Enhancer-promoter Specificity Research Articles

Alu elements confer enhancer-promoter specificity.

Alu elements confer enhancer-promoter specificity.

Enhancers have more cofactor specificity than we think: A potential new way to classify enhancers based on their functional cofactor requirements

By systematically assessing the effects of depleting eight cofactors on enhancer activity, Neumayr etal. (2022) found that different enhancers have different requirements for some perceived universal cofactors. While some cofactors influence enhancer strength, others affect enhancer-promoter specificity.

An insulator blocks access to enhancers by an illegitimate promoter, preventing repression by transcriptional interference.

Several distinct activities and functions have been described for chromatin insulators, which separate genes along chromosomes into functional units. Here, we describe a novel mechanism of functional separation whereby an insulator prevents gene repression. When the homie insulator is deleted from the end of a Drosophila even skipped (eve) locus, a flanking P-element promoter is activated in a partial eve pattern, causing expression driven by enhancers in the 3’ region to be repressed. The mechanism involves transcriptional read-through from the flanking promoter. This conclusion is based on the following. Read-through driven by a heterologous enhancer is sufficient to repress, even when homie is in place. Furthermore, when the flanking promoter is turned around, repression is minimal. Transcriptional read-through that does not produce anti-sense RNA can still repress expression, ruling out RNAi as the mechanism in this case. Thus, transcriptional interference, caused by enhancer capture and read-through when the insulator is removed, represses eve promoter-driven expression. We also show that enhancer-promoter specificity and processivity of transcription can have decisive effects on the consequences of insulator removal. First, a core heat shock 70 promoter that is not activated well by eve enhancers did not cause read-through sufficient to repress the eve promoter. Second, these transcripts are less processive than those initiated at the P-promoter, measured by how far they extend through the eve locus, and so are less disruptive. These results highlight the importance of considering transcriptional read-through when assessing the effects of insulators on gene expression.

Red Light/Green Light, a Dual Fluorescent Protein Reporter System To Study Enhancer-Promoter Specificity in Drosophila.

Enhancers activate gene transcription in spatial and temporal patterns by interactions with gene promoters. These elements typically reside distal to their target promoter, with which they must interact selectively. Additional elements may contribute to enhancer-promoter specificity, including remote control element sequences within enhancers, tethering elements near promoters, and insulator/boundary elements that disrupt off-target interactions. However, few of these elements have been mapped, and as a result, the mechanisms by which these elements interact remain poorly understood. One impediment is their method of study, namely reporter transgenes in which enhancers are placed adjacent to a heterologous promoter, which may circumvent mechanisms controlling enhancer-promoter specificity and long-range interactions. Here, we report an optimized dual reporter transgene system in Drosophila melanogaster that allows the simultaneous comparison of an enhancer’s ability to activate proximal and distal fluorescent reporter genes. Testing a panel of fluorescent transgenes in vivo, we found a two-protein combination that allows simultaneous measurement with minimal detection interference. We note differences among four tested enhancers in their ability to regulate a distally placed reporter transgene. These results suggest that enhancers differ in their requirements for promoter interaction and raise important practical considerations when studying enhancer function.

Optimization of reporter gene architecture for quantitative measurements of gene expression in the Drosophila embryo

Quantitative assessment of gene regulation is critical for mathematical modeling of transcriptional systems for systems biology efforts. Enhancers, also termed cis-regulatory modules (CRMs), are the primary mediators of transcriptional regulation in higher eukaryotes; transcription factors binding to CRMs dictate the likelihood and frequency of promoter activation. To provide a suitable platform for in-depth CRM analysis, we adapted a targeted integration vector to compare action of basal promoters with diverse combination of TATA, Inr and DPE motifs, as well as a set of 3'-UTRs representative of those used in different reporter vectors. This "Honda" series of reporter gene vectors was activated by a regulatory element binding Dorsal and Twist activators suitable for transcription in the early Drosophila embryo. The diverse promoters functioned in a similar manner with minor quantitative differences, consistent with a lack of enhancer-promoter specificity. Constructs bearing SV40 3'-UTR sequences appeared to produce somewhat higher levels of mRNA. Confocal laser scanning microscopy revealed that the mRNA distribution produced by these constructs was punctate; this pattern appears to be dependent on 5'-UTR sequences, as an optimized vector including an alternate 5'-UTR produced a more even distribution, which may be preferable for quantitative imaging. This set of Honda vectors contains convenient sites for modification of basal promoter, 3' UTR, and enhancer, and will be useful for analysis of CRMs and quantitative studies of gene expression.

A novel promoter-tethering element regulates enhancer-driven gene expression at the bithorax complex in theDrosophilaembryo

A key question in our understanding of the cis-regulation of gene expression during embryonic development has been the molecular mechanism that directs enhancers to specific promoters within a gene complex. Promoter competition and insulators are thought to play a role in regulating these interactions. In the bithorax complex of Drosophila, the IAB5 enhancer is located 55 kb 3' of the Abdominal-B (Abd-B) promoter and 48 kb 5' of the abdominal-A (abd-A) promoter. Although roughly equidistant from the two promoters, IAB5 specifically interacts only with the Abdominal-B promoter, even though the enhancer and promoter are separated by at least two insulators. Here we demonstrate that a 255 bp element, located 40 bp 5' of the Abd-B transcriptional start site, has a novel cis-regulatory activity as it is able to tether IAB5 to the Abd-B promoter in transgenic embryos. The tethering element is sufficient to direct IAB5 to an ectopic promoter in competition assays. Deletion of the promoter-tethering element results in the redirection of enhancer-driven gene expression on transgenes. Taken together, these results provide evidence that specific long-range enhancer-promoter interactions in the bithorax complex are regulated by a tethering element 5' of the Abd-B promoter. We discuss a bioinformatic analysis of the tethering element across different Drosophila species and a possible molecular mechanism by which this element functions. We also examine existing evidence that this novel class of cis-regulatory elements might regulate enhancer-promoter specificity at other gene complexes.

Enhancer choice in cis and in trans in Drosophila melanogaster: role of the promoter.

Eukaryotic enhancers act over very long distances, yet still show remarkable specificity for their own promoter. To better understand mechanisms underlying this enhancer-promoter specificity, we used transvection to analyze enhancer choice between two promoters, one located in cis to the enhancer and the other in trans to the enhancer, at the yellow gene of Drosophila melanogaster. Previously, we demonstrated that enhancers at yellow prefer to act on the cis-linked promoter, but that mutation of core promoter elements in the cis-linked promoter releases enhancers to act in trans. Here, we address the mechanism by which these elements affect enhancer choice. We consider and explicitly test three models that are based on promoter competency, promoter pairing, and promoter identity. Through targeted gene replacement of the endogenous yellow gene, we show that competency of the cis-linked promoter is a key parameter in the cis-trans choice of an enhancer. In fact, complete replacement of the yellow promoter with both TATA-containing and TATA-less heterologous promoters maintains enhancer action in cis.

A novel boundary element may facilitate independent gene regulation in the Antennapedia complex of Drosophila.

The intrinsic enhancer-promoter specificity and chromatin boundary/insulator function are two general mechanisms that govern enhancer trafficking in complex genetic loci. They have been shown to contribute to gene regulation in the homeotic gene complexes from fly to mouse. The regulatory region of the Scr gene in the Drosophila Antennapedia complex is interrupted by the neighboring ftz transcription unit, yet both genes are specifically activated by their respective enhancers from such juxtaposed positions. We identified a novel insulator, SF1, in the Scr-ftz intergenic region that restricts promoter selection by the ftz-distal enhancer in transgenic embryos. The enhancer-blocking activity of the full-length SF1, observed in both embryo and adult, is orientation- and enhancer-independent. The core region of the insulator, which contains a cluster of GAGA sites essential for its activity, is highly conserved among other Drosophila species. SF1 may be a member of a conserved family of chromatin boundaries/insulators in the HOM/Hox complexes and may facilitate the independent regulation of the neighboring Scr and ftz genes, by insulating the evolutionarily mobile ftz transcription unit.