NLR family members, NLRP3 is the most sensitive pathogens by generating abundant pro-inflammatory cytokines, receptor that can be activated by a broad range of pathogen- including interleukin-1b (IL-1b), which exerts a wide range of associated molecular patterns [13,15,16]. The NLRP3 inflamma- inflammatory and antimicrobial activities [1–3]. Production of some is particularly important for IL-1b production. Although IL-1b is under tight regulation and requires a two-step process: several stimuli effectively trigger NLRP3 [17,18], one established (1) Toll-like receptor (TLR) and NF-jB signaling-dependent mechanism for regulating its expression involves the TLR/MyD88 transcription and translation resulting in an inactive precursor, pathway, which is tightly controlled by signals culminating into pro-IL-1b, and (2) inflammasome-mediated cleavage of pro-IL-1b activation of NF-jB [19,20]. into mature biologically active IL-1b [4,5]. Since NLRP3 expression is a critical checkpoint for inflamma- Inflammasome is a multiprotein complex critical for IL-1b mat- some activation [19,21], negative regulatory mechanisms that sup- uration and release [6,7]. The complex is generally composed of press NLRP3 are necessary to control excessive IL-1b production pro-caspase-1, adaptor protein apoptosis-associated speck-like during sepsis [22,23]. Recently, miR-223 was shown to suppress protein (ASC) and one of the NOD-like receptor (NLR) family mem- NLRP3 expression. However, since miR-223 itself is not regulated bers, such as NLRP1 (NOD-like receptor containing pyrin 1), NLRP3 by pro-inflammatory signals, it may act like a rheostat to tightly and NLRC4 (NOD-like receptor containing CARD) [8–10]. Upon control the NLRP3 message for basal protection [24]. stimulation with a myriad of microbial and endogenous signals, Growth factor independence 1 (Gfi1) encodes a 55 kDa nuclear NLR is activated to assemble the inflammasome and sequentially zinc finger transcription factor with an N-terminal SNAG domain primes auto-catalysis of pro-caspase-1 into active caspase-1, and a six C-terminal zinc finger motifs linked by an intermediary region [25,26]. Gfi1 acts as a transcriptional by binding to a DNA recognition sequence in target promoters [26–28]. ⇑ Corresponding author at: Institute of Infectious Diseases, Beijing Ditan Hospital, Transcriptional repression activity of Gfi1 is dependent on the zinc Capital Medical University, No. 8, Jing Shun East Street, Beijing 100015, China. Fax: +86 10 84322606. finger motifs required for DNA binding as well as the intact E-mail address: [email protected] (H. Zeng). N-terminal SNAG domain responsible for nuclear localization 1 These authors contributed equally to this work. [26,28]. In addition, Gfi1 harbors functions independent of its

http://dx.doi.org/10.1016/j.febslet.2014.10.025 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 4514 L. Zhu et al. / FEBS Letters 588 (2014) 4513–4519 activity as a transcriptional regulator. Gfi1 is reported to interact treatment with 5 mM ATP (Sigma–Aldrich) for a further 45 min. with the STAT3 (signal transducer and activator of transcription Supernatant fractions were collected for ELISA, and the cells lysed 3) inhibitor PIAS3 (protein inhibitor of activated STAT 3) and the for RNA and protein isolation. p65 subunit of NF-jB via its intermediary region [29,30]. Earlier studies revealed that Gfi1-deficient mice exhibit defects 2.4. Cell culture, transfection and dual-luciferase reporter assay in development of hematopoietic and immune systems as well as excessive endotoxin-initiated innate immune responses [31–35]. 293ET cells were cultured in DMEM with 10% FBS and 1% pen- Gfi1-deficient mice are highly susceptible to lipopolysaccharide icillin–streptomycin. For the luciferase reporter assay, 5 104 cells (LPS)-induced septic shock due to overproduction of pro- were cultured on a 24-well plate and subsequently transfected inflammatory cytokines [31]. Recently, Gfi1 was shown to inhibit with a mixture of the indicated expression plasmids, promoter fire- TLR-mediated production of TNF-a through binding p65 of NF-jB fly luciferase reporters and Renilla luciferase plasmid (pRL-TK) and consequently interfering with p65 binding to target gene pro- using VigoFect reagent (Vigorous Biotechnology). After 24 h, lucif- moter DNA [30]. In addition, the finding that Gfi1-deficient macro- erase assays were performed using a dual-Luciferase Reporter phages display higher levels of IL-1b mRNA than their wild-type Assay System (Promega), according to the manufacturer’s instruc- counterparts after LPS stimulation suggests that Gfi1 negatively tions. Relative luciferase activity was calculated by normalizing the regulates IL-1b production at the transcriptional level [30]. ratio of firefly/Renilla luciferase. In the present study, we investigated whether Gfi1 regulates TLR-mediated production of IL-1b at the post-transcriptional 2.5. Electrophoretic mobility shift assay (EMSA) stages. The promoter sequence of Nlrp3 contains two NF-jB bind- ing elements [20]. Screening of the promoter sequence revealed In total, 2 106 293ET cells were cultured in a 60 mm dish and two putative Gfi1 binding sites containing the consensus motif transfected with 5 lg Gfi1 expression plasmids or the vehicle con- AATC. Our data suggest that Gfi1 inhibits NLRP3 expression via trol for 48 h. WT and Gfi1-deficient BMDMs were stimulated with two different pathways, either by binding directly to the promoter 100 ng/ml LPS for 4 h before nuclear protein extraction. Nuclear cis-element or antagonizing NF-jB p65 transcriptional activity, extracts were prepared from 293ET cells or BMDMs using the leading to subsequent effects on IL-1b maturation. NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce). EMSA was conducted with Light Shift Chemiluminescent EMSA Kit 2. Materials and methods (Pierce) using the manufacturer’s protocol. Oligonucleotide probes were labeled with biotin. The sequences of the GRE1 and NRE1 2.1. Mice probes were 50-CAGATGAGTAACTGCCAATCCGTCTTTGACAGTG-30 and 50-AGGCTTCTGCAGGGAACCCCCGGGAATTCT-30, respectively. Gfi1/ mice were kindly provided by Professor Tarik Möröy In competitive binding assays, unlabeled oligonucleotides were (Institut de recherches cliniques de Montréal, Canada). Mice were added at 200-fold molar excess. For supershift assay, 3 lg housed under specific pathogen-free conditions at the Institute of anti-Gfi1 antibody (Santa Cruz) was added. Laboratory Animal Science, Chinese Academy of Medical Sciences, and Peking Union Medical College. Mouse treatments were 2.6. Chromatin immunoprecipitation assay and PCR (ChIP–PCR) approved by the local animal research committee at the Institute of Laboratory Animal Sciences. BMDMs were stimulated with 100 ng/ml LPS for 1 h and ChIP– PCR assay performed as described previously [36]. Input samples 2.2. Plasmids corresponded to 0.5% (v/v) of the total solution before immunopre- cipitation. Purified chromatin was immunoprecipitated with 4 lg WT and NRE mutant Nlrp3 promoters were a kind gift from anti-Gfi1 antibody (Santa Cruz) or IgG. After DNA purification, Professor Chengjiang Gao, including the full-length Nlrp3 promoter the presence of specific DNA sequences was assessed using PCR. (nt 3033/+116), functional promoter (nt 1434/1113), mut- The primers used were 50-CTCCACACACTGGTTCTGCTTG-30 (for- NRE1 (nt 1434/1113, referred to as mutA in their report), mut- ward) and 50-AGCAGCCTTGGGTACAAATAAAG-30 (reverse) for the NRE2 (nt 1434/1113, referred to as mutB) and mutNRE1/2 (nt GRE1 element (proximal region), and 50-TTCTTCAAGGTCATTA 1434/1113, referred to as mutAB) [20]. The mutGRE1, mutGRE2 GTGGCTTC-30 (forward) and 50-AGTGCTAAAGGCCTAAGCAGATC-30 and mutGRE1/2 promoters were constructed by mutation of GRE1 (reverse) for the negative control (distal region). The intensities and/or GRE2 sequence based on the full-length Nlrp3 promoter (nt of bands were quantified with Image J software (National Institutes 3033/+116). The mutNRE1/GRE1 promoter (nt 1434/1113) of Health, Maryland, USA). was generated by mutation of GRE1 (AATC to GGAG) based on the mutNRE1 promoter using the GBclonart Seamless Cloning 2.7. Real-time PCR and Assembly Kit (Genebank Biosciences Inc). All promoters were cloned into the pGL3-basic reporter vector. NF-jB p65 and various Total RNA was isolated with TRIzol reagent (Life Technology) Gfi1 expression plasmids were kindly provided by Professor Tarik according to the manufacturer’s instructions. RNA (2 lg) was Möröy, including Gfi1-FL (full-length), DSNAG (SNAG domain dele- reverse-transcribed to cDNA using the High Capacity cDNA Reverse tion), DIM (intermediate region deletion) and DZF (zinc finger Transcription kit (Life Technology), and specific transcripts quanti- Ò motif deletion). All plasmids were confirmed by DNA sequencing. fied using the Applied Biosystems 7500 Real-Time PCR System (Life Technology). Quantitative real-time PCR was performed using Ò Ò 2.3. Stimulation of bone marrow-derived macrophages TaqMan Master Mix and TaqMan probes according to the manufacturer’s instructions (Life Technology). Bone marrow-derived macrophages (BMDM) were differenti- ated from bone marrow cells of 6- to 8-week-old C57BL/6 2.8. Western blot Gfi1-deficient mice and their wild-type (WT) littermates, as described previously [30]. Unless otherwise indicated, cells were Whole cell extracts (20 lg) were used for Western blot analysis primed for 4 h with 100 ng/ml LPS (Sigma–Aldrich) before with primary antibodies against IL-1b (Abcam), NLRP3 (AdipoGen), L. Zhu et al. / FEBS Letters 588 (2014) 4513–4519 4515

Fig. 1. Gfi1 inhibits NLRP3 inflammasome activity and NLRP3 expression in macrophages. WT and Gfi1/ macrophages were primed with 100 ng/ml LPS for 4 h and stimulated with or without 5 mM ATP for 45 min. (A) IL-1b secretion in supernatants was measured using ELISA. (B) Whole-cell proteins were subjected to Western blot for pro-IL-1b, mature IL-1b, pro-caspase-1, cleaved caspase-1 (p10), ASC and NLRP3. b-Actin was detected as the loading control. (C) The mRNA levels of Nlrp3, Nlrp1a and Asc were quantified with real-time PCR. Values were normalized to those of Gadph and expressed relative to WT control cells (Ctrl). Representative results from three independent experiments are shown. ⁄P < 0.05; ⁄⁄⁄P < 0.001.

NF-jB p65 (CST), Flag (Sigma), caspase-1, ASC, Gfi1 and b-Actin increase in IL-1b production in culture medium than WT BMDMs (Santa Cruz). in response to LPS stimulation alone, and a more dramatic release of IL-1b when stimulated with LPS and ATP (Fig. 1A). Moreover, 2.9. Measurement of cytokine production Western blot analysis revealed a significant increase in mature IL-1b in Gfi1-deficient BMDMs (Fig. 1B and Suppl. 1). The data col- BMDMs were seeded in 96-well plates and cultured overnight. lectively suggest that Gfi1 inhibits maturation of IL-1b. After priming with 100 ng/ml LPS for 4 h and stimulating with 5 mM ATP for another 45 min, supernatant fractions were collected 3.2. Gfi1 inhibits inflammasome activation and NLRP3 expression and IL-1b concentrations measured using the mouse IL-1b ELISA set (BD Biosciences Pharmingen), according to the manufacturer’s Since caspase-1 activity is essential for cleavage of pro-IL-1b instructions. into the mature form, we assessed cleavage of caspase-1 upon LPS/ATP stimulation in WT and Gfi1-deficient macrophages. 2.10. Statistical analysis Compared with WT, Gfi1/ BMDMs exhibited increased levels of caspase-1 after LPS/ATP stimulation, indicating elevated auto- Data are presented as means ± S.D. of three independent exper- catalysis of pro-caspase-1. Consistent with enhanced caspase-1 iments. Statistical differences were determined with the Student’s cleavage, we detected higher levels of NLRP3, but unaltered ASC t-test, and P values < 0.05 considered statistically significant. expression in Gfi1/ BMDMs (Fig. 1B). Real-time PCR revealed increased Nlrp3 mRNA levels in Gfi1-deficient macrophages 3. Results and discussion (Fig. 1C), implying that Gfi1 affects expression of Nlrp3 at the tran- scriptional level. 3.1. Secretion of IL-1b is increased in Gfi1-deficient macrophages 3.3. Gfi1 inhibits Nlrp3 transcription through direct binding to Our previous experiments disclosed significantly higher levels promoter DNA of IL-1b in sera of Gfi1-deficient mice treated with LPS [31], indi- cating that Gfi1 inhibits cytokine production upon endotoxin chal- In view of the finding that Gfi1 represses target by bind- lenge. To confirm the potential regulatory activity of Gfi1 on IL-1b ing the response element in the promoter region, we further exam- production, WT and Gfi1/ BMDMs were primed with LPS and ined whether Gfi1 acts as a transcriptional regulator of Nlrp3 subsequently stimulated with ATP. Consistent with previous through binding to its promoter DNA. 293ET cells were transfected in vivo findings [31], Gfi1-deficient BMDMs exhibited a slight with the mouse Nlrp3 promoter reporter, Gfi1 and activator NF-jB 4516 L. Zhu et al. / FEBS Letters 588 (2014) 4513–4519

Fig. 2. Gfi1 inhibits Nlrp3 transcription by binding the GRE1 element in the promoter. (A) 293ET cells were transfected with 3 ng pRL-TK, 150 ng full-length Nlrp3 promoter reporter (nt 3033/+116) or pGL3-basic vehicle vector, 300 ng Gfi1 expression plasmid or pcDNA3.1 and 150 ng p65 expression plasmid for 24 h. Luciferase activities were measured and normalized to that of Renilla luciferase. (B) Alignment of the Gfi1 binding site consensus with the two potential GRE sites. The core sequence is in uppercase. Schematic of GRE1 and GRE2 location within the Nlrp3 promoter. (C) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 plasmid, 300 ng Gfi1 expression plasmid or pcDNA3.1 and 150 ng full-length Nlrp3 promoter or the mutants (nt 3033/+116) for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. (D) Nuclear extracts were prepared with Gfi1-transfected 293ET cells (left) and LPS-stimulated BMDMs (right). EMSA was performed with biotin-labeled Nlrp3 oligonucleotides containing the Gfi1 binding site, GRE1. In competitive binding assays, 200-fold molar excess cold probe was added. The supershift bands indicate the complexes of Gfi1 antibody, Gfi1 protein and GRE1 element. (E) ChIP–PCR analysis of the promoter region of GRE1. BMDMs were stimulated with 100 ng/ml LPS for 1 h, protein–DNA complexes were immunoprecipitated with anti-Gfi1 or control IgG antibody. The 1434/1113 region harboring GRE1 element was amplified by PCR assay. An irrelevant distal region (nt 2269/2067) was amplified as the negative control. The relative intensities of the PCR bands were quantified and normalized to input control. ⁄P < 0.05; ⁄⁄⁄P < 0.001. p65 expression plasmids. As expected, the dual luciferase report To further establish the effects of binding between GRE1 and assay revealed significant suppression of Nlrp3 transcription by Gfi1, EMSA was performed with 293ET nuclear extracts and a Gfi1 (Fig. 2A). Screening of the Nlrp3 promoter sequence further biotin-labeled probe containing GRE1. Compared with the Gfi1- disclosed two putative binding sites for Gfi1 located at positions deficient nuclear protein (lane 2 in the left panel and lane 3 in 1210/1207 and 408/405 of the transcription start site. To the right panel), a shift band was observed in the Gfi1-expressing validate the functions of these two putative binding sites, mutant nuclear protein (lane 3 in the left panel and lane 2 in the right reporter promoters (mutGRE1, mutGRE2 and mutGRE1/2) were panel). The protein–DNA complex formation was remarkably constructed and compared with the Gfi1 consensus binding site reduced with the unlabeled probe competition (Lane 4). The bind- [27], as depicted in Fig. 2B. Notably, mutation of GRE1 reversed ing specificity was further verified by the supershift band with the inhibitory effect of Gfi1 while mutation of GRE2 had no influ- anti-Gfi1 antibody (Lane 5). Consistent with this finding, a binding ence on Nlrp3 transcription (Fig. 2C). shift was observed with nuclear extracts from BMDMs (Fig. 2D). L. Zhu et al. / FEBS Letters 588 (2014) 4513–4519 4517

Fig. 3. Gfi1 suppresses Nlrp3 transcription by antagonizing the binding of NF-jB p65 and NRE1 elements. (A) Schematic of NRE1 and NRE2 positions in the Nlrp3 promoter. (B) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 expression plasmid or pcDNA3.1 and 150 ng functional Nlrp3 promoter mutants (nt 1434/1113) for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. (C) 293ET cells were transfected with p65 and Gfi1 plasmids, and nuclear extracts identified via Western blot. EMSA was performed with biotin-labeled Nlrp3 oligonucleotides containing the NF-jB binding site, NRE1. In competitive binding assays, 200-fold molar excess cold probe was added. ⁄⁄⁄P < 0.001.

Fig. 4. Functions of different domains in Gfi1. (A) Schematic diagram depicts the deletion of various regions in the Gfi1 protein. All of the mutants were recombinant with a Flag-tag, and the expressions of them were detected by Western blot. (B–D) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 expression plasmid, 300 ng full-length or mutant Gfi1 expression plasmids and 150 ng WT Nlrp3 promoter (nt 3033/+116) (B), 150 ng mutNRE1 promoter (nt 1434/1113) (C) or 150 ng mutGRE1 promoter (nt 3033/+116) (D) for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.

These results suggest a combination of Gfi1 and GRE1 elements. in vivo and in vitro, and support direct transcriptional repression The ChIP–PCR assay was additionally performed to determine of Nlrp3 by Gfi1. whether Gfi1 occupies the endogenous promoter site at GRE1. The Nlrp3 promoter sequence containing GRE1 (proximal region) 3.4. Gfi1 inhibits Nlrp3 transcription indirectly by interfering with NF- was successfully amplified from precipitates with the anti-Gfi1 jB p65 activity antibody, but not the IgG control sample. For the negative control, the distal promoter region without GRE1 was not amplified from As specified earlier, mutation of both GRE1 and GRE2 did not any of the immunoprecipitates (Fig. 2E). Our results collectively counteract inhibition by Gfi1 completely (Fig. 2C), signifying the validate interactions between Gfi1 and GRE1 elements, both existence of other regulatory mechanisms. Accordingly, we further 4518 L. Zhu et al. / FEBS Letters 588 (2014) 4513–4519

mutation of NRE1 almost completely suppressed activation of NF- jB in the Nlrp3 promoter, mutation of NRE2 had no effect, and mutation of both NRE1 and 2 induced a similar level of inhibition as mutNRE1 (Fig. 3A and B). Our data indicate that NF-jB interacts specifically with NRE1 for activation in this system. To determine whether Gfi1 interferes with NF-jB binding to NRE1, EMSA was performed with nuclear NF-jB p65 and Gfi1. As shown in Fig. 3C, NF-jB p65 strongly interacted with the labeled probe (lane 2). This binding was competed by Gfi1 (lane 3) and excess cold probes (lane 4), suggesting that Gfi1 interferes with NF-jB p65 binding to the Nlrp3 promoter.

3.5. SNAG domain and zinc-finger motif of Gfi1are indispensable for regulation of Nlrp3

To clarify the specific domains that function in regulation of Nlrp3 transcription, a series of Gfi1 mutants (depicted in Fig. 4A) were adopted for the dual luciferase reporter assay. Significantly, all three domains were involved in the regulation of Nlrp3 tran- scription (Fig. 4B). With the mutNRE1 promoter containing only the GRE1 element, suppression by Gfi1 was abolished upon muta- tion of either SNAG (Gfi1 DSNAG) or zinc finger (Gfi1 DZF) domains, suggesting that intact SNAG domain and zinc finger

Fig. 5. Both GRE1 and NRE1 elements are involved in negative regulation by Gfi1. motifs are indispensable for GRE1-mediated regulation (Fig. 4C). (A) A map of GRE1/NRE1 mutations in the Nlrp3 promoter region. (B) 293ET cells In contrast, with the mutGRE1 promoter containing only NRE1, were transfected with 3 ng pRL-TK, 150 ng p65 plasmid, 300 ng Gfi1 plasmid or the inhibitory effect of Gfi1 was counteracted by deletion of the pcDNA3.1 and 150 ng Nlrp3 promoter mutants for 24 h. Luciferase activity was intermediate region (Gfi1 DIM), indicating that this region is nec- ⁄ measured and normalized to that of Renilla luciferase. P < 0.05. essary for NF-jB-dependent inhibition (Fig. 4D). investigated whether Gfi1 modulates Nlrp3 transcription by inter- 3.6. Gfi1 regulates the NLRP3 inflammasome via a ‘‘dual repression fering with NF-jB p65. Earlier, Qiao et al. identified two NF-jB model’’ mechanism binding sites in the Nlrp3 promoter [20]. To avoid endogenous influence, the dual luciferase reporter assay was performed with Next, we constructed an Nlrp3 promoter reporter with a NRE1/ 293ET cells instead of macrophages. The Nlrp3 promoter was GRE1 double mutant (Fig. 5A). As shown in Fig. 5B, upon mutation transfected along with NF-jB binding site mutants (mutNRE1, of NRE1 and GRE1 separately, Gfi1 suppressed NF-jB-mediated mutNRE2 and mutNRE1/2) into 293ET cells. Surprisingly, although transcription of Nlrp3. Notably, maximal inhibition was attained

Fig. 6. Model for regulation of the NLRP3 inflammasome in macrophages. (A) LPS/ATP induce robust activation of the NLRP3 inflammasome. The inflammatory response may be unregulated in the absence of Gfi1. (B) Gfi1 plays an essential role in controlling NLRP3 expression and inflammasome activation by binding the GRE1 element directly and blocking the transcriptional activity of NF-jB p65. L. Zhu et al. / FEBS Letters 588 (2014) 4513–4519 4519 upon mutation of both NRE1 and GRE1 elements. Our results col- increased activity in Muckle–Wells autoinflammatory disorder. Immunity 20, lectively imply that Gfi1 inhibits Nlrp3 transcription through dual 319–325. [7] Dinarello, C.A. (2004) Unraveling the NALP-3/IL-1beta inflammasome: a big mechanisms: (1) direct binding of promoter DNA and (2) antago- lesson from a small mutation. Immunity 20, 243–244. nizing NF-jB transcriptional activity. Upon mutation of the NRE1 [8] Schroder, K. and Tschopp, J. (2010) The inflammasomes. 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(1996) Gfi-1 encodes a nuclear zinc finger protein that binds DNA and functions as a We thank Professor Chengjiang Gao at Key Laboratory for transcriptional repressor. Mol. Cell. Biol. 16, 4024–4034. [27] Duan, Z. and Horwitz, M. (2003) Targets of the transcriptional repressor Experimental Teratology of the Ministry of Education, Shandong oncoprotein Gfi-1. Proc. Natl. Acad. Sci. U.S.A. 100, 5932–5937. University Medical School, Jinan, Shandong, China, for providing [28] Grimes, H.L., Chan, T.O., Zweidler-McKay, P.A., Tong, B. and Tsichlis, P.N. Nlrp3 promoter reporter genes. We are additionally grateful to Pro- (1996) The Gfi-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal. fessor Tarik Möröy at Institut de recherches cliniques de Montréal, Mol. Cell. Biol. 16, 6263–6272. Canada, for p65 and Gfi1 expression plasmids, Gfi1/ mice, and [29] Rodel, B. et al. (2000) The zinc finger protein Gfi-1 can enhance STAT3 critical comments on the manuscript. signaling by interacting with the STAT3 inhibitor PIAS3. EMBO J. 19, 5845– 5855. [30] Sharif-Askari, E. et al. (2010) Zinc finger protein Gfi1 controls the endotoxin- Appendix A. Supplementary data mediated Toll-like receptor inflammatory response by antagonizing NF- kappaB p65. Mol. Cell. Biol. 30, 3929–3942. [31] Karsunky, H., Zeng, H., Schmidt, T., Zevnik, B., Kluge, R., Schmid, K.W., Duhrsen, Supplementary data associated with this article can be found, U. and Moroy, T. (2002) Inflammatory reactions and severe neutropenia in in the online version, at http://dx.doi.org/10.1016/j.febslet.2014. mice lacking the transcriptional repressor Gfi1. Nat. Genet. 30, 295–300. 10.025. [32] Zeng, H., Yucel, R., Kosan, C., Klein-Hitpass, L. and Moroy, T. (2004) Transcription factor Gfi1 regulates self-renewal and engraftment of hematopoietic stem cells. EMBO J. 23, 4116–4125. 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