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LoopingRegulates Transcription through Gene Lymphoid Enhancer Binding Factor 1

Kangsun Yun, Jae-Seon So, Arijita Jash and Sin-Hyeog Im

http://www.jimmunol.org/content/183/8/5129doi: 10.4049/jimmunol.0802744September 2009;

2009; 183:5129-5137; Prepublished online 25J Immunol 

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4.DC1http://www.jimmunol.org/content/suppl/2009/09/25/jimmunol.080274

Referenceshttp://www.jimmunol.org/content/183/8/5129.full#ref-list-1

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Lymphoid Enhancer Binding Factor 1 Regulates Transcriptionthrough 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 targetgenes COX2 and MMP13 in mouse chondrocytes by binding to the Lef1 binding sites located in the 3� region. In this study, weinvestigated how the 3� region-bound Lef1 regulates expression of target genes. IL-1� stimulation induced gene looping in COX2and 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 chromosome conformation capture (3C) assay, the 5� and 3� genomic regions of these genes werejuxtaposed in an IL-1�-stimulation dependent manner. Lef1 played a pivotal role in this gene looping; Lef1 knockdown decreasedthe incidence of gene looping, while Lef1 overexpression induced it. Physical interactions between the 3� region-bound Lef1 andpromoter-bound transcription factors AP-1 or NF-�B in COX2 and MMP13, respectively, were increased upon stimulation,leading to synergistic up-regulation of gene expression. Knockdown of RelA or c-Jun decreased the formation of gene loop anddown-regulated cyclooxygenase 2 (COX2) or matrix metalloproteinase 13 (MMP13) transcription levels. However, overexpressionof 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 induciblegene transcription of mammalian cells. The Journal of Immunology, 2009, 183: 5129–5137.

T ranscription is a continuous process in which each step isconnected physically and functionally (1). Physical cross-talk between promoter and terminator regions of a gene

through formation of a DNA loop serves to stabilize physical as-sociations between elements of the transcriptional machinery andto increase transcriptional efficiency (2–4). Recently, rRNA andtRNA synthesis by RNA polymerase I and III, respectively, wereshown to involve physical connections between promoter and ter-minator regions (5, 6). Moreover, the formation of a gene loop inRNA polymerase II-mediated transcription has been suggested inyeast (7–9) and in mammalian cells (10, 11). However, the exactmolecular functions of specific transcription factors in gene loop-ing have not yet been elucidated.

Interactions between promoter and enhancers regulate gene tran-scription by intrachromosomal interaction. In the immune system, Tcell activation increases TNF levels by inducing intrachromosomallooping in the TNF locus (12). NFAT-containing nucleoprotein com-

plexes mediate this interaction by inducing the association of HSS-9/HSS�3 with TNF promoter (12). CCCTC-binding factor (CTCF)5

is known to regulate the transcription of numerous target genes bymodulating epigenetic states (13). At the maternal allele of the Igf2/H19 locus, CTCF binds to the unmethylated imprinting control regionand mediates inactive looping by intrachromosomal interactions withpromoter (14). CTCF also mediates interchromosomal association be-tween Igf2/H19 and Wsb1/Nf1 by bridging distant DNA segments toa common transcription factor (15).

The lymphoid enhancer binding factor 1 (Lef1) is a DNA-bindingtranscription factor that plays important roles in organogenesis, coloncancer progression (16), and cartilage degeneration (17). Notably,Lef1 is an “architectural” transcription factor that binds to DNA via itshigh-mobility group domain and induces a sharp bend in the DNAhelix (18). The bend induced by Lef1 binding facilitates the assemblyof nucleoproteins bound at nonadjacent sites (16, 19). In the center ofthe TCR-� enhancer, the high-mobility group domain of Lef1 bendsthe DNA helix and facilitates interactions between activating tran-scription factor/CREB and E26 transformation-specific sequence-1(20). The Lef1-�-catenin interaction has been well characterized inthe context of the canonical Wnt signaling pathway (21). �-Cateninconnects with the RNA polymerase II transcription machinerythrough Pygo and Lgs, which interact with the N-terminal region (22–24); C-terminal regions interact with parafibromin/hyrax via polymer-ase-associated factor 1 (25). �-catenin is involved in the modificationof chromatin structure through its association with CREB-bindingprotein/p300 (26) and the brahma-related gene 1-containing switch/sucrose nonfermentable chromatin remodeling complex (27).

Department of Life Sciences, Gwangju Institute of Science and Technology,Gwangju, Korea

Received for publication August 20, 2008. Accepted for publication August 15, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This research was supported by grants from the 21C Frontier Functional HumanGenome Project, Grant RTI05-01-01 from the Regional Technology Innovation Pro-gram of the Ministry of Commerce, Industry and Energy, by the Korea ResearchFoundation funded by the Korean government (KRF-2007-313-C00507), by the Ko-rea Healthcare Technology R&D Project, Ministry for Health, Welfare and FamilyAffairs (A080588-5), by the Center for Distributed Sensor Network at the GwangjuInstitute 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.3 Current address: Genetic Disease Research Branch, National Human GenomeResearch Institute, National Institutes of Health, 49 Convent Drive, Bethesda,MD 20892.4 Address correspondence and reprint requests to Dr. Sin-Hyeog Im, Department ofLife Sciences, Gwangju Institute of Science and Technology, 1 Oryong-dong, Puk-ku,Gwangju 500-712, Korea. E-mail address: imsh@gist.ac.kr

5 Abbreviations used in this paper: CTCF, CCCTC-binding factor; 3C, chromatinconformation capture; ChIP, chromatin immunoprecipitation; COX2, cyclooxygenase2; Lef1, lymphoid enhancer binding factor 1; MMP13, matrix metalloproteinase 13;siRNA, small interfering RNA; HA, hemagglutinin; BMP2, bone morphogenetic pro-tein 2.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802744

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IL-1� is a proinflammatory cytokine that increases expressionof the COX2 and MMP13 genes in arthritic cartilage (28, 29) aswell as in mouse chondrocytes (30, 31). We previously reportedthat IL-1� regulates the expression of two transcription factors,Lef1 and its interacting transcriptional coactivator �-catenin, inprimary mouse chondrocytes (17, 30, 31). In turn, these transcrip-tion factors regulate COX2 and MMP13 transcription (17, 30–32).Unusually, both COX2 and MMP13 genes feature evolutionarilyconserved binding sites for Lef1 in 3� regions and Lef1 binds tothese sites in a manner dependent on IL-1� stimulation (30, 31).

To understand how Lef1 bound to the 3� regions of the COX2 orMMP13 genes might regulate transcription of these genes in re-sponse to IL-1�, we hypothesized that Lef1 was involved in me-diating long-range interactions with IL-1�-induced transcriptionfactors that associate in a conventional manner with the 5� regionsof the COX2 or MMP13 genes. In this study, we show that NF-�Band AP-1 are induced in chondrocytes in response to IL-1� sig-naling and Lef1 interacts with both transcription factors in chon-drocyte nuclear lysates. Furthermore, we demonstrate that the 5�and 3� regions of the COX2 or MMP13 genes interact in an IL-1�stimulation-dependent manner, and Lef1 plays a crucial role in thisprocess. Lef1 depletion by small interfering RNA (siRNA) de-creased the incidence of DNA looping at both the COX-2 andMMP13 genes, whereas Lef1 overexpression increased the effi-ciency of loop formation. In addition, knockdown of NF-�B orAP-1 also suppressed the looping, whereas overexpression of thosefactors along with Lef1 increased formation of the DNA loop. Wesuggest that Lef1-mediated transcriptional regulation involves theformation of long-range DNA loops through interactions with tran-scription factors bound to distal regulatory elements of Lef1 targetgenes.

Materials and MethodsMaterials and reagents

The following Abs were used: anti-Lef1, anti-p65/RelA, anti-phospho-c-Jun, anti-MMP13, anti-�-tubulin, and anti-lamin B, all from Santa CruzBiotechnology; anti-�-catenin from BD Transduction Laboratories; anti-COX2 from Cayman Chemical; anti-hemagglutinin (HA) from Covance;and anti-IgG from Sigma-Aldrich. The following materials were pur-chased: rIL-1� from Calbiochem; control siRNA, Lef1 siRNA, RelAsiRNA, and c-Jun siRNA from Santa Cruz Biotechnology and AseI, NcoI,BglII, and PvuII restriction enzymes from New England Biolabs.

Culture of mouse rib chondrocytes

Primary chondrocytes were isolated from ribs of 4-day-old imprinting con-trol region mice and cultured as previously described (17). The chondro-cytes released from cartilage were suspended in DMEM supplementedwith FBS (10% (v/v); HyClone), streptomycin (50 �g/ml), and penicillin(50 U/ml) and then plated on culture dishes at 2–4 � 104 cells/cm2. Thecells reached confluence in 4–5 days.

Preparation of nuclear fractions, immunoprecipitation, andimmunoblotting

Preparation of nuclear fractions was done as described previously (17). Theresulting supernatant, containing the nuclear fraction, was retained for Lef1,RelA, and phospho-c-Jun analysis. Lamin B or �-tubulin was used as controlsfor preparation of nuclear fractions. Immunoprecipitation was performed asdescribed previously (17). Briefly, after transfecting constitutively active S37A�-catenin, HA-tagged Lef1, RelA, or c-Jun or some combination of these intoHEK293T cells or primary chondrocytes using Lipofectamine Plus (Invitro-gen), cell lysates were prepared. The lysates were precipitated with specificAbs against HA, Lef1, RelA, or phospho-c-Jun. Then the immune complexeswere collected with protein A-Sepharose beads. Immunoblotting was per-formed as described previously (32). The proteins were detected with specificAbs against �-catenin, HA, Lef1, RelA, phospho-c-Jun, COX2, and MMP13.Blots were developed using a peroxidase-conjugated secondary Ab (Sigma-Aldrich) and an ECL kit (Amersham Biosciences).

Chromosome conformation capture (3C) assay

The 3C assay was performed as described previously (8, 33). Subcon-fluent mouse chondrocytes were incubated with IL-1� (5 ng/ml) for 1 h.Chromatin cross-linking of whole cells was achieved by treating themwith formaldehyde (2% v/v) for 10 min before extracting nuclei. Thecross-linked DNA was restricted using AseI, NcoI, BglII, or PvuII andligated using T4 DNA ligase. The ligated products were analyzed byPCR. PCR conditions were 94°C for 30 s, 60°C for 30 s, and 72°C for30 s for a total of 40 or 45 cycles. PCR primers for the 3C assay wereas follows: AseI-restricted a, 5�-GGAAGTGCATGGTGTCAAAC-3�;b, 5�-ACTTAGGCTGTTGGAATTTACG-3�; a�, 5�-GAAACGTGCTCTGTGGACC-3�; NcoI-restricted A, 5�-TGCTGAGCTCCACTTCATCG-3�; B, 5�-CCCTGTCCTTCATTCGTGTG-3�; C, 5�-CATTCTTTGCCCAGCACTTCAC-3�; and C�, 5�-ATGGGTGTGATTTGTTTGGC-3�.The sizes of the products from the 3C assay were as follows: a � b (268bp), a� � a (316 bp), A � B (420 bp), A � C (442 bp), and C � C� (165bp). In the case of NcoI-restricted A � D, the primers and product sizewere follows: A, 5�-CTGTGTTTGTGTTGGCCATGGTC-3�; D, 5�-GAGAAGGAAATGGCTGCAGAATTG-3� (138-bp product); PvuII-restricted a,5�-TTAAGAGAATGCCCAAGAGTGGGTGG-3�; b, 5�-ATCCAGCTAAGACACAGCAAGCCA-3�; b�, 5�-AAGCAGAGAGGGATTAACAAACATGG-3�; BglII- restricted A, 5�-GAATGTATTCTCAGTGGGAGCTGTCG-3�; B, 5�-TGTGCAGGTGTGGTTGTATTTATGAGAG-3�; C, 5�-GTGTGGCAAACTGGTAAAGCAAAGTC-3�; and C�, 5�-GCCATCTTTCTGTTCTCACTGCTGTC-3�. The sizes of the products from the 3C assaywere as follows: a � b (100 bp), b � b� (108 bp), A � B (138 bp), A �C (143 bp), and C � C� (259 bp). In the case of BglII- restricted A � D,the primers and product size were follows: A, 5�-CCAGGAGCAGGTTAATAGATCTGTTG-3� and D, 5�-TTCCCTGGAATTGGCAACAAAGTAGA-3� (278-bp product). As a loading control, PCR was conducted withprimers for the NF-�B binding site of genomic COX2. PCR products wereresolved on 2% agarose gels or cloned into pGEM-T easy vector and se-quenced. Quantitative levels of 3C assay were analyzed by real-time PCRand normalized by input.

Chromatin immunoprecipitation (ChIP) and siRNA-coupledChIP assays

The ChIP assay was conducted as previously described (17) using subconflu-ent mouse rib chondrocytes treated with IL-1� (5 ng/ml) for 1 h. The primersused in the ChIP assays were as follows: Lef1 binding site of genomic COX2,forward 5�-AATGCTGGTGTGGAAGGTG-3� and reverse 5�-CCTATTGCATTGAGAGATGGAC-3� (325 bp product); NF-�B binding site of genomicCOX2, forward 5�-ATTAACCGGTAGCTGTGTGCG-3� and reverse 5�-AGGTGGTGCCAAGAGAGCTG-3� (263 bp product); Lef1 binding site ofgenomic MMP13, forward 5�-CATGCCAACAAATTCCATATTG-3� and re-verse 5�-CCAGCCACGCATAGTCATATAG-3� (195 bp product); AP-1binding site of genomic MMP13, forward 5�-CATTTCCCTCAGATTCTGCCAC-3� and reverse 5�-GGGAGTCCAGCTCAACAAGAAG-3� (231 bp prod-uct); and control binding site for the ChIP assays with Lef1, RelA, and phos-pho-c-Jun, forward 5�-TGGTTATAATGAAGCAAGTGGC3� and reverse 5�-TGTATGTCATGTGCTTGTCTGG-3� (281 bp product). As a loadingcontrol, the PCR was conducted directly on input DNA purified from chro-matin before immunoprecipitation. The PCR products were resolved on 2%agarose gels. For the analysis of physiological interaction between Lef1 andRelA or c-Jun, anti-p65/RelA or anti-phospho-c-Jun Abs were used for im-munoprecipitation, and then PCR was performed with primers for the Lef1binding site of genomic COX2 or MMP13. Otherwise, PCR was performedwith primers for the NF-�B binding site of genomic COX2 or for the AP-1binding site of genomic MMP13 after immunoprecipitation using anti-Lef1Ab. For the siRNA-coupled ChIP assay, cells were transiently transfected withsiRNAs using Lipofectamine Plus for 24 h before IL-1� treatment. Quantita-tive levels of ChIP assay were analyzed by real-time PCR and normalized byinput DNA.

RT-PCR

RT-PCR was performed as previously described (17). PCR conditionswere 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s for a total of 35cycles. PCR primers used were as follows: COX2, forward 5�-TGGCTGCAGAATTGAAAGCCCT-3� and reverse 5�-AAAGGTGCTCGGCTTCCAGTAT-3� (191 bp product); MMP13, forward 5�-TGATGGACCTTCTGGTCTTCTGGC-3� and reverse 5�-CATCCACATGGTTGGGAAGTTCTG-3� (473 bp product); Lef1, forward 5�-ACAGCGACCCGTACATGTCAAA-3� and reverse 5�-TGGACATGCCTTGCTTGGAGTT-3� (195 bp product); GAPDH, forward 5�-TCACTGCCACCCAGAAGAC-3� and reverse 5�-TGTAGGCCATGAGGTCCAC-3� (450 bpproduct); and L32, forward 5�-GCCCAAGATCGTCAAAAAGA-3�

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and reverse 5�-GTGAGCAATCTCAGCACAGT-3� (217 bp product).PCR products were resolved on 2% agarose gels with GAPDH as aloading control or PCR products were analyzed by real-time PCR nor-malized with L32 products.

Bioinformatic analysis

Mouse and human COX2 or MMP13 loci were aligned, and the extent ofDNA sequence homology was computed with the web-based programVISTA (http://www-gsd.lbl.gov/vista) (34, 35) or ECR browser (36). Lef1and NF-�B or AP-1 binding sites were predicted with rVISTA 2.0 (http://rvista.dcode.org) using the optimum matrix similarity.

Statistical analysis

For the statistical analysis of the data, Student’s t tests were applied onquantification experiments. A value of p � 0.05 was considered signifi-cantly different.

ResultsIL-1� increases the binding of transcription factors in the 5�and 3� regions

We previously reported that IL-1� induces NF-�B activation inprimary chondrocytes and that NF-�B is involved in the up-regu-lation of Lef1 transcription (17). Lef1 is required for IL-1�-me-diated up-regulation of COX2 expression, and bioinformatic anal-ysis showed that the 5� and 3� regions of the COX2 (Ptgs2) genecontain conserved elements for binding of NF-�B (black arrow)and Lef1 (gray arrow), respectively (Fig. 1A, top) (30, 31). Thesedata suggested a functional cooperation between Lef1 and NF-�B

for COX2 gene transcription and led us to ask whether there wasa physical interaction between these two transcription factorsbound to either end of the COX2 gene.

Since MMP13 expression is also up-regulated by IL-1� in aLef1-dependent manner (31), we performed a similar analysis forthe MMP13 gene. We had previously shown that, similar to theCOX2 (Ptgs2) gene, the MMP13 gene also contains a conservedLef1 binding site in the 3� region (30, 31). However, bioinformaticanalysis did not reveal conserved binding sites for NF-�B in the 5�region of the MMP13 gene. We therefore examined the role ofother inducible transcription factors that could potentially be in-volved in IL-1�-mediated up-regulation of MMP13. The AP-1transcription factor, which is composed of Jun-Jun or Fos-Jundimers, was an obvious candidate, since the 5� region of theMMP13 gene contains a conserved AP-1 binding site (black ar-row) as described previously (Fig. 1A, bottom) (37).

We first assessed the activation of NF-�B and AP-1 in IL-1�-stimulated primary mouse chondrocytes by examining the nucleartranslocation of RelA and phosphorylated c-Jun (phospho-c-Jun)(38). Indeed, immunoblotting of nuclear extracts prepared frommouse chondrocytes showed a clear increase in nuclear RelA andphospho-c-Jun upon IL-1� stimulation for 1 h (Fig. 1B). Further-more, ChIP assays showed that IL-1� stimulation of mouse chon-drocytes dramatically increased the levels of RelA and phospho-c-Jun binding under physiological conditions (i.e., in intact cells)to the 5� region of COX2 (Ptgs2) and MMP13 gene, respectively

FIGURE 1. IL-1� increases the binding of transcription factors in the 5� and 3� regions. A, Vista analysis of the NF-�B and Lef1 binding sites on COX2(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-�Bbinding site of COX2 or AP-1 binding site of MMP13. The gray arrows denote the conserved Lef1 binding sites of each genome. B, Mouse chondrocyteswere 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 withRelA 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 MMP13and 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 orphospho-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 withprimers specific for the conserved Lef1 binding site of COX2 or MMP13. PCR products were resolved on 2% agarose gels. Input denotes the PCR productsobtained from genomic DNA without immunoprecipitation. The data shown in B–D are representative of three independent experiments with similar results.In each experiment, primary chondrocytes were isolated from ribs of 10 mice.

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(Fig. 1C, left and middle, and supplemental Fig. 1A6 for quantita-tive analysis). There was no detectable binding of these transcrip-tion factors to a control region of the COX2 (Ptgs2) gene (Fig. 1A),which contains neither a NF-�B nor AP-1 binding motif (Fig. 1C,right). We have previously reported that Lef1 binds to the con-served 3� binding elements in the COX2 (Ptgs2) and MMP13genes by ChIP (30, 31). IL-1� stimulation increased Lef1 bindingto the 3� region of COX2 or MMP13 genes (Fig. 1D and supple-mental Fig. 1B) (30, 31) as well as the binding of RelA or c-Jun tothe 5� region of the COX2 or MMP13 gene (Fig. 1C). These resultssuggest the possibility of functional cooperation between tran-scription factors for the regulation of Lef1 target genes.

Interactions of Lef1 with RelA or c-Jun up-regulatetranscription of Lef1 target genes COX2 or MMP13

We next examined the interactions among Lef1, �-catenin, RelA,and c-Jun by coimmunoprecipitation assays in HEK293 cells andprimary chondrocytes. Immunoprecipitates of HA-tagged Lef1 aswell as RelA contained �-catenin as expected (Fig. 2A, lanes 3 and4) (39). RelA were also coimmunoprecipitated with Lef1-HA (Fig.2A, lane 5). Consistent with a previous report (40), immunopre-cipitates of phospho-c-Jun contained Lef1-HA as well (Fig. 2B).

To confirm whether these interactions also occur in mouse chon-drocytes, we performed coimmunoprecipitation assays after over-expression of Lef1 and RelA or c-Jun. Immunoprecipitates ofRelA or Lef1 revealed their coexistence with Lef1 or RelA, re-spectively (Fig. 2C, lanes 3 and 4). In vivo association betweenphospho-c-Jun and Lef1 was also confirmed in chondrocytes (Fig.2D, lanes 3 and 4).

We also assessed the functional relevance of these in vivo in-teractions in mouse chondrocytes, in which target gene expressionwas monitored by real-time PCR. When Lef1 and a constitutivelyactive �-catenin were coexpressed with RelA in mouse chondro-cytes, we observed an up-regulation of COX2 mRNA within 24 h,much greater than that elicited by Lef1/�-catenin or RelA alone(Fig. 2E). Similarly, coexpression of Lef1/�-catenin and c-Jun inprimary mouse chondrocytes resulted in synergistic up-regulationof MMP13 mRNA expression within 24 h (Fig. 2F). These resultssuggest that physical interactions between Lef1 and RelA or phos-pho-c-Jun cooperatively mediate the transcription of Lef1 targetgenes COX2 or MMP13, respectively.

IL-1� induces juxtaposition of the 5� region and the 3� region ofgenomic COX2 and MMP13

The above data suggest physical as well as functional interactionsbetween Lef1-binding 3� elements and NF-�B or AP-1-binding 5�6 The online version of this article contains supplemental material.

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 wereimmunoprecipitated (IP). Coprecipitated �-catenin or Lef1-HA was analyzed by immunoblotting (IB) with �-catenin or HA Ab. B, c-Jun with or withoutHA-tagged Lef1 was transiently overexpressed in HEK293T cells, and the lysates were immunoprecipitated with phospho-c-Jun Ab. CoprecipitatedLef1-HA was analyzed by immunoblotting with a HA Ab. C, Lef1 and RelA were transiently transfected into chondrocytes and then Lef1 and RelA wereimmunoprecipitated. Coprecipitated RelA or Lef1 were analyzed by immunoblotting with RelA or Lef1 Ab. D, Lef1 and c-Jun were transiently transfectedinto chondrocytes and then Lef1 and phospho-c-Jun were immunoprecipitated. Coprecipitated c-Jun or Lef1 were analyzed by immunoblotting withphospho-c-Jun or Lef1 Ab. The data shown in A–D are representative of three independent experiments with similar results. E, Chondrocytes weretransfected with empty vector (mock) or HA-tagged Lef1 and constitutively active �-catenin (�-cat) with or without RelA for 24 h. COX2 mRNA wasanalyzed by real-time PCR and expressed relative to the mock sample after normalizing with mouse L32. F, Chondrocytes were transfected with emptyvector (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. Thedata shown in E and F are the mean of three independent experiments, and error bars indicate SDs. Primary chondrocytes were isolated from ribs of 10mice in each independent experiment.

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regions of the COX2 or MMP13 genes, respectively (Fig. 1A). Wetherefore proceeded to evaluate our hypothesis that during IL-1�-driven transcription of the COX2 and MMP13 genes, looping in-teractions mediated by these transcription factors occurred be-tween the two ends of these transcribed genes. To test theformation of these postulated gene loops, we performed chromo-some conformation capture (3C) assays (8, 33). We selected therestriction enzymes Ase1 and NcoI for COX2 or PvuII and BglIIfor MMP13, since they can cut appropriate restriction sites, in-cluding the 3� and 5� regions of each genomic locus (Fig. 3A).Chondrocytes were stimulated with IL-1� or left unstimulated,then treated with formaldehyde to fix their DNA conformation.Then the cross-linked DNA complexes were digested with the re-striction enzymes and treated with ligase to join together ends ofDNA which were in reasonable physical proximity. The ligatedproducts were analyzed by PCR using primer pairs specific for therestriction enzyme-containing regions (Fig. 3B). The identity of allof the products was confirmed by DNA sequencing (data notshown).

In the 3C assay, ligated products can be obtained only when therestriction sites are close to one another (33). As a result, regard-less of IL-1� stimulation, we obtained ligated products betweenthe A and B in the NcoI sites of COX2 or BglII sites of MMP13genes (Fig. 3, B and C, left top panels), presumably due to theirphysical proximity in the primary DNA sequence. We did not ob-tain ligated products between the more distant A and C sites underthe same conditions (Fig. 3, B and C, left second panels). In con-trast, ligated products between the A and D sites, located at the 5�and 3� ends of these genes, respectively, were obtained for bothgenes only in the sample stimulated with IL-1� (Fig. 3, B and C,

left third panels). The amount of input DNA was the same in bothIL-1�-untreated and treated samples (Fig. 3, B and C, left fourthpanels).

To further confirm the formation of an IL-1�-dependent loopbetween the 5� and 3� ends of the COX2 or MMP13 genes, werepeated the 3C assay with the restriction enzymes AseI or PvuII,which cut near the Lef1 binding sites (Fig. 3A, site b) as well as inthe 5� regions of both genes (Fig. 3A, site a). Again, the AseI- orPvuII-restricted products of the 3C assay were obtained only uponIL-1� stimulation (Fig. 3, B and C, right panels). As a control (41),we proceeded the 3C assay in the absence of cross-linking or li-gation. The PCR products were dramatically decreased in the ab-sence of cross-linking (supplemental Fig. 2A) or ligase treatment(supplemental Fig. 2B). Taken together, these results demonstratethat IL-1� stimulation induces a conformational change in COX2and MMP13 gene loci to form gene loops, such that the 3� regionwhich binds Lef1 is in close proximity to the 5� region.

Lef1 plays crucial roles in the regulation of transcription andgene looping of Lef1 target genes COX2 or MMP13

To elucidate the role of Lef1 in the gene looping, we knockeddown Lef1 expression using Lef1-directed siRNA and tested itseffect on gene looping. Transfection of Lef1 siRNA into primarychondrocytes decreased Lef1 transcripts and also diminished the

FIGURE 3. IL-1� induces juxtaposition of the 5� and the 3� regionsof genomic COX2 and MMP13. A, AseI or NcoI cleavage sites in themouse genomic COX2 locus and PvuII or BglII cleavage sites in themouse genomic MMP13 are illustrated: the black horizontal arrows de-note the 3C primers for the AseI restriction sites (COX2) or PvuII restric-tion sites (MMP13); the empty horizontal arrows denote the 3C primers forthe NcoI restriction sites (COX2) or BglII restriction sites (MMP13), re-spectively. B, Mouse chondrocytes were incubated with IL-1� for 1 h andthe 3C assays restricted by NcoI or AseI were performed in the COX2locus. C, 3C assays restricted by BglII or PvuII was performed in theMMP13 locus. Input represents products of PCRs using primers specific forthe conserved NF-�B binding site of COX2 performed to establish thatequal amounts of template DNA were present in the samples. The datashown in B and C are from one of at least three independent experiments,all of which yielded similar results. Primary chondrocytes were isolatedfrom ribs of 10 mice in each independent experiment.

FIGURE 4. Lef1 plays a crucial role in the gene looping. A, Chondro-cytes were transfected with Lef1 or control siRNA and then stimulatedwith IL-1� (left). Chondrocytes were transfected with empty or a Lef1expression vector for 24 h (right). The mRNA levels of Lef1, COX2, andMMP13 were analyzed by RT-PCR. GAPDH was analyzed as a loadingcontrol. B, Left, chondrocytes were transfected with Lef1 or control siRNAfor 24 h and IL-1� was further added for 1 h. The 3C assay was performedwith AseI restriction for COX2 or PvuII restriction for MMP13. Right,Chondrocytes were transfected with empty or Lef1 expression vector for24 h and the 3C assay was performed with AseI restriction for COX2 orPvuII restriction for MMP13. Input represents PCR products of a conservedNF-�B binding site of COX2. C, After incubation with IL-1�, physiolog-ical interaction of Lef1 and RelA or c-Jun were shown by ChIP assays: left(upper), RelA or Lef1 was immunoprecipitated and PCR was performedwith primers specific for the conserved Lef1 or NF-�B binding site ofCOX2. Right (upper), phospho-c-Jun or Lef1 was immunoprecipitated andPCR was performed with primers specific for the conserved Lef1 or AP-1binding site of MMP13. Input denotes the PCR products obtained withgenomic DNA without immunoprecipitation. Left (lower), Lef1 was im-munoprecipitated and PCR was performed with primers specific for theconserved 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 productsobtained with genomic DNA without immunoprecipitation. The datashown in A–C are from one of at least three independent experiments thatyielded similar results. Primary chondrocytes were isolated from ribs of 10mice in each independent experiment.

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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 quantitativeanalysis). 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 PvuIIrestriction for MMP13 (Fig. 4B, left panels, and supplemental Fig.3B for quantitative analysis). We also evaluated the effect of Lef1overexpression, in the absence of IL-1� stimulation, on transcriptlevels and gene looping of COX2 and MMP13. Cells transfectedwith Lef1 showed an increase in both gene expression and 3Cproduct, whereas cells transfected with empty vector showed nei-ther (Fig. 4, A and B, right panels, and supplemental Fig. 3, A andB, for quantitative analysis). Together, these data indicate a crucialrole of Lef1 in gene looping and also suggest that gene looping iscorrelated with active transcription.

We next asked whether the IL-1�-mediated gene looping couldbe mediated by the physical interaction of 3� region-bound Lef1with 5� region-bound NF-�B or AP-1 in COX2 and MMP13 loci,respectively. Accordingly, we performed ChIP analysis. After im-munoprecipitation with a RelA or phospho-c-Jun Ab, PCR wasperformed with primers specific for each Lef1 binding site in the3� regions of the COX2 or MMP13 genes. Conversely, after im-munoprecipitation with a Lef1 Ab, PCR was performed with prim-ers specific for the 5� NF-�B or AP-1 binding sites of the COX2 orMMP13 genes, respectively. The amount of Lef1-bound DNA onthe 3� region at the COX2 locus was enriched after IL-1� treat-ment, as shown following immunoprecipitation with a RelA Ab(Fig. 4C, upper left top, and supplemental Fig. 3C for quantitativeanalysis). Likewise, NF-�B (RelA)-bound DNA at the promoterwas also increased after immunoprecipitation with a Lef1 Ab (Fig.4C, upper left middle, and supplemental Fig. 3C for quantitativeanalysis). We observed similar results from ChIP analysis in theMMP13 locus. The ChIP products in the MMP13 genomic locuswere increased after IL-1� stimulation (Fig. 4C, right, and sup-plemental Fig. 3C for quantitative analysis). To show the speci-ficity of Lef1 binding to the RelA-binding 5� region at the COX2locus or c-Jun binding to Lef1-binding 3� region at the MMP13locus, ChIP assay was performed. ChIP analysis in Bone morpho-genetic protein 2 (BMP2) or cyclin D1 loci was also performed asnegative controls (Fig. 4C, bottom). The BMP2 gene contains aNF-�B-binding element in the �838/�829 region (42). The Cy-clin D1 gene has a Lef1 binding site in the �81/-73 region (43).After immunoprecipitation with a Lef1 or c-Jun Ab, PCR wasperformed with primers specific for the NF-�B (RelA) or Lef1binding site of the BMP2 or cyclin D1 gene, respectively. Lef1 didnot interact with the NF-�B binding site of the 5� region in theBMP2 gene, and c-Jun did not bind to the Lef1 binding sites of the5� region in the cyclin D1 gene, respectively. Taken together, thesedata suggest that the 3� region-bound Lef1 interacts with the RelA-binding 5� region of the genomic COX2 locus. In the MMP13transcription, the 3� region-bound Lef1 physiologically cooperateswith the AP-1-binding 5� region in an IL-1� stimulation-depen-dent manner. Indeed, co-overexpression of RelA and Lef1/�-cate-nin resulted in synergistic up-regulation of COX2 expression (Fig.2E). MMP13 expression was also increased by co-overexpressionof c-Jun and Lef1/�-catenin (Fig. 2F).

Knockdown of RelA or c-Jun reduces the interactions of Lef1with 5� region of COX2 or MMP13

Interaction of Lef1 with the 5� region-bound RelA or c-Jun inCOX2 or MMP13 locus enhanced their transcription (Fig. 2)through gene looping (Figs. 3 and 4). To further elucidate the roleof RelA or c-Jun in these associations, we analyzed their knock-

down effect on COX2 or MMP13 transcription and gene looping.Transfection of RelA siRNA or c-Jun siRNA into primary chon-drocytes decreased the levels of COX2 or MMP13 gene expressionand protein levels, respectively, (Fig. 5A and supplemental Fig. 5,B and C). Lef1 siRNA treatment reduced gene looping between the5� region and 3� region of COX2 or MMP13 genes (Fig. 4B). Wefurther tested whether treatments of siRNAs for Lef1, RelA, orc-Jun directly diminish the physical interaction of the 5� regionbinding RelA or c-Jun with the 3� region binding Lef1 in COX2and MMP13 loci, respectively. After Lef1 siRNA treatment andthen immunoprecipitation with RelA or phospho-c-Jun Ab, PCRwas performed with primers specific for the Lef1 binding site inthe 3� regions of the COX2 or MMP13 genes. As expected, Lef1siRNA treatment diminished the interactions between 5�-boundtranscription factors and the Lef1-bound 3� region (Fig. 5B andsupplemental Fig. 4A for quantitative analysis). Next, we assessedthe effect of siRNA treatment for RelA or c-Jun on these interac-tions. After RelA siRNA or c-Jun siRNA treatment, ChIP wasperformed with a Lef1 Ab. Enriched DNA was then analyzed byPCR with primers specific for the 5� NF-�B or AP-1 binding sitesof the COX2 or MMP13 genes, respectively. Treatment with RelAsiRNA or c-Jun siRNA reduced NF-�B or AP-1-bound DNA lev-els at the 5� region (Fig. 5C, top, and supplemental Fig. 4B for

FIGURE 5. Knockdown of RelA or c-Jun reduces Lef1 binding to the 5�region of COX2 or MMP13. A, Chondrocytes were transfected withsiRNAs for RelA, c-Jun, or with control siRNA and then stimulated withIL-1�. The mRNA levels of COX2 or MMP13 were analyzed by real-timePCR and expressed relative to control siRNA after normalizing with mouseL32. The data shown are mean of three independent experiments and errorbars indicate SDs. Significance was determined by Student’s t test. �, p �0.05 and ��, p � 0.01. B, Physiological binding of RelA or c-Jun to the 3�region was shown by ChIP assays: left, RelA or Lef1 was immunoprecipi-tated (IP) after treating Lef1 siRNA and PCR was performed with primersspecific for the conserved Lef1 binding site of COX2. Right, Phospho-c-Junor Lef1 was immunoprecipitated after treating Lef1 siRNA and PCR wasperformed with primers specific for the conserved Lef1 binding site ofMMP13. Input denotes the PCR products obtained with genomic DNAwithout immunoprecipitation. C, Physiological binding of Lef1 to the 5�region was shown by ChIP assays: left, Lef1 or RelA was immunoprecipi-tated after treating RelA siRNA and PCR was performed with primersspecific for the conserved NF-�B binding site of COX2. Right, Lef1 orphospho-c-Jun was immunoprecipitated after treating c-Jun siRNA andPCR was performed with primers specific for the conserved AP-1 bindingsite of MMP13. The data shown in B and C are representative of threeindependent experiments with similar results. Primary chondrocytes wereisolated from ribs of 10 mice in each independent experiment.

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quantitative analysis). Taken together, these data suggest that the3� region-bound Lef1 interacts with the 5� region-bound RelA orc-Jun on the COX2 or MMP13 locus, respectively.

Interaction of Lef1 with RelA or c-Jun cooperatively regulatesgene looping

Knock-down of Lef1, RelA or c-Jun suppressed the expression ofLef1 target genes in chondrocytes, and reduced the associationbetween 5�-bound RelA or c-Jun and 3�-bound Lef1 (Fig. 5). Totest the role of RelA or c-Jun in the formation of gene loops, 3Cassays was performed after siRNA treatment for RelA or c-Jun.We used the restriction enzymes AseI or PvuII, which cut near theRelA or c-Jun binding sites of COX2 or MMP13, respectively (Fig.3A). Knock-down of RelA reduced the efficiency of gene loopingassessed by formation of the ligation product between sites a andb in AseI restriction for COX2 (Fig. 6A). Similarly, c-Jun siRNAsuppressed the loop formation as assessed by PvuII restriction forMMP13 (Fig. 6A). These data showed that gene looping in COX2or MMP13 is also mediated by RelA or c-Jun as well as Lef1.

The above data also suggest that intrachromosomal interactionsare mediated through the interaction of transcription factors bind-ing to the 5� and 3� region of COX2 or MMP13 genes. To confirmthe cooperative role of Lef1, RelA or c-Jun in the formation of aloop between the 5� and 3� region of the COX2 or MMP13 genes,chondrocytes were transfected with expression vectors of Lef1,RelA or c-Jun in the absence of IL-1� stimulation. In the COX2gene locus, loop formation was increased by over-expression ofLef1 or RelA. In addition, this loop formation was further in-creased by over-expression of Lef1 and RelA together (Fig. 6B),which mediated additive up-regulation of COX2 transcription (Fig.

2E). Likewise, gene looping between 5� and 3� region of MMP13was increased by coexpression of Lef1 and c-Jun together com-pared with individual over-expression of either alone (Fig. 6C).This result was also in accordance with expression pattern ofMMP13 (Fig. 2F). Taken together, these results demonstrate thatIL-1� stimulation induces intrachromosomal interaction in COX2or MMP13 gene loci to form gene loops, which is mediated byjuxtaposition between the Lef1-binding 3� region and the RelA orc-Jun-binding 5� region. Collectively, these results suggest the crit-ical role of transcription factors in gene looping and the closecorrelation of gene looping with active gene expression.

DiscussionCOX2 and MMP13 are critical molecules involved in the immuneresponse and inflammation of rheumatoid arthritis and osteoarthri-tis. They are primarily produced by chondrocytes of arthritic car-tilage. COX2 catalyzes the production of PGE2 which mediatesjoint inflammation. MMP13 is a proteolytic enzyme that degradesthe extracellular matrix of cartilage tissues (44). We previouslyreported that Lef1 is highly expressed in osteoarthritic cartilageand induced by IL-1� in mouse chondrocytes (17). We alsoshowed that proinflammatory cytokine IL-1� up-regulates COX2and MMP13 levels by increasing Lef1 binding to the 3� regions oftarget genes (30, 31). In this study, we further elucidated how the3� region-binding Lef1 regulates its target genes COX2 andMMP13 in chondrocytes.

We have demonstrated that Lef1-mediated regulation of targetgenes in chondrocytes COX-2 or MMP13 correlates with the loop-ing contact of the Lef-1-binding 3� region with 5� regions which

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. Inputdenotes the PCR products obtained with genomic DNA without immunoprecipitation using primers specific for the conserved NF-�B binding site of COX2.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 transfectedwith 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 byreal-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 yieldedsimilar results. The error bars show the SDs of the PCR performed in triplicate. Primary chondrocytes were isolated from ribs of 10 mice in eachindependent experiment.

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bound by the inducible transcription factors NF-�B or AP-1, re-spectively. It has been suggested that gene looping is associatedwith transcription reinitiation, a cyclic process of RNA synthesis inactive genes (5). To maintain the accelerated transcription rate,RNA polymerases and transcription factors need to be recycled(45). The formation of a gene loop, in which both ends of thetranscription unit are juxtaposed, could physically stabilize the ac-tive transcriptional machinery and direct new rounds of transcrip-tion. This would eventually result in an accelerated transcriptionrate and/or an increase in transcriptional efficiency (5).

The formation of gene loops in RNA polymerase II-mediatedtranscription has been previously reported. The incidence of genelooping was determined by the phosphorylation status of RNApolymerase II (8, 10). Cleavage and polyadenylation factor 3�-endprocessing machinery also functions in RNA polymerase II-depen-dent gene looping (7). TFIIB is suggested as another connector ofgene looping (9). Our study suggests that an architectural tran-scription factor, Lef1, is associated with the gene looping in mam-malian cells; 3� region-bound Lef1 induced gene looping throughthe interaction of 5� region-bound transcription factors NF-�B orAP-1. In addition, our results also suggest that gene looping inmammalian cells is involved in the early stages of transcriptionalactivation (8), since transcription factors usually bind to DNA be-fore formation of the preinitiation complex formation, necessitat-ing recycling.

Binding of Lef1 to its consensus DNA sequence induces a sharpbending in DNA structure (46). Interactions of 3� region-boundLef1 with other factors can direct the bending of DNA. Mostknown transcription factors bind to promoter regions. Therefore,the bent DNA in the 3� region will result in the formation of a geneloop by the turn toward the promoter bound by diverse transcrip-tion factors. Such a role of the 3� region-bound Lef1 is consistentwith the formation of a gene loop which is mediated by interactionwith other transcription factors. Indeed, in immune cells, Lef1/�-catenin regulate TCR-� and HIV-1 transcription in a chromatin-dependent manner by recruiting chromatin remodeling complexes(47). �-Catenin, a well-known Lef1-binding transcriptional coac-tivator, plays crucial roles both in connecting DNA to RNA poly-merase II transcription machinery and in chromatin remodeling;Pygo and Lgs link �-catenin to the RNA transcription machinery(22–24) and parafibromin/hyrax is physically connected to the C-terminal of �-catenin with platelet-activating factor 1 (25). Thesefunctions of Lef1/�-catenin might be appropriate for gene loopingand thus recycling of RNA transcription machinery. Thus, theremight be cooperative roles between Lef1/�-catenin and remodel-ing factors for inducing gene looping. Initiation of local DNAbending by Lef1 might lead to the formation of a gene loopthrough chromatin remodeling, which is mediated by �-catenin-interacting proteins. Taken together, locus-specific looping medi-ated by Lef1 binding can provide the mechanism for specific andefficient transcription of Lef1 target genes. Furthermore, the con-servation of the Lef1 binding site in the 3� region might be evo-lutionarily developed to facilitate gene looping.

Stimulation induces more efficient transcription by inducing theinteractions between transcription factors through looping mech-anisms. IL-1� stimulation up-regulated the protein level of Lef1 or�-catenin in chondrocytes (30). Interestingly, at the same time, wealso observed an increase of NF-�B or AP-1 translocation into thenucleus upon IL-1� stimulation (Fig. 1B). These imply that IL-1�stimulation may facilitate the cooperative interaction between di-verse transcription factors, resulting in efficient expression of tar-get genes. Interactions between proteins bound at nonadjacent sitesmediate bending or looping of the DNA and generate the higher-order nucleoprotein complex (48). Architectural proteins induce

the formation of these complexes by bending DNA to facilitate theinteractions between cis-acting elements. Widely separated cis-act-ing regulatory elements at gene loci evidence the functional inter-action between regulatory elements (49) and provide a mechanismfor specific and efficient transcription of genes. Stimulation-depen-dent DNA looping mediated by activated transcription factorsshould be efficient in terms of energy expenditure and rate of tran-scription. We showed the pivotal role of Lef1 in gene looping ofCOX2 or MMP13 under IL-1� signaling. Besides Lef1/�-catenin,NF-�B, and AP-1, other transcription factors may also be involvedin gene looping following IL-1� stimulation. Indeed, knockdownof Lef1 (Fig. 4B), RelA, or c-Jun (Fig. 6A) decreased the incidenceof gene looping while overexpression of them induced looping(Figs. 4B and 6, B and C). In addition, overexpression of Lef1along with its 5� region-binding partner RelA or c-Jun additivelyup-regulated gene looping (Fig. 6, B and C) as well as up-regula-tion of Lef1 target genes COX2 and MMP13 (Fig. 2, E and F).However, further studies are needed to determine how the associ-ation between Lef1 and RelA or c-Jun is mediated for inducing agene loop.

Interactions between DNA-bound transcription factors directformation of an enhanceosome, which induces transcriptional syn-ergy throughout the gene from the promoter to the terminator (50,51). Multiple interactions between transcription factors maystrengthen the assembly of enhanceosomes and facilitate the for-mation of gene loops. As suggested in yeast, RNA polymeraseII-mediated transcription may be facilitated by gene looping.Long-distance associations between regulatory elements may playan important role in eukaryotic gene regulation. Inactivation ordetachment of transcription factors from enhanceosomes could de-stabilize gene loops, leading to termination of transcription. There-fore, the transcription factors involved in gene looping may formthe mechanism whereby transcription is switched on and off. In-vestigation of 3�-bound transcription factors may reveal furtherdiverse gene looping systems.

In conclusion, Lef1 mediates COX2 and MMP13 gene loopingduring RNA polymerase II-mediated transcription in mammaliancells. Formation of a DNA loop as a transcription unit could in-crease the efficiency of RNA polymerase II-mediated transcriptionby increasing the transcription rate while conserving energy, aneffect that could have particular significance in the mammaliansystem, where the typical transcription unit is relatively long. Genelooping may constitute a mechanism for inducible transcriptionalregulation in mammalian cells and 3� region-bound transcriptionfactors may function as connectors forming integrated transcrip-tion units.

AcknowledgmentsWe thank specially Dr. Anjana Rao for priceless advice on manuscriptorganization. We also thank Darren Reece Williams for critical proof read-ing of this manuscript and Dr. E.-J. Cho for kind discussion in experimentaldesign.

DisclosuresThe authors have no financial conflict of interest.

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