Lymphoid Enhancer Binding Factor 1 Regulates Transcription through Looping

This information is current as Kangsun Yun, Jae-Seon So, Arijita Jash and Sin-Hyeog Im of October 2, 2021. J Immunol 2009; 183:5129-5137; Prepublished online 25 September 2009; doi: 10.4049/jimmunol.0802744 http://www.jimmunol.org/content/183/8/5129 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Lymphoid Enhancer Binding Factor 1 Regulates Transcription through Gene Looping1

Kangsun Yun,2,3 Jae-Seon So,2 Arijita Jash, and Sin-Hyeog Im4

Efficient transcription depends upon efficient physical and functional interactions between transcriptosome complexes and DNA. We have previously shown that IL-1␤-induced lymphoid enhancer binding factor 1 (Lef1) regulates the transcription of its target COX2 and MMP13 in mouse chondrocytes by binding to the Lef1 binding sites located in the 3؅ region. In this study, we investigated how the 3؅ region-bound Lef1 regulates expression of target genes. IL-1␤ stimulation induced gene looping in COX2 and MMP13 genomic loci, which is mediated by the physical interaction of Lef1 with its binding partners, including ␤-catenin, AP-1, and NF-␬B. As shown by conformation capture (3C) assay, the 5؅ and 3؅ genomic regions of these genes were juxtaposed in an IL-1␤-stimulation dependent manner. Lef1 played a pivotal role in this gene looping; Lef1 knockdown decreased the incidence of gene looping, while Lef1 overexpression induced it. Physical interactions between the 3؅ region-bound Lef1 and promoter-bound transcription factors AP-1 or NF-␬BinCOX2 and MMP13, respectively, were increased upon stimulation, Downloaded from leading to synergistic up-regulation of . Knockdown of RelA or c-Jun decreased the formation of gene loop and down-regulated cyclooxygenase 2 (COX2) or matrix metalloproteinase 13 (MMP13) transcription levels. However, overexpression of RelA or c-Jun along with Lef1 increased the looping and their expression levels. Our results indicate a novel function of Lef1, as a mediator of gene looping between 5؅ and 3؅ regions. Gene looping may serve to delineate the transcription unit in the inducible gene transcription of mammalian cells. The Journal of Immunology, 2009, 183: 5129–5137. http://www.jimmunol.org/ ranscription is a continuous process in which each step is plexes mediate this interaction by inducing the association of HSS- connected physically and functionally (1). Physical cross- 9/HSSϩ3 with TNF promoter (12). CCCTC-binding factor (CTCF)5 T talk between promoter and terminator regions of a gene is known to regulate the transcription of numerous target genes by through formation of a DNA loop serves to stabilize physical as- modulating epigenetic states (13). At the maternal allele of the Igf2/ sociations between elements of the transcriptional machinery and H19 locus, CTCF binds to the unmethylated imprinting control region to increase transcriptional efficiency (2–4). Recently, rRNA and and mediates inactive looping by intrachromosomal interactions with tRNA synthesis by RNA polymerase I and III, respectively, were promoter (14). CTCF also mediates interchromosomal association be- shown to involve physical connections between promoter and ter- tween Igf2/H19 and Wsb1/Nf1 by bridging distant DNA segments to by guest on October 2, 2021 minator regions (5, 6). Moreover, the formation of a gene loop in a common (15). RNA polymerase II-mediated transcription has been suggested in The lymphoid enhancer binding factor 1 (Lef1) is a DNA-binding yeast (7–9) and in mammalian cells (10, 11). However, the exact transcription factor that plays important roles in organogenesis, colon molecular functions of specific transcription factors in gene loop- cancer progression (16), and cartilage degeneration (17). Notably, ing have not yet been elucidated. Lef1 is an “architectural” transcription factor that binds to DNA via its Interactions between promoter and enhancers regulate gene tran- high-mobility group domain and induces a sharp bend in the DNA scription by intrachromosomal interaction. In the immune system, T helix (18). The bend induced by Lef1 binding facilitates the assembly cell activation increases TNF levels by inducing intrachromosomal of nucleoproteins bound at nonadjacent sites (16, 19). In the center of looping in the TNF locus (12). NFAT-containing nucleoprotein com- the TCR-␣ enhancer, the high-mobility group domain of Lef1 bends the DNA helix and facilitates interactions between activating tran- scription factor/CREB and E26 transformation-specific sequence-1 Department of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea (20). The Lef1-␤-catenin interaction has been well characterized in ␤ Received for publication August 20, 2008. Accepted for publication August 15, 2009. the context of the canonical Wnt signaling pathway (21). -Catenin The costs of publication of this article were defrayed in part by the payment of page connects with the RNA polymerase II transcription machinery charges. This article must therefore be hereby marked advertisement in accordance through Pygo and Lgs, which interact with the N-terminal region (22– with 18 U.S.C. Section 1734 solely to indicate this fact. 24); C-terminal regions interact with parafibromin/hyrax via polymer- 1 This research was supported by grants from the 21C Frontier Functional Human ase-associated factor 1 (25). ␤-catenin is involved in the modification Genome Project, Grant RTI05-01-01 from the Regional Technology Innovation Pro- gram of the Ministry of Commerce, Industry and Energy, by the Korea Research of chromatin structure through its association with CREB-binding Foundation funded by the Korean government (KRF-2007-313-C00507), by the Ko- /p300 (26) and the brahma-related gene 1-containing switch/ rea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family sucrose nonfermentable chromatin remodeling complex (27). Affairs (A080588-5), by the Center for Distributed Sensor Network at the Gwangju Institute of Science and Technology, and by a Systems Biology Infrastructure Estab- lishment Grant provided by the Gwangju Institute of Science and Technology. 2 K.Y. and J.-S.S. equally contributed to this work. 5 3 Abbreviations used in this paper: CTCF, CCCTC-binding factor; 3C, chromatin Current address: Genetic Disease Research Branch, National conformation capture; ChIP, chromatin immunoprecipitation; COX2, cyclooxygenase Research Institute, National Institutes of Health, 49 Convent Drive, Bethesda, 2; Lef1, lymphoid enhancer binding factor 1; MMP13, matrix metalloproteinase 13; MD 20892. siRNA, small interfering RNA; HA, hemagglutinin; BMP2, bone morphogenetic pro- 4 Address correspondence and reprint requests to Dr. Sin-Hyeog Im, Department of tein 2. Life Sciences, Gwangju Institute of Science and Technology, 1 Oryong-dong, Puk-ku, Gwangju 500-712, Korea. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802744 5130 Lef1 MEDIATES GENE LOOPING

IL-1␤ is a proinflammatory cytokine that increases expression Chromosome conformation capture (3C) assay of the COX2 and MMP13 genes in arthritic cartilage (28, 29) as The 3C assay was performed as described previously (8, 33). Subcon- well as in mouse chondrocytes (30, 31). We previously reported fluent mouse chondrocytes were incubated with IL-1␤ (5 ng/ml) for 1 h. that IL-1␤ regulates the expression of two transcription factors, Chromatin cross-linking of whole cells was achieved by treating them Lef1 and its interacting transcriptional coactivator ␤-catenin, in with formaldehyde (2% v/v) for 10 min before extracting nuclei. The primary mouse chondrocytes (17, 30, 31). In turn, these transcrip- cross-linked DNA was restricted using AseI, NcoI, BglII, or PvuII and ligated using T4 DNA ligase. The ligated products were analyzed by tion factors regulate COX2 and MMP13 transcription (17, 30–32). PCR. PCR conditions were 94°C for 30 s, 60°C for 30 s, and 72°C for Unusually, both COX2 and MMP13 genes feature evolutionarily 30 s for a total of 40 or 45 cycles. PCR primers for the 3C assay were conserved binding sites for Lef1 in 3Ј regions and Lef1 binds to as follows: AseI-restricted a, 5Ј-GGAAGTGCATGGTGTCAAAC-3Ј; these sites in a manner dependent on IL-1␤ stimulation (30, 31). b, 5Ј-ACTTAGGCTGTTGGAATTTACG-3Ј;aЈ,5Ј-GAAACGTGCTCT Ј GTGGACC-3Ј; NcoI-restricted A, 5Ј-TGCTGAGCTCCACTTCATCG- To understand how Lef1 bound to the 3 regions of the COX2 or 3Ј;B,5Ј-CCCTGTCCTTCATTCGTGTG-3Ј;C,5Ј-CATTCTTTGCCC MMP13 genes might regulate transcription of these genes in re- AGCACTTCAC-3Ј; and CЈ,5Ј-ATGGGTGTGATTTGTTTGGC-3Ј. sponse to IL-1␤, we hypothesized that Lef1 was involved in me- The sizes of the products from the 3C assay were as follows: a ϩ b (268 diating long-range interactions with IL-1␤-induced transcription bp), aЈϩa (316 bp), A ϩ B (420 bp), A ϩ C (442 bp), and C ϩ CЈ (165 Ј bp). In the case of NcoI-restricted A ϩ D, the primers and product size factors that associate in a conventional manner with the 5 regions Ј Ј Ј ␬ were follows: A, 5 -CTGTGTTTGTGTTGGCCATGGTC-3 ;D,5-GAGA of the COX2 or MMP13 genes. In this study, we show that NF- B AGGAAATGGCTGCAGAATTG-3Ј (138-bp product); PvuII-restricted a, and AP-1 are induced in chondrocytes in response to IL-1␤ sig- 5Ј-TTAAGAGAATGCCCAAGAGTGGGTGG-3Ј;b,5Ј-ATCCAGCTAA naling and Lef1 interacts with both transcription factors in chon- GACACAGCAAGCCA-3Ј;bЈ,5Ј-AAGCAGAGAGGGATTAACAAAC drocyte nuclear lysates. Furthermore, we demonstrate that the 5Ј ATGG-3Ј; BglII- restricted A, 5Ј-GAATGTATTCTCAGTGGGAGCTGT Ј ␤ CG-3Ј;B,5Ј-TGTGCAGGTGTGGTTGTATTTATGAGAG-3Ј;C,5Ј-GT Downloaded from and 3 regions of the COX2 or MMP13 genes interact in an IL-1 GTGGCAAACTGGTAAAGCAAAGTC-3Ј; and CЈ,5Ј-GCCATCTTTC stimulation-dependent manner, and Lef1 plays a crucial role in this TGTTCTCACTGCTGTC-3Ј. The sizes of the products from the 3C assay process. Lef1 depletion by small interfering RNA (siRNA) de- were as follows: a ϩ b (100 bp), b ϩ bЈ (108 bp), A ϩ B (138 bp), A ϩ creased the incidence of DNA looping at both the COX-2 and C (143 bp), and C ϩ CЈ (259 bp). In the case of BglII- restricted A ϩ D, the primers and product size were follows: A, 5Ј-CCAGGAGCAGGTTA MMP13 genes, whereas Lef1 overexpression increased the effi- Ј Ј ␬ ATAGATCTGTTG-3 and D, 5 -TTCCCTGGAATTGGCAACAAAGTA ciency of loop formation. In addition, knockdown of NF- Bor GA-3Ј (278-bp product). As a loading control, PCR was conducted with AP-1 also suppressed the looping, whereas overexpression of those primers for the NF-␬B binding site of genomic COX2. PCR products were http://www.jimmunol.org/ factors along with Lef1 increased formation of the DNA loop. We resolved on 2% agarose gels or cloned into pGEM-T easy vector and se- suggest that Lef1-mediated transcriptional regulation involves the quenced. Quantitative levels of 3C assay were analyzed by real-time PCR and normalized by input. formation of long-range DNA loops through interactions with tran- scription factors bound to distal regulatory elements of Lef1 target Chromatin immunoprecipitation (ChIP) and siRNA-coupled genes. ChIP assays The ChIP assay was conducted as previously described (17) using subconflu- Materials and Methods ent mouse rib chondrocytes treated with IL-1␤ (5 ng/ml) for 1 h. The primers

Materials and reagents used in the ChIP assays were as follows: Lef1 binding site of genomic COX2, by guest on October 2, 2021 forward 5Ј-AATGCTGGTGTGGAAGGTG-3Ј and reverse 5Ј-CCTATTGC The following Abs were used: anti-Lef1, anti-p65/RelA, anti-phospho-c- ATTGAGAGATGGAC-3Ј (325 bp product); NF-␬B binding site of genomic Jun, anti-MMP13, anti-␤-tubulin, and anti-lamin B, all from Santa Cruz COX2, forward 5Ј-ATTAACCGGTAGCTGTGTGCG-3Ј and reverse 5Ј-AG Biotechnology; anti-␤-catenin from BD Transduction Laboratories; anti- GTGGTGCCAAGAGAGCTG-3Ј (263 bp product); Lef1 binding site of COX2 from Cayman Chemical; anti-hemagglutinin (HA) from Covance; genomic MMP13, forward 5Ј-CATGCCAACAAATTCCATATTG-3Ј and re- and anti-IgG from Sigma-Aldrich. The following materials were pur- verse 5Ј-CCAGCCACGCATAGTCATATAG-3Ј (195 bp product); AP-1 chased: rIL-1␤ from Calbiochem; control siRNA, Lef1 siRNA, RelA binding site of genomic MMP13, forward 5Ј-CATTTCCCTCAGATTCTGCC siRNA, and c-Jun siRNA from Santa Cruz Biotechnology and AseI, NcoI, AC-3Ј and reverse 5Ј-GGGAGTCCAGCTCAACAAGAAG-3Ј (231 bp prod- BglII, and PvuII restriction enzymes from New England Biolabs. uct); and control binding site for the ChIP assays with Lef1, RelA, and phos- pho-c-Jun, forward 5Ј-TGGTTATAATGAAGCAAGTGGC3Ј and reverse 5Ј- TGTATGTCATGTGCTTGTCTGG-3Ј (281 bp product). As a loading Culture of mouse rib chondrocytes control, the PCR was conducted directly on input DNA purified from chro- Primary chondrocytes were isolated from ribs of 4-day-old imprinting con- matin before immunoprecipitation. The PCR products were resolved on 2% trol region mice and cultured as previously described (17). The chondro- agarose gels. For the analysis of physiological interaction between Lef1 and cytes released from cartilage were suspended in DMEM supplemented RelA or c-Jun, anti-p65/RelA or anti-phospho-c-Jun Abs were used for im- with FBS (10% (v/v); HyClone), streptomycin (50 ␮g/ml), and penicillin munoprecipitation, and then PCR was performed with primers for the Lef1 (50 U/ml) and then plated on culture dishes at 2–4 ϫ 104 cells/cm2. The binding site of genomic COX2 or MMP13. Otherwise, PCR was performed ␬ cells reached confluence in 4–5 days. with primers for the NF- B binding site of genomic COX2 or for the AP-1 binding site of genomic MMP13 after immunoprecipitation using anti-Lef1 Ab. For the siRNA-coupled ChIP assay, cells were transiently transfected with Preparation of nuclear fractions, immunoprecipitation, and siRNAs using Lipofectamine Plus for 24 h before IL-1␤ treatment. Quantita- immunoblotting tive levels of ChIP assay were analyzed by real-time PCR and normalized by input DNA. Preparation of nuclear fractions was done as described previously (17). The resulting supernatant, containing the nuclear fraction, was retained for Lef1, RT-PCR RelA, and phospho-c-Jun analysis. Lamin B or ␤-tubulin was used as controls for preparation of nuclear fractions. Immunoprecipitation was performed as RT-PCR was performed as previously described (17). PCR conditions described previously (17). Briefly, after transfecting constitutively active S37A were 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s for a total of 35 ␤-catenin, HA-tagged Lef1, RelA, or c-Jun or some combination of these into cycles. PCR primers used were as follows: COX2, forward 5Ј-TGGC HEK293T cells or primary chondrocytes using Lipofectamine Plus (Invitro- TGCAGAATTGAAAGCCCT-3Ј and reverse 5Ј-AAAGGTGCTCGGCT gen), cell lysates were prepared. The lysates were precipitated with specific TCCAGTAT-3Ј (191 bp product); MMP13, forward 5Ј-TGATGGAC Abs against HA, Lef1, RelA, or phospho-c-Jun. Then the immune complexes CTTCTGGTCTTCTGGC-3Ј and reverse 5Ј-CATCCACATGGTTGGG were collected with protein A-Sepharose beads. Immunoblotting was per- AAGTTCTG-3Ј (473 bp product); Lef1, forward 5Ј-ACAGCGACCC formed as described previously (32). The were detected with specific GTACATGTCAAA-3Ј and reverse 5Ј-TGGACATGCCTTGCTTGGAG Abs against ␤-catenin, HA, Lef1, RelA, phospho-c-Jun, COX2, and MMP13. TT-3Ј (195 bp product); GAPDH, forward 5Ј-TCACTGCCACCCAG Blots were developed using a peroxidase-conjugated secondary Ab (Sigma- AAGAC-3Ј and reverse 5Ј-TGTAGGCCATGAGGTCCAC-3Ј (450 bp Aldrich) and an ECL kit (Amersham Biosciences). product); and L32, forward 5Ј-GCCCAAGATCGTCAAAAAGA-3Ј The Journal of Immunology 5131 Downloaded from

FIGURE 1. IL-1␤ increases the binding of transcription factors in the 5Ј and 3Ј regions. A, Vista analysis of the NF-␬B and Lef1 binding sites on COX2 http://www.jimmunol.org/ (upper) or ECR browser analysis of the AP-1 and Lef1 binding site on MMP13 (lower). The arrow in black above the plot denotes the conserved NF-␬B binding site of COX2 or AP-1 binding site of MMP13. The gray arrows denote the conserved Lef1 binding sites of each genome. B, Mouse chondrocytes were incubated with IL-1␤ for 1 h and levels of RelA or phospho-c-Jun protein in the nucleus were determined by immunoblot analysis. Lamin B and ␤-tubulin were analyzed as controls for nuclear extraction and loading. C, After treatment with IL-1␤ (5 ng/ml) for 1 h, ChIP assays were performed with RelA or phospho-c-Jun Ab. PCR was performed with primers specific for the conserved NF-␬B binding site of COX2 or the AP-1 binding site of MMP13 and the control binding site. IgG was used as a control for the specificity of the Ab and the control binding site was analyzed as a control for RelA or phospho-c-Jun binding specificity. D, After IL-1␤ treatment for 1 h, ChIP assays were performed with Lef1 Ab or normal IgG. PCR was performed with primers specific for the conserved Lef1 binding site of COX2 or MMP13. PCR products were resolved on 2% agarose gels. Input denotes the PCR products

obtained from genomic DNA without immunoprecipitation. The data shown in B–D are representative of three independent experiments with similar results. by guest on October 2, 2021 In each experiment, primary chondrocytes were isolated from ribs of 10 mice.

and reverse 5Ј-GTGAGCAATCTCAGCACAGT-3Ј (217 bp product). for COX2 gene transcription and led us to ask whether there was PCR products were resolved on 2% agarose gels with GAPDH as a a physical interaction between these two transcription factors loading control or PCR products were analyzed by real-time PCR nor- bound to either end of the COX2 gene. malized with L32 products. Since MMP13 expression is also up-regulated by IL-1␤ in a Bioinformatic analysis Lef1-dependent manner (31), we performed a similar analysis for Mouse and human COX2 or MMP13 loci were aligned, and the extent of the MMP13 gene. We had previously shown that, similar to the DNA was computed with the web-based program COX2 (Ptgs2) gene, the MMP13 gene also contains a conserved VISTA (http://www-gsd.lbl.gov/vista) (34, 35) or ECR browser (36). Lef1 Lef1 binding site in the 3Ј region (30, 31). However, bioinformatic ␬ and NF- B or AP-1 binding sites were predicted with rVISTA 2.0 (http:// analysis did not reveal conserved binding sites for NF-␬B in the 5Ј rvista.dcode.org) using the optimum matrix similarity. region of the MMP13 gene. We therefore examined the role of Statistical analysis other inducible transcription factors that could potentially be in- ␤ For the statistical analysis of the data, Student’s t tests were applied on volved in IL-1 -mediated up-regulation of MMP13. The AP-1 quantification experiments. A value of p Ͻ 0.05 was considered signifi- transcription factor, which is composed of Jun-Jun or Fos-Jun cantly different. dimers, was an obvious candidate, since the 5Ј region of the MMP13 gene contains a conserved AP-1 binding site (black ar- Results row) as described previously (Fig. 1A, bottom) (37). IL-1␤ increases the binding of transcription factors in the 5Ј We first assessed the activation of NF-␬B and AP-1 in IL-1␤- and 3Ј regions stimulated primary mouse chondrocytes by examining the nuclear We previously reported that IL-1␤ induces NF-␬B activation in translocation of RelA and phosphorylated c-Jun (phospho-c-Jun) primary chondrocytes and that NF-␬B is involved in the up-regu- (38). Indeed, immunoblotting of nuclear extracts prepared from lation of Lef1 transcription (17). Lef1 is required for IL-1␤-me- mouse chondrocytes showed a clear increase in nuclear RelA and diated up-regulation of COX2 expression, and bioinformatic anal- phospho-c-Jun upon IL-1␤ stimulation for 1 h (Fig. 1B). Further- ysis showed that the 5Ј and 3Ј regions of the COX2 (Ptgs2) gene more, ChIP assays showed that IL-1␤ stimulation of mouse chon- contain conserved elements for binding of NF-␬B (black arrow) drocytes dramatically increased the levels of RelA and phospho- and Lef1 (gray arrow), respectively (Fig. 1A, top) (30, 31). These c-Jun binding under physiological conditions (i.e., in intact cells) data suggested a functional cooperation between Lef1 and NF-␬B to the 5Ј region of COX2 (Ptgs2) and MMP13 gene, respectively 5132 Lef1 MEDIATES GENE LOOPING Downloaded from http://www.jimmunol.org/ FIGURE 2. Interaction of Lef1 with RelA or c-Jun leads to up-regulation of Lef1 target genes COX2 and MMP13. A, Interactions of Lef1/␤-catenin, ␤-catenin/RelA, and Lef1/RelA. HA-tagged Lef1, ␤-catenin, and RelA were transiently transfected into HEK293T cells and then RelA and Lef1-HA were immunoprecipitated (IP). Coprecipitated ␤-catenin or Lef1-HA was analyzed by immunoblotting (IB) with ␤-catenin or HA Ab. B, c-Jun with or without HA-tagged Lef1 was transiently overexpressed in HEK293T cells, and the lysates were immunoprecipitated with phospho-c-Jun Ab. Coprecipitated Lef1-HA was analyzed by immunoblotting with a HA Ab. C, Lef1 and RelA were transiently transfected into chondrocytes and then Lef1 and RelA were immunoprecipitated. Coprecipitated RelA or Lef1 were analyzed by immunoblotting with RelA or Lef1 Ab. D, Lef1 and c-Jun were transiently transfected into chondrocytes and then Lef1 and phospho-c-Jun were immunoprecipitated. Coprecipitated c-Jun or Lef1 were analyzed by immunoblotting with phospho-c-Jun or Lef1 Ab. The data shown in A–D are representative of three independent experiments with similar results. E, Chondrocytes were transfected with empty vector (mock) or HA-tagged Lef1 and constitutively active ␤-catenin (␤-cat) with or without RelA for 24 h. COX2 mRNA was analyzed by real-time PCR and expressed relative to the mock sample after normalizing with mouse L32. F, Chondrocytes were transfected with empty by guest on October 2, 2021 vector (mock) or HA-tagged Lef1 and constitutively active ␤-catenin with or without c-Jun for 24 h. MMP13 mRNA was analyzed by real-time PCR. The data shown in E and F are the mean of three independent experiments, and error bars indicate SDs. Primary chondrocytes were isolated from ribs of 10 mice in each independent experiment.

(Fig. 1C, left and middle, and supplemental Fig. 1A6 for quantita- To confirm whether these interactions also occur in mouse chon- tive analysis). There was no detectable binding of these transcrip- drocytes, we performed coimmunoprecipitation assays after over- tion factors to a control region of the COX2 (Ptgs2) gene (Fig. 1A), expression of Lef1 and RelA or c-Jun. Immunoprecipitates of which contains neither a NF-␬B nor AP-1 binding motif (Fig. 1C, RelA or Lef1 revealed their coexistence with Lef1 or RelA, re- right). We have previously reported that Lef1 binds to the con- spectively (Fig. 2C, lanes 3 and 4). In vivo association between served 3Ј binding elements in the COX2 (Ptgs2) and MMP13 phospho-c-Jun and Lef1 was also confirmed in chondrocytes (Fig. genes by ChIP (30, 31). IL-1␤ stimulation increased Lef1 binding 2D, lanes 3 and 4). to the 3Ј region of COX2 or MMP13 genes (Fig. 1D and supple- We also assessed the functional relevance of these in vivo in- mental Fig. 1B) (30, 31) as well as the binding of RelA or c-Jun to teractions in mouse chondrocytes, in which target gene expression the 5Ј region of the COX2 or MMP13 gene (Fig. 1C). These results was monitored by real-time PCR. When Lef1 and a constitutively suggest the possibility of functional cooperation between tran- active ␤-catenin were coexpressed with RelA in mouse chondro- scription factors for the regulation of Lef1 target genes. cytes, we observed an up-regulation of COX2 mRNA within 24 h, much greater than that elicited by Lef1/␤-catenin or RelA alone Interactions of Lef1 with RelA or c-Jun up-regulate (Fig. 2E). Similarly, coexpression of Lef1/␤-catenin and c-Jun in transcription of Lef1 target genes COX2 or MMP13 primary mouse chondrocytes resulted in synergistic up-regulation We next examined the interactions among Lef1, ␤-catenin, RelA, of MMP13 mRNA expression within 24 h (Fig. 2F). These results and c-Jun by coimmunoprecipitation assays in HEK293 cells and suggest that physical interactions between Lef1 and RelA or phos- primary chondrocytes. Immunoprecipitates of HA-tagged Lef1 as pho-c-Jun cooperatively mediate the transcription of Lef1 target well as RelA contained ␤-catenin as expected (Fig. 2A, lanes 3 and genes COX2 or MMP13, respectively. 4) (39). RelA were also coimmunoprecipitated with Lef1-HA (Fig. 2A, lane 5). Consistent with a previous report (40), immunopre- IL-1␤ induces juxtaposition of the 5Ј region and the 3Ј region of cipitates of phospho-c-Jun contained Lef1-HA as well (Fig. 2B). genomic COX2 and MMP13 The above data suggest physical as well as functional interactions 6 The online version of this article contains supplemental material. between Lef1-binding 3Ј elements and NF-␬B or AP-1-binding 5Ј The Journal of Immunology 5133

FIGURE 4. Lef1 plays a crucial role in the gene looping. A, Chondro- cytes were transfected with Lef1 or control siRNA and then stimulated with IL-1␤ (left). Chondrocytes were transfected with empty or a Lef1 expression vector for 24 h (right). The mRNA levels of Lef1, COX2, and MMP13 were analyzed by RT-PCR. GAPDH was analyzed as a loading FIGURE 3. IL-1␤ induces juxtaposition of the 5Ј and the 3Ј regions control. B, Left, chondrocytes were transfected with Lef1 or control siRNA of genomic COX2 and MMP13. A, AseIorNcoI cleavage sites in the for 24 h and IL-1␤ was further added for 1 h. The 3C assay was performed Downloaded from mouse genomic COX2 locus and PvuII or BglII cleavage sites in the with AseI restriction for COX2 or PvuII restriction for MMP13. Right, mouse genomic MMP13 are illustrated: the black horizontal arrows de- Chondrocytes were transfected with empty or Lef1 expression vector for note the 3C primers for the AseI restriction sites (COX2)orPvuII restric- 24 h and the 3C assay was performed with AseI restriction for COX2 or tion sites (MMP13); the empty horizontal arrows denote the 3C primers for PvuII restriction for MMP13. Input represents PCR products of a conserved the NcoI restriction sites (COX2)orBglII restriction sites (MMP13), re- NF-␬B binding site of COX2. C, After incubation with IL-1␤, physiolog- spectively. B, Mouse chondrocytes were incubated with IL-1␤ for 1 h and ical interaction of Lef1 and RelA or c-Jun were shown by ChIP assays: left the 3C assays restricted by NcoIorAseI were performed in the COX2 (upper), RelA or Lef1 was immunoprecipitated and PCR was performed http://www.jimmunol.org/ locus. C, 3C assays restricted by BglII or PvuII was performed in the with primers specific for the conserved Lef1 or NF-␬B binding site of MMP13 locus. Input represents products of PCRs using primers specific for COX2. Right (upper), phospho-c-Jun or Lef1 was immunoprecipitated and the conserved NF-␬B binding site of COX2 performed to establish that PCR was performed with primers specific for the conserved Lef1 or AP-1 equal amounts of template DNA were present in the samples. The data binding site of MMP13. Input denotes the PCR products obtained with shown in B and C are from one of at least three independent experiments, genomic DNA without immunoprecipitation. Left (lower), Lef1 was im- all of which yielded similar results. Primary chondrocytes were isolated munoprecipitated and PCR was performed with primers specific for the from ribs of 10 mice in each independent experiment. conserved NF-␬B binding site of BMP2. Right (lower), c-Jun was immu- noprecipitated and PCR was performed with primers specific for the con-

served Lef1 binding site of cyclin D1. Input denotes the PCR products by guest on October 2, 2021 regions of the COX2 or MMP13 genes, respectively (Fig. 1A). We obtained with genomic DNA without immunoprecipitation. The data therefore proceeded to evaluate our hypothesis that during IL-1␤- shown in A–C are from one of at least three independent experiments that driven transcription of the COX2 and MMP13 genes, looping in- yielded similar results. Primary chondrocytes were isolated from ribs of 10 teractions mediated by these transcription factors occurred be- mice in each independent experiment. tween the two ends of these transcribed genes. To test the formation of these postulated gene loops, we performed chromo- some conformation capture (3C) assays (8, 33). We selected the left third panels). The amount of input DNA was the same in both restriction enzymes Ase1 and NcoI for COX2 or PvuII and BglII IL-1␤-untreated and treated samples (Fig. 3, B and C, left fourth for MMP13, since they can cut appropriate restriction sites, in- panels). cluding the 3Ј and 5Ј regions of each genomic locus (Fig. 3A). To further confirm the formation of an IL-1␤-dependent loop Chondrocytes were stimulated with IL-1␤ or left unstimulated, between the 5Ј and 3Ј ends of the COX2 or MMP13 genes, we then treated with formaldehyde to fix their DNA conformation. repeated the 3C assay with the restriction enzymes AseIorPvuII, Then the cross-linked DNA complexes were digested with the re- which cut near the Lef1 binding sites (Fig. 3A, site b) as well as in striction enzymes and treated with ligase to join together ends of the 5Ј regions of both genes (Fig. 3A, site a). Again, the AseI- or DNA which were in reasonable physical proximity. The ligated PvuII-restricted products of the 3C assay were obtained only upon products were analyzed by PCR using primer pairs specific for the IL-1␤ stimulation (Fig. 3, B and C, right panels). As a control (41), restriction enzyme-containing regions (Fig. 3B). The identity of all we proceeded the 3C assay in the absence of cross-linking or li- of the products was confirmed by DNA sequencing (data not gation. The PCR products were dramatically decreased in the ab- shown). sence of cross-linking (supplemental Fig. 2A) or ligase treatment In the 3C assay, ligated products can be obtained only when the (supplemental Fig. 2B). Taken together, these results demonstrate restriction sites are close to one another (33). As a result, regard- that IL-1␤ stimulation induces a conformational change in COX2 less of IL-1␤ stimulation, we obtained ligated products between and MMP13 gene loci to form gene loops, such that the 3Ј region the A and B in the NcoI sites of COX2 or BglII sites of MMP13 which binds Lef1 is in close proximity to the 5Ј region. genes (Fig. 3, B and C, left top panels), presumably due to their physical proximity in the primary DNA sequence. We did not ob- Lef1 plays crucial roles in the regulation of transcription and tain ligated products between the more distant A and C sites under gene looping of Lef1 target genes COX2 or MMP13 the same conditions (Fig. 3, B and C, left second panels). In con- To elucidate the role of Lef1 in the gene looping, we knocked trast, ligated products between the A and D sites, located at the 5Ј down Lef1 expression using Lef1-directed siRNA and tested its and 3Ј ends of these genes, respectively, were obtained for both effect on gene looping. Transfection of Lef1 siRNA into primary genes only in the sample stimulated with IL-1␤ (Fig. 3, B and C, chondrocytes decreased Lef1 transcripts and also diminished the 5134 Lef1 MEDIATES GENE LOOPING levels of COX2 and MMP13 gene expression (Fig. 4A, left panels) and protein levels (supplemental Fig. 5A) in response to IL-1␤ stimulation as expected (supplemental Fig. 3A for quantitative analysis). In a 3C assay, Lef1 depletion also diminished the effi- ciency of gene looping assessed by formation of the ligation prod- uct between sites a and b in AseI restriction for COX2 or PvuII restriction for MMP13 (Fig. 4B, left panels, and supplemental Fig. 3B for quantitative analysis). We also evaluated the effect of Lef1 overexpression, in the absence of IL-1␤ stimulation, on transcript levels and gene looping of COX2 and MMP13. Cells transfected with Lef1 showed an increase in both gene expression and 3C product, whereas cells transfected with empty vector showed nei- ther (Fig. 4, A and B, right panels, and supplemental Fig.3,Aand B, for quantitative analysis). Together, these data indicate a crucial role of Lef1 in gene looping and also suggest that gene looping is correlated with active transcription. We next asked whether the IL-1␤-mediated gene looping could be mediated by the physical interaction of 3Ј region-bound Lef1 with 5Ј region-bound NF-␬B or AP-1 in COX2 and MMP13 loci,

Ј Downloaded from respectively. Accordingly, we performed ChIP analysis. After im- FIGURE 5. Knockdown of RelA or c-Jun reduces Lef1 binding to the 5 region of COX2 or MMP13. A, Chondrocytes were transfected with munoprecipitation with a RelA or phospho-c-Jun Ab, PCR was siRNAs for RelA, c-Jun, or with control siRNA and then stimulated with performed with primers specific for each Lef1 binding site in the IL-1␤. The mRNA levels of COX2 or MMP13 were analyzed by real-time Ј 3 regions of the COX2 or MMP13 genes. Conversely, after im- PCR and expressed relative to control siRNA after normalizing with mouse munoprecipitation with a Lef1 Ab, PCR was performed with prim- L32. The data shown are mean of three independent experiments and error p Ͻ ,ء .ers specific for the 5Ј NF-␬B or AP-1 binding sites of the COX2 or bars indicate SDs. Significance was determined by Student’s t test /p Ͻ 0.01. B, Physiological binding of RelA or c-Jun to the 3Ј http://www.jimmunol.org ,ءء MMP13 genes, respectively. The amount of Lef1-bound DNA on 0.05 and the 3Ј region at the COX2 locus was enriched after IL-1␤ treat- region was shown by ChIP assays: left, RelA or Lef1 was immunoprecipi- ment, as shown following immunoprecipitation with a RelA Ab tated (IP) after treating Lef1 siRNA and PCR was performed with primers (Fig. 4C, upper left top, and supplemental Fig. 3C for quantitative specific for the conserved Lef1 binding site of COX2. Right, Phospho-c-Jun analysis). Likewise, NF-␬B (RelA)-bound DNA at the promoter or Lef1 was immunoprecipitated after treating Lef1 siRNA and PCR was performed with primers specific for the conserved Lef1 binding site of was also increased after immunoprecipitation with a Lef1 Ab (Fig. MMP13. Input denotes the PCR products obtained with genomic DNA 4C, upper left middle, and supplemental Fig. 3C for quantitative without immunoprecipitation. C, Physiological binding of Lef1 to the 5Ј analysis). We observed similar results from ChIP analysis in the region was shown by ChIP assays: left, Lef1 or RelA was immunoprecipi- MMP13 locus. The ChIP products in the MMP13 genomic locus tated after treating RelA siRNA and PCR was performed with primers by guest on October 2, 2021 were increased after IL-1␤ stimulation (Fig. 4C, right, and sup- specific for the conserved NF-␬B binding site of COX2. Right, Lef1 or plemental Fig. 3C for quantitative analysis). To show the speci- phospho-c-Jun was immunoprecipitated after treating c-Jun siRNA and ficity of Lef1 binding to the RelA-binding 5Ј region at the COX2 PCR was performed with primers specific for the conserved AP-1 binding locus or c-Jun binding to Lef1-binding 3Ј region at the MMP13 site of MMP13. The data shown in B and C are representative of three locus, ChIP assay was performed. ChIP analysis in Bone morpho- independent experiments with similar results. Primary chondrocytes were genetic protein 2 (BMP2) or cyclin D1 loci was also performed as isolated from ribs of 10 mice in each independent experiment. negative controls (Fig. 4C, bottom). The BMP2 gene contains a NF-␬B-binding element in the Ϫ838/Ϫ829 region (42). The Cy- down effect on COX2 or MMP13 transcription and gene looping. clin D1 gene has a Lef1 binding site in the Ϫ81/-73 region (43). Transfection of RelA siRNA or c-Jun siRNA into primary chon- After immunoprecipitation with a Lef1 or c-Jun Ab, PCR was drocytes decreased the levels of COX2 or MMP13 gene expression performed with primers specific for the NF-␬B (RelA) or Lef1 and protein levels, respectively, (Fig. 5A and supplemental Fig. 5, binding site of the BMP2 or cyclin D1 gene, respectively. Lef1 did B and C). Lef1 siRNA treatment reduced gene looping between the not interact with the NF-␬B binding site of the 5Ј region in the 5Ј region and 3Ј region of COX2 or MMP13 genes (Fig. 4B). We BMP2 gene, and c-Jun did not bind to the Lef1 binding sites of the further tested whether treatments of siRNAs for Lef1, RelA, or 5Ј region in the cyclin D1 gene, respectively. Taken together, these c-Jun directly diminish the physical interaction of the 5Ј region data suggest that the 3Ј region-bound Lef1 interacts with the RelA- binding RelA or c-Jun with the 3Ј region binding Lef1 in COX2 binding 5Ј region of the genomic COX2 locus. In the MMP13 and MMP13 loci, respectively. After Lef1 siRNA treatment and transcription, the 3Ј region-bound Lef1 physiologically cooperates then immunoprecipitation with RelA or phospho-c-Jun Ab, PCR with the AP-1-binding 5Ј region in an IL-1␤ stimulation-depen- was performed with primers specific for the Lef1 binding site in dent manner. Indeed, co-overexpression of RelA and Lef1/␤-cate- the 3Ј regions of the COX2 or MMP13 genes. As expected, Lef1 nin resulted in synergistic up-regulation of COX2 expression (Fig. siRNA treatment diminished the interactions between 5Ј-bound 2E). MMP13 expression was also increased by co-overexpression transcription factors and the Lef1-bound 3Ј region (Fig. 5B and of c-Jun and Lef1/␤-catenin (Fig. 2F). supplemental Fig. 4A for quantitative analysis). Next, we assessed the effect of siRNA treatment for RelA or c-Jun on these interac- Knockdown of RelA or c-Jun reduces the interactions of Lef1 tions. After RelA siRNA or c-Jun siRNA treatment, ChIP was Ј with 5 region of COX2 or MMP13 performed with a Lef1 Ab. Enriched DNA was then analyzed by Interaction of Lef1 with the 5Ј region-bound RelA or c-Jun in PCR with primers specific for the 5Ј NF-␬B or AP-1 binding sites COX2 or MMP13 locus enhanced their transcription (Fig. 2) of the COX2 or MMP13 genes, respectively. Treatment with RelA through gene looping (Figs. 3 and 4). To further elucidate the role siRNA or c-Jun siRNA reduced NF-␬B or AP-1-bound DNA lev- of RelA or c-Jun in these associations, we analyzed their knock- els at the 5Ј region (Fig. 5C, top, and supplemental Fig. 4B for The Journal of Immunology 5135 Downloaded from

FIGURE 6. Crucial role of Lef1 and its binding partners in gene looping. A, Chondrocytes were transfected with siRNA for RelA, c-Jun, or control (Cont) siRNA and then stimulated with IL-1␤. Left, The 3C assay was performed with AseI restriction for COX2 or PvuII restriction for MMP13. Input denotes the PCR products obtained with genomic DNA without immunoprecipitation using primers specific for the conserved NF-␬B binding site of COX2. http://www.jimmunol.org/ Right, The 3C values were analyzed by real-time PCR using 3C primer and expressed relative to control siRNA after normalizing with input DNA. B, Upper, chondrocytes were transfected with Lef1 or RelA expression vector for 24 h. The 3C assay was performed with AseI restriction for COX2. Lower, The 3C values were analyzed by real-time PCR using 3C primer and expressed relative to control (empty vector). C, Upper, Chondrocytes were transfected with Lef1 or c-Jun expression vector for 24 h. ⌻he 3C assay was performed with PvuII restriction for MMP13. Lower, The 3C values were analyzed by real-time PCR using 3C primer and expressed relative to control. The data shown in A–C are from one of at least three independent experiments that yielded similar results. The error bars show the SDs of the PCR performed in triplicate. Primary chondrocytes were isolated from ribs of 10 mice in each independent experiment. by guest on October 2, 2021 quantitative analysis). Taken together, these data suggest that the 2E). Likewise, gene looping between 5Ј and 3Ј region of MMP13 3Ј region-bound Lef1 interacts with the 5Ј region-bound RelA or was increased by coexpression of Lef1 and c-Jun together com- c-Jun on the COX2 or MMP13 locus, respectively. pared with individual over-expression of either alone (Fig. 6C). This result was also in accordance with expression pattern of Interaction of Lef1 with RelA or c-Jun cooperatively regulates MMP13 (Fig. 2F). Taken together, these results demonstrate that gene looping IL-1␤ stimulation induces intrachromosomal interaction in COX2 Knock-down of Lef1, RelA or c-Jun suppressed the expression of or MMP13 gene loci to form gene loops, which is mediated by Lef1 target genes in chondrocytes, and reduced the association juxtaposition between the Lef1-binding 3Ј region and the RelA or between 5Ј-bound RelA or c-Jun and 3Ј-bound Lef1 (Fig. 5). To c-Jun-binding 5Ј region. Collectively, these results suggest the crit- test the role of RelA or c-Jun in the formation of gene loops, 3C ical role of transcription factors in gene looping and the close assays was performed after siRNA treatment for RelA or c-Jun. correlation of gene looping with active gene expression. We used the restriction enzymes AseI or PvuII, which cut near the RelA or c-Jun binding sites of COX2 or MMP13, respectively (Fig. 3A). Knock-down of RelA reduced the efficiency of gene looping Discussion assessed by formation of the ligation product between sites a and COX2 and MMP13 are critical molecules involved in the immune b in AseI restriction for COX2 (Fig. 6A). Similarly, c-Jun siRNA response and inflammation of rheumatoid arthritis and osteoarthri- suppressed the loop formation as assessed by PvuII restriction for tis. They are primarily produced by chondrocytes of arthritic car-

MMP13 (Fig. 6A). These data showed that gene looping in COX2 tilage. COX2 catalyzes the production of PGE2 which mediates or MMP13 is also mediated by RelA or c-Jun as well as Lef1. joint inflammation. MMP13 is a proteolytic enzyme that degrades The above data also suggest that intrachromosomal interactions the extracellular matrix of cartilage tissues (44). We previously are mediated through the interaction of transcription factors bind- reported that Lef1 is highly expressed in osteoarthritic cartilage ing to the 5Ј and 3Ј region of COX2 or MMP13 genes. To confirm and induced by IL-1␤ in mouse chondrocytes (17). We also the cooperative role of Lef1, RelA or c-Jun in the formation of a showed that proinflammatory cytokine IL-1␤ up-regulates COX2 loop between the 5Ј and 3Ј region of the COX2 or MMP13 genes, and MMP13 levels by increasing Lef1 binding to the 3Ј regions of chondrocytes were transfected with expression vectors of Lef1, target genes (30, 31). In this study, we further elucidated how the RelA or c-Jun in the absence of IL-1␤ stimulation. In the COX2 3Ј region-binding Lef1 regulates its target genes COX2 and gene locus, loop formation was increased by over-expression of MMP13 in chondrocytes. Lef1 or RelA. In addition, this loop formation was further in- We have demonstrated that Lef1-mediated regulation of target creased by over-expression of Lef1 and RelA together (Fig. 6B), genes in chondrocytes COX-2 or MMP13 correlates with the loop- which mediated additive up-regulation of COX2 transcription (Fig. ing contact of the Lef-1-binding 3Ј region with 5Ј regions which 5136 Lef1 MEDIATES GENE LOOPING bound by the inducible transcription factors NF-␬B or AP-1, re- the formation of these complexes by bending DNA to facilitate the spectively. It has been suggested that gene looping is associated interactions between cis-acting elements. Widely separated cis-act- with transcription reinitiation, a cyclic process of RNA synthesis in ing regulatory elements at gene loci evidence the functional inter- active genes (5). To maintain the accelerated transcription rate, action between regulatory elements (49) and provide a mechanism RNA polymerases and transcription factors need to be recycled for specific and efficient transcription of genes. Stimulation-depen- (45). The formation of a gene loop, in which both ends of the dent DNA looping mediated by activated transcription factors transcription unit are juxtaposed, could physically stabilize the ac- should be efficient in terms of energy expenditure and rate of tran- tive transcriptional machinery and direct new rounds of transcrip- scription. We showed the pivotal role of Lef1 in gene looping of tion. This would eventually result in an accelerated transcription COX2 or MMP13 under IL-1␤ signaling. Besides Lef1/␤-catenin, rate and/or an increase in transcriptional efficiency (5). NF-␬B, and AP-1, other transcription factors may also be involved The formation of gene loops in RNA polymerase II-mediated in gene looping following IL-1␤ stimulation. Indeed, knockdown transcription has been previously reported. The incidence of gene of Lef1 (Fig. 4B), RelA, or c-Jun (Fig. 6A) decreased the incidence looping was determined by the phosphorylation status of RNA of gene looping while overexpression of them induced looping polymerase II (8, 10). Cleavage and polyadenylation factor 3Ј-end (Figs. 4B and 6, B and C). In addition, overexpression of Lef1 processing machinery also functions in RNA polymerase II-depen- along with its 5Ј region-binding partner RelA or c-Jun additively dent gene looping (7). TFIIB is suggested as another connector of up-regulated gene looping (Fig. 6, B and C) as well as up-regula- gene looping (9). Our study suggests that an architectural tran- tion of Lef1 target genes COX2 and MMP13 (Fig. 2, E and F). scription factor, Lef1, is associated with the gene looping in mam- However, further studies are needed to determine how the associ- malian cells; 3Ј region-bound Lef1 induced gene looping through ation between Lef1 and RelA or c-Jun is mediated for inducing a the interaction of 5Ј region-bound transcription factors NF-␬Bor gene loop. Downloaded from AP-1. In addition, our results also suggest that gene looping in Interactions between DNA-bound transcription factors direct mammalian cells is involved in the early stages of transcriptional formation of an enhanceosome, which induces transcriptional syn- activation (8), since transcription factors usually bind to DNA be- ergy throughout the gene from the promoter to the terminator (50, fore formation of the preinitiation complex formation, necessitat- 51). Multiple interactions between transcription factors may ing recycling. strengthen the assembly of enhanceosomes and facilitate the for- Binding of Lef1 to its consensus DNA sequence induces a sharp mation of gene loops. As suggested in yeast, RNA polymerase http://www.jimmunol.org/ bending in DNA structure (46). Interactions of 3Ј region-bound II-mediated transcription may be facilitated by gene looping. Lef1 with other factors can direct the bending of DNA. Most Long-distance associations between regulatory elements may play known transcription factors bind to promoter regions. Therefore, an important role in eukaryotic gene regulation. Inactivation or the bent DNA in the 3Ј region will result in the formation of a gene detachment of transcription factors from enhanceosomes could de- loop by the turn toward the promoter bound by diverse transcrip- stabilize gene loops, leading to termination of transcription. There- tion factors. Such a role of the 3Ј region-bound Lef1 is consistent fore, the transcription factors involved in gene looping may form with the formation of a gene loop which is mediated by interaction the mechanism whereby transcription is switched on and off. In- with other transcription factors. Indeed, in immune cells, Lef1/␤- vestigation of 3Ј-bound transcription factors may reveal further by guest on October 2, 2021 catenin regulate TCR-␣ and HIV-1 transcription in a chromatin- diverse gene looping systems. dependent manner by recruiting chromatin remodeling complexes In conclusion, Lef1 mediates COX2 and MMP13 gene looping (47). ␤-Catenin, a well-known Lef1-binding transcriptional coac- during RNA polymerase II-mediated transcription in mammalian tivator, plays crucial roles both in connecting DNA to RNA poly- cells. Formation of a DNA loop as a transcription unit could in- merase II transcription machinery and in chromatin remodeling; crease the efficiency of RNA polymerase II-mediated transcription Pygo and Lgs link ␤-catenin to the RNA transcription machinery by increasing the transcription rate while conserving energy, an (22–24) and parafibromin/hyrax is physically connected to the C- effect that could have particular significance in the mammalian terminal of ␤-catenin with platelet-activating factor 1 (25). These system, where the typical transcription unit is relatively long. Gene functions of Lef1/␤-catenin might be appropriate for gene looping looping may constitute a mechanism for inducible transcriptional and thus recycling of RNA transcription machinery. Thus, there regulation in mammalian cells and 3Ј region-bound transcription might be cooperative roles between Lef1/␤-catenin and remodel- factors may function as connectors forming integrated transcrip- ing factors for inducing gene looping. Initiation of local DNA tion units. bending by Lef1 might lead to the formation of a gene loop through chromatin remodeling, which is mediated by ␤-catenin- Acknowledgments interacting proteins. Taken together, locus-specific looping medi- We thank specially Dr. Anjana Rao for priceless advice on manuscript ated by Lef1 binding can provide the mechanism for specific and organization. We also thank Darren Reece Williams for critical proof read- efficient transcription of Lef1 target genes. Furthermore, the con- ing of this manuscript and Dr. E.-J. Cho for kind discussion in experimental servation of the Lef1 binding site in the 3Ј region might be evo- design. lutionarily developed to facilitate gene looping. 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