DNA supercoiling allows enhancer action over a large distance

Abstract

Enhancers are regulatory DNA elements that can activate their genomic targets over a large distance. The mechanism of enhancer action over large distance is unknown. Activation of the glnAp2 promoter by NtrC-dependent enhancer in Escherichia coli was analyzed by using a purified system supporting multiple-round transcription in vitro. The data suggest that DNA supercoiling is an essential requirement for enhancer action over a large distance (2,500 bp) but not over a short distance (110 bp). DNA supercoiling facilitates functional enhancer-promoter communication over a large distance, probably by bringing the enhancer and promoter into close proximity.T ranscriptional enhancers are relatively short (30 -200 bp) DNA sequences usually composed of several binding sites for activator protein(s). The landmark of enhancers is their ability to communicate with promoters and activate genes over a large distance (up to 60 kb; see ref. 1 for a review). Prokaryotic and eukaryotic enhancers share several key properties such as ability to activate transcription over a large distance, tight coupling of DNA melting with ATP hydrolysis and high stability of the initiation complexes (see refs. 2 and 3 for recent reviews). In contrast to eukaryotic enhancers, prokaryotic enhancers can activate transcription over a large distance in vitro. This ability allows detailed analysis of the mechanism of enhancer action impossible in the case of eukaryotic enhancers.The mechanism of enhancer action over a large distance is unknown. It is most likely that proteins bound at the enhancer and promoter directly interact such that intervening DNA forms a loop (see refs. 4 and 5 for reviews). The main problem for communication over a large distance is low concentration of the DNA regions in the vicinity of each other (see ref. 4 for a review). Measurements of local concentration of linear DNA ends in the vicinity of each other suggest that it is relatively high (10 Ϫ7 M) only when the distance between DNA ends is 100-900 bp (6). When distance between the DNA ends is increased to 3 kb, their local concentration is decreased by an order of magnitude (6). Thus, it is remarkable that DNA regions positioned far away from each other (such as an enhancer and a promoter) can communicate efficiently.Several models have been proposed to explain the mechanism of enhancer action over a large distance. One class of models suggests that initial communication of an enhancer with a promoter leads to formation of a stable DNA-protein complex in the vicinity of the promoter. This stable complex may facilitate subsequent rounds of transcription serving as a ''memory'' of initial enhancer-promoter interaction (see ref. 1 for a review). Alternatively, the average distance between promoter and enhancer could be considerably decreased if the intervening DNA is supercoiled (sc) or bent (see ref. 4 for a review). It has also been shown that sequence-dependent superhelical DNA inserts can facilitate enhancer action (7).The mechanism of action of the NtrC-dependent, 54...

Biochemical analysis of enhancer–promoter communication in chromatin

“…At the same time, bacterial transcriptional enhancers can work efficiently over a large distance (up to at least 3.5 kb) both in vivo and in vitro [17][18][19]. Moreover, proand eukaryotic transcriptional enhancers share many key properties [1], and our preliminary data suggest that bacterial enhancers can also work efficiently over a large distance in chromatin environment in vitro.…”

“…To analyze enhancer action over a large distance in chromatin environment, the 7637 bp pLY10 plasmid was used [18]. It contains the glnAp2 promoter that is strongly (more than 100-fold) activated by NtrC-dependent enhancer positioned 2.5-kb away from the promoter [18,37].…”

“…It contains the glnAp2 promoter that is strongly (more than 100-fold) activated by NtrC-dependent enhancer positioned 2.5-kb away from the promoter [18,37]. It was shown previously that DNA supercoiling can greatly (up to 50-fold) facilitate enhancerpromoter (E-P) communication over a large distance on histone-free pLY10, which is the ratelimiting step during distantly activated transcription of the relaxed or linear plasmid [18].…”

“…Activity of the promoter itself in this system does not depend on the level of negative DNA supercoiling (Y.S.P., unpublished data) that makes possible to analyze communication properties of linear, relaxed or supercoiled DNA, as well as chromatin templates. The stage of physical interaction between enhancer and promoter is the rate-limiting step for the whole process of enhancer-dependent transcription [18]. Therefore, the overall efficiency of transcript accumulation can serve as a direct measure of E-P communication and allows quantitative analysis of the rate of communication.…”

“…Using this experimental technique we have recently shown that action of NtrC-dependent enhancer over a large (2.5 kb) distance is greatly stimulated by negative DNA supercoiling [18]. Moreover, for the first time it was shown that enhancer-blocking insulator-like activity can be recapitulated in vitro in a highly purified system using a sequence-specific DNA-binding protein that makes stable DNA loops [17].…”