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  • Transcription factors mediate long-rangeenhancerpromoter interactionsIlias K. Nolisa,1, Daniel J. McKayb,1, Eva Mantouvaloua, Stavros Lomvardasc,2, Menie Merikaa, and Dimitris Thanosa,3

    aInstitute of Molecular Biology, Genetics and Biotechnology, Biomedical Research Foundation, Academy of Athens, 4 Soranou Efesiou Street, Athens 11527Greece; and bIntegrated Program in Cellular, Molecular and Biophysical Studies and cDepartment of Biochemistry and Molecular Biophysics, ColumbiaUniversity, New York, NY 10032

    Edited by Tom Maniatis, Harvard University, Cambridge, MA, and approved October 9, 2009 (received for review March 5, 2009)

    We examined how remote enhancers establish physical commu-nication with target promoters to activate gene transcription inresponse to environmental signals. Although the natural IFN-enhancer is located immediately upstream of the core promoter, italso can function as a classical enhancer element conferring virusinfection-dependent activation of heterologous promoters, evenwhen it is placed several kilobases away from these promoters. Wedemonstrated that the remote IFN- enhancer loops out theintervening DNA to reach the target promoter. These chromatinloops depend on sequence-specific transcription factors bound tothe enhancer and the promoter and thus can explain the specificityobserved in enhancerpromoter interactions, especially in complexgenetic loci. Transcription factor binding sites scattered betweenan enhancer and a promoter can work as decoys trapping theenhancer in nonproductive loops, thus resembling insulator ele-ments. Finally, replacement of the transcription factor binding sitesinvolved in DNA looping with those of a heterologous prokaryoticprotein, the repressor, which is capable of loop formation,rescues enhancer function from a distance by re-establishingenhancerpromoter loop formation.

    DNA looping transcription transcription factors

    The accurate execution of gene expression programs duringdevelopment and differentiation and in response to envi-ronmental cues requires 3 types of regulatory DNA elements inhigher eukaryotes: core promoters, upstream promoter ele-ments, and enhancers (1). Core promoters function by providingthe blueprints for the assembly of functional pre-initiationcomplexes executing both the mechanics and the accurateinitiation of mRNA synthesis (2). Upstream promoter elementsare localized within the first 100200 bp upstream of the corepromoter and contain transcription factor binding sites, func-tioning to increase the rate of transcription by promotingassembly of pre-initiation complexes (3). Enhancers, on theother hand, can be located either upstream or downstream ofthe promoter, on the same or on different chromosomes (4).Enhancer elements do not simply fine-tune promoter activitybut rather are critical for defining the expression patterns ofgenes (5, 6).

    Recent studies have shown that gene activation by remoteenhancers is associated with long-range interactions betweenregulatory elements (chromatin loop formation) (710). It ishypothesized that proteins bound to remote enhancers interactdirectly with proteins bound to promoters, with the interveningDNA being looped out (1114). Furthermore, it also has beenproposed that enhancer complexes migrate along the chromatinfiber until they encounter a functional promoter (facilitatedtracking model) (15, 16). The intervening chromatin betweenthe enhancer and the promoter loops out as the enhancercomplex moves progressively along the chromatin fiber towardthe promoter. The physical proximity between the enhancer andthe target promoter stimulates assembly of a functional pre-initiation complex on the promoter, thus causing activation oftranscription. Interestingly, some studies suggest that RNA

    polymerase II (PolII) is recruited to the enhancers and thenvia DNA looping and/or facilitated tracking appears on thepromoter (17).

    The molecular basis for DNA looping is not yet clear, althoughinteractions between structural proteins, transcription factors, orgeneral transcription factors within transcription factories havebeen implicated in enhancerpromoter interactions (18). We donot know, in molecular terms, how these interactions are estab-lished and maintained. This paper reports our investigations ofthe mechanisms by which a remote enhancer reaches a targetpromoter; that is, whether the transcription factors bound toenhancers and promoters are the ones that provide the contactsurfaces for these interactions.

    We addressed these issues by examining how the IFN-enhancer activates transcription when positioned away from itspromoter. Although, the natural IFN- enhancer is locatedimmediately upstream of the core promoter, it also can functionas a classical enhancer element conferring virus inducibility toheterologous promoters, even when it is placed several kilobasesaway from these promoters (19, 20). The nearly completemolecular picture of the mechanisms by which the IFN-enhancer activates transcription in its natural context (2124)provided a useful tool for investigating the nature of the inter-actions between enhancers and promoters. Our experimentssuggest that sequence-specific transcription factors bound toenhancers and promoters mediate loop formation and can thusexplain the specificity observed in enhancerpromoter interac-tions, especially in complex genetic loci.

    ResultsEnhancer Action from a Distance Requires Upstream Promoter Ele-ments. To investigate whether the IFN- enhancer can activatetranscription at a distance from the core promoter, we trans-fected HeLa cells with the constructs shown in Fig. 1. In thenatural cis arrangement, the IFN- enhancer/core promoterresponds to virus infection by stimulating transcription 100-fold (line 1). Roughly equivalent levels of transcriptional acti-vation were obtained when the IFN- enhancer was positioned75 bp away from the core promoter (line 2). However, when theIFN- enhancer was placed 220 bp upstream of the corepromoter, the levels of activated transcription were significantlyreduced (line 3). The IFN- enhancer was practically inactive

    Author contributions: I.K.N., D.J.M., E.M., S.L., M.M., and D.T. designed research; I.K.N.,D.J.M., E.M., S.L., and M.M. performed research; I.K.N., D.J.M., E.M., S.L., M.M., and D.T.analyzed data; and D.T. wrote the paper.

    The authors declare no conflict of interest.

    This article is a PNAS Direct Submission.

    Freely available online through the PNAS open access option.

    1I.K.N. and D.J.M. contributed equally to this work.

    2Present address: Department of Anatomy, UCSF Mission Bay, 1550 4th Street, UCSF, SanFrancisco, CA 94158.

    3To whom correspondence should be addressed. E-mail: thanos@bioacademy.gr.

    This article contains supporting information online at www.pnas.org/cgi/content/full/0902454106/DCSupplemental.

    2022220227 PNAS December 1, 2009 vol. 106 no. 48 www.pnas.orgcgidoi10.1073pnas.0902454106

    http://www.pnas.org/cgi/content/full/0902454106/DCSupplementalhttp://www.pnas.org/cgi/content/full/0902454106/DCSupplemental

  • when placed 560 bp away (line 4) or 2.3 kbp away (line 5). Theseresults indicate that the IFN- enhancer requires proximalpromoter elements to operate at a distance. A similar prereq-uisite was observed for the Ig enhancer, which depends on aproximal (promoter) bound octamer factor to mediate enhancerfunction from a distance (25). Therefore, we replaced the IFN-core promoter with the thymidine kinase (TK) promoter, which,in addition to the core promoter elements, bears upstreamregulatory binding sites for the Sp1 and CCAAT enhancer-binding protein (C/EBP) transcription factors (3). As seen in Fig.1 (line 6), the IFN- enhancer strongly activated transcriptionfrom the TK promoter from a distance. The rescue of the IFN-enhancer action from a distance requires intact upstream pro-moter transcription factor binding sites, because deletion of theSp1 and C/EBP sites eliminated enhancer-dependent transcrip-tional activation (line 7). The experiment shown in line 8indicates that the IFN- enhancer can activate transcriptionfrom the IFN- core promoter when an Sp1 site is placedupstream of the TATA box. Taken together, these experimentssupport the notion that the IFN- enhancer can communicatewith either the native or a heterologous core promoter from adistance; however, the information decoded by the enhancer canbe conveyed only when transcription factor binding sites arepresent upstream of the target core promoters.

    DNA Looping Mediates the Interaction Between a Remote Enhancerand a Promoter. To test whether enhancerpromoter communi-cation involves contacts between the distant regulatory ele-ments, we carried out chromosome conformation capture (3C)assays (26) in which enhancer DNA sequences are ligated topromoter DNA sequences only if they are in close physicalproximity. HeLa cells were transfected with the Distal templatebearing the IFN- enhancer 2.3 kbp upstream of the TKpromoter (Fig. 1, line 6, and Fig. 2B) or with the DistalSp1-deleted template (DistalSp1 template) (Fig. 1, line 7, andFig. 2B), followed by virus infection and formaldehyde cross-linking. The cross-linked chromatin was digested with NlaIII(f lanking the IFN- enhancer and the TK promoter) (Fig. S1).The digested chromatin was diluted, and DNA ligase was added

    to link covalently DNA sequences that are in physical proximity.After the proteins were removed, PCR primers specific for theenhancer and the promoter were used to detect the interactingsequences. Fig. 2 A (lane 1) shows that there is no detectablePCR product in uninfected cells. A PCR product was generatedwhen the chromatin DNA used was prepared from virus-infectedcells (lane 2) but was not generated in any of the controls,including genomic DNA (lanes 5 and 6) and NlaIII-cleaved butnot ligated chromatin DNA (lanes 3 and 4). The size of the PCRp

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