Several copies of insulator from MDG4 can determine the interaction between positively and negatively acting regulatory elements and promoter of the miniwhite gene of Drosophila melanogaster

Fine regulation of complex gene loci in higher eukaryotes is realized through the interaction of promoters with enhancers and repressors, which can be located long distance from the promoter regulated. A question arises, what mechanisms determine proper contacts between the regulatory elements over large distances in the genome. It is suggested that the important role in this process is played by a special class of regulatory elements, insulators, which block the interaction of enhancer and promoter, if they are positioned between them. Furthermore, enhancers do not directly inactivate the activities of enhancer and promoter. Nevertheless, an enhancer, isolated from one of the promoters by an insulator, can activate another, not isolated promoter. The best studied insulator of Drosophila melanogaster was found in the 5′ regulatory region of retrotransposon MDG4. It consists of 12 binding sites for the Su(Hw) protein, which is critical for the activity of this insulator. It was demonstrated that Su(Hw) insulator could protect the gene expression from the negative influence of heterochromatin and from repression, induced by the Polycomb group proteins (Pc proteins). In the present study, it was demonstrated that in transgenic lines, two or three copies of the Su(Hw) insulator could determine the interaction of the miniwhite enhancer and Pc dependant silencer with the miniwhite promoter. Thus, it was first demonstrated that insulators could participate in the regulation of the contacts between promoter and functionally opposite elements, responsible for either gene activation, or repression. Original Russian Text ? M.V. Kostyuchenko, E.E. Savitskaya, M.N. Krivega, P.G. Georgiev, 2008, published in Genetika, 2008, Vol. 44, No. 12, pp. 1693–1697.     

Drosophila gypsy insulator and yellow enhancers regulate activity of yellow promoter through the same regulatory element

There is ample evidence that the enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other chromosome where the enhancers are inactive or deleted, which is indicative of a high specificity of the enhancer–promoter interaction in yellow. In this paper, we have found that the yellow sequence from −100 to −69 is essential for stimulation of the heterologous eve (TATA-containing) and white (TATA-less) promoters by the yellow enhancers from a distance. However, the presence of this sequence is not required when the yellow enhancers are directly fused to the heterologous promoters or are activated by the yeast GAL4 activator. Unexpectedly, the same promoter proximal region defines previously described promoter-specific, long-distance repression of the yellow promoter by the gypsy insulator on the mod(mdg4) ^u1 background. These finding suggest that proteins bound to the −100 to −69 sequence are essential for communication between the yellow promoter and upstream regulatory elements.     

Effects of functional interactions between nonhomologous insulators wari and Su(Hw)

Insulators are regulatory DNA elements restricting gene activation by enhancers. Interactions between insulators can lead to both insulation and activation of promoters by enhancers. In this work, we analyzed the effects of interaction of two Drosophila insulators, Wari and Su(Hw). The functional interaction between these insulators was found to enhance the activity of the Su(Hw) insulator only, but not of the Wari insulator. This suggests that the formation of a chromatin loop between interacting insulators is not a key factor for enhancement of insulation, which is in disagreement with the main idea of structural models. In addition, the effect of interaction between Wari and Su(Hw) depends on a distance between them and on the position in the system relative to other regulatory elements.     

The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication

Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3' cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.     

1A2 Insulator can interact with promoter of hsp70 gene in D. melanogaster

Insulators are regulatory DNA elements that participate in the modulation of the interactions between enhancers and promoters. Depending on the situation, insulators can either stabilize or destroy the contacts between enhancers and promoters. A possible explanation for the activity of insulators is their ability to directly interact with gene promoters. In the present study, it was demonstrated that, in model systems, a 1A2 insulator could interact with the core sequence of an hsp70 promoter. In this case, the insulator protein CP190 is found on the hsp70 promoter, which depends on the presence of an insulator in the transgene. The data obtained are consistent with the model, which implies that direct contacts between insulators and promoters make a considerable contribution to the modulation of the interactions between insulators and promoters.     

Multiple Promoter Targeting Sequences exist in Abdominal-B to regulate long-range gene activation

In complex genomes, insulators set up chromatin domain boundaries and protect promoters from inappropriate activation by enhancers from neighboring genes. The Drosophila Abdominal-B locus uses insulator elements to organize its large regulatory region into several body segment-specific chromatin domains. This organization leads to a problem in enhancer-promoter communication, that is, how do distal enhancers activate the Abd-B promoter when there are several insulators in between? This issue is partially resolved by the Promoter Targeting Sequence, which can overcome the enhancer blocking effect of an insulator. In this study, we describe a new Promoter Targeting Sequence, PTS-6, from the Abd-B 3' regulatory region. PTS-6, comprised of approximately 200 bp, was found to bypass both homologous Abdominal-B insulators, such as Fab-7 and Fab-8, and a heterologous insulator, suHw. Most importantly, it also overcomes a combination of two insulators such as Fab-7/Fab-8. Thus, PTS-6 could, in principle, target remote enhancers that are separated from the Abd-B promoter by multiple insulators. In addition, PTS-6 selectively targets the distal enhancer to only one transgenic promoter, and it strongly facilitates Abd-B enhancers. These results suggest that promoter targeting is necessary for long-range enhancer-promoter communication in Abd-B, and PTS elements could be a common occurrence in large, complex genetic loci.     

Highly interactive nature of flower‐specific enhancers and promoters,and its potential impact on tissue‐specific expression and engineering of multiple genes or agronomic traits

Molecular stacking enables multiple traits to be effectively engineered in crops using a single vector. However, the co‐existence of distinct plant promoters in the same transgenic unit might, like their mammalian counterparts, interfere with one another. In this study, we devised a novel approach to investigate enhancer–promoter and promoter–promoter interactions in transgenic plants and demonstrated that three of four flower‐specific enhancer/promoters were capable of distantly activating a pollen‐ and stigma‐specific Pps promoter (fused to the cytotoxic DT‐A gene) in other tissues, as revealed by novel tissue ablation phenotypes in transgenic plants. The NtAGI1 enhancer exclusively activated stamen‐ and carpel‐specific DT‐A expression, thus resulting in tissue ablation in an orientation‐independent manner; this activation was completely abolished by the insertion of an enhancer‐blocking insulator (EXOB) between the NtAGI1 enhancer and Pps promoter. Similarly, AGL8 and AP1Lb1, but not AP1La, promoters also activated distinct tissue‐specific DT‐A expression and ablation, with the former causing global growth retardation and the latter ablating apical inflorescences. While the tissue specificity of the enhancer/promoters generally defined their activation specificities, the strength of their activity in particular tissues or developmental stages appeared to determine whether activation actually occurred. Our findings provide the first evidence that plant‐derived enhancer/promoters can distantly interact/interfere with one another, which could pose potential problems for the tissue‐specific engineering of multiple traits using a single‐vector stacking approach. Therefore, our work highlights the importance of adopting enhancer‐blocking insulators in transformation vectors to minimize promoter–promoter interactions. The practical and fundamental significance of these findings will be discussed.     

Composite cis-acting epigenetic switches in eukaryotes: lessons from Drosophila Fab-7 for the Igf2-H19 imprinted domain

One of the central problems of eukaryotic gene regulation is to understand the mechanism(s) by which the activity of enhancer elements is circumscribed such that they only act upon their cognate promoter sequences. Studies on the bithorax gene complex (BX-C) in Drosophila have highlighted the potential problem of enhancer promiscuity and detailed molecular and genetic analyses are now providing insight into how this gene complex resolves the problem through the activity of boundary/silencer elements that can block the communication between enhancers and promoters. Analysis of the mouse Igf2–H19 imprinted locus also suggests a role for boundary/silencer elements, but in this case these elements are invoked to account for the preferential expression of Igf2 and H19 from the paternally and maternally inherited chromosomes respectively despite the presence of functional downstream enhancers. We discuss recent work that has illuminated both of these systems and consider what parallels exist between them.