Transcription factors mediate long-range enhancer-promoter interactions

Abstract

We examined how remote enhancers establish physical communication with target promoters to activate gene transcription in response to environmental signals. Although the natural IFN-beta enhancer is located immediately upstream of the core promoter, it also can function as a classical enhancer element conferring virus infection-dependent activation of heterologous promoters, even when it is placed several kilobases away from these promoters. We demonstrated that the remote IFN-beta enhancer "loops out" the intervening DNA to reach the target promoter. These chromatin loops depend on sequence-specific transcription factors bound to the enhancer and the promoter and thus can explain the specificity observed in enhancer-promoter interactions, especially in complex genetic loci. Transcription factor binding sites scattered between an enhancer and a promoter can work as decoys trapping the enhancer in nonproductive loops, thus resembling insulator elements. Finally, replacement of the transcription factor binding sites involved in DNA looping with those of a heterologous prokaryotic protein, the lambda repressor, which is capable of loop formation, rescues enhancer function from a distance by re-establishing enhancer-promoter loop formation.

Fig. 1.

Enhancer action from a distance requires upstream promoter elements. HeLa cells were transfected with the indicated chloramphenicol acetyl transferase (CAT) reporter plasmids. The cells were mock or virus infected for 24 h before being harvested. Then CAT activity was determined.

Fig. 2.

DNA looping mediates the interaction between a remote enhancer and a promoter. (A) Shown is a 3C experiment depicting the PCR products using primers specific for the enhancer and the promoter as seen in Fig. S1. PCR was performed on NlaIII-digested chromatin derived from HeLa cells mock or virus infected for 6 h harboring the Distal (lanes 1 and 2) or the DistalΔSp1 (lanes 7 and 8) plasmids. Genomic DNA (lanes 5, 6, 11, and 12) and cross-linked digested but not ligated chromatin (lanes 3, 4, 9, and 10) derived from mock- or virus-infected (6 h) cells were used as controls. Lane 13 is a negative PCR control, and lane 14 is the size marker. (B) Schematic representation of the CAT constructs used to determine mechanisms of enhancer function. The arrows indicate the position of the primers used in the PCR reactions with immunoprecipitated DNA. The wild-type TK promoter contains an Sp1 site (Proximal and Distal constructs), whereas in the DistalΔSp1 construct the Sp1 site has been mutated. In the Distal p50 construct, the Sp1 site was replaced by a consensus p50 homodimer site. (C) Stable HeLa cells bearing the indicated CAT reporter plasmids were mock or virus infected for 12 h before being harvested; then CAT activity was determined. The error bars indicate SD. (D) Cross-linked chromatin prepared from mock- or virus-infected (6 h) HeLa cells stably transfected with the indicated CAT constructs was immunoprecipitated with the indicated antibodies. The precipitated DNA was subjected to PCR analysis using ³²P-dCTP and plasmid-specific primers. (E) The process is as described in (D), except that p65 and IRF-3 antibodies were used, and the Distal p50 construct instead of the proximal construct was included in the experiment.

Fig. 3.

Enhancer–promoter interactions are facilitated by heterologous proteins capable of DNA looping. (A) HeLa cells were cotransfected with the indicated CAT reporter plasmids in the presence or absence of a λ expression vector; 24 h later, the cells were virus infected for 12 h before being harvested; then CAT activity was determined. The error bars indicate SD. (B) HeLa cells were cotransfected with the indicated CAT reporter plasmids and the λ expression vector; 24 h later, the cells were mock or virus infected for different amounts of time before being harvested. Then CAT activity was determined. The error bars indicate SD. (C) HeLa cells were transfected with the indicated plasmids as described in (A), except that the cells were mock or virus infected for increasing amounts of time. Cross-linked chromatin was immunoprecipitated with the p65 antibody. The precipitated DNA was subjected to PCR analysis using ³²P-dCTP and plasmid-specific primers as indicated. (D) HeLa cells were cotransfected with the indicated CAT reporter plasmids in the presence or in the absence of a λ expression vector; 24 h later, the cells were virus infected for 12 h before being harvested. Then CAT activity was determined. The error bars indicate SD.

Fig. 4.

The effect of inserting transcription factor binding sites between an enhancer and a promoter. (A) HeLa cells were transfected with the Distal reporter plasmid, and 24 h later the cells were mock or virus infected for increasing amounts of time. Cross-linked chromatin was immunoprecipitated with the p65 antibody. The precipitated DNA was subjected to PCR analysis using ³²P-dCTP and primers specific for plasmid sequences located between the enhancer and the promoter (intervening #1, #2, and #3), and enhancer- or promote-specific primers as shown at the diagram at the left of the gel. (B) HeLa cells were transfected with the constructs indicated (Left). The cells were infected with virus for different amounts of time, and the isolated RNA was used as a template for RT-PCR analysis using CAT-specific primers. The radioactive bands were quantitated using PhosphorImager (Typhoon), and the data from 3 independent experiments were plotted (Right). Shown are mean values ± SD.

Fig. 5.

Transcription factor binding sites trap enhancer complexes. (A) A 3C experiment depicts the PCR products using pairs of primers specific for enhancer and promoter (Top) or enhancer and Sp1 (Bottom). PCR was performed on NlaIII-digested chromatin (Fig. S1) derived from HeLa cells mock- or virus-infected for the indicated times transfected with the Distal or Distal+Sp1 (lanes 1–6). Genomic DNA (lanes 13–18) and cross-linked digested but not ligated chromatin (lanes 7–12) derived from mock- or virus-infected cells were used as controls. (B) HeLa cells were transfected with the Distal reporter plasmid; 24 h later the cells were mock or virus infected for increasing amounts of time. Cross-linked chromatin was immunoprecipitated with the indicated antibodies. The precipitated DNA was subjected to PCR analysis using ³²P-dCTP and primers specific for the enhancer (lanes 1–6), the intervening #2 (lanes 7–12), and the promoter (lanes 13–18). (C) The process was as described in (B), except the cells were transfected with the Distal+TKcore promoter plasmid. In this case, the intervening primers amplify the inserted TK core promoter. (D) The process was as described in (B), except that the cells were transfected with the Distal+Sp1 plasmid. In this case, the intervening primers amplify the inserted Sp1 DNA binding site.