Effective blocking of the white enhancer requires cooperation between two main mechanisms suggested for the insulator function.

Chromatin insulators block the action of transcriptional enhancers when interposed between an enhancer and a promoter. In this study, we examined the role of chromatin loops formed by two unrelated insulators, gypsy and Fab-7, in their enhancer-blocking activity. To test for this activity, we selected the white reporter gene that is activated by the eye-specific enhancer. The results showed that one copy of the gypsy or Fab-7 insulator failed to block the eye enhancer in most of genomic sites, whereas a chromatin loop formed by two gypsy insulators flanking either the eye enhancer or the reporter completely blocked white stimulation by the enhancer. However, strong enhancer blocking was achieved due not only to chromatin loop formation but also to the direct interaction of the gypsy insulator with the eye enhancer, which was confirmed by the 3C assay. In particular, it was observed that Mod(mdg4)-67.2, a component of the gypsy insulator, interacted with the Zeste protein, which is critical for the eye enhancer-white promoter communication. These results suggest that efficient enhancer blocking depends on the combination of two factors: chromatin loop formation by paired insulators, which generates physical constraints for enhancer-promoter communication, and the direct interaction of proteins recruited to an insulator and to the enhancer-promoter pair.

Kyrchanova O ，Maksimenko O ，Stakhov V ，Ivlieva T ，Parshikov A ，Studitsky VM ，Georgiev P ... -  《PLoS Genetics》

New properties of Drosophila fab-7 insulator.

In the Abd-B 3' cis-regulatory region, which is subdivided into a series of iab domains, boundary elements have previously been detected, including the Fab-7 element providing for the autonomous functioning of the iab-6 and iab-7 cis-regulatory domains. Here, it has been shown that a single copy of the 860-bp Fab-7 insulator effectively blocks the yellow and white enhancers. The eye and testis enhancers can stimulate the white promoter across the pair of Fab-7, which is indicative of a functional interaction between the insulators. Unexpectedly, Fab-7 has proved to lose the enhancer-blocking activity when placed near the white promoter. It seems likely that Fab-7 strengthens the relatively weak white promoter, which leads to the efficient enhancer-promoter interaction and insulator bypass.

Rodin S ，Kyrchanova O ，Pomerantseva E ，Parshikov A ，Georgiev P ... -  《GENETICS》

Interaction between the GAGA factor and Mod(mdg4) proteins promotes insulator bypass in Drosophila.

Chromatin insulators or boundaries are proposed to structure the chromatin fiber into functionally independent domains by promoting the formation of chromatin loops. These elements can block the communication between an enhancer and a gene when placed between them. Interestingly, it has been previously observed that two tandem copies of the Drosophila Su(Hw) insulator abolish this enhancer-blocking activity, presumably through pairing. This bypass effect has not been described with other insulators, however. In this report, we show that the insertion of binding sites for the GAGA factor (GAF) between an enhancer and the Su(Hw) insulator allows bypassing of the insulator. This bypass relies on the activity of both the GAF protein and the Mod(mdg4)-67.2 protein, a factor required for Su(Hw) insulator activity. We show that these two proteins interact in vitro and in vivo, providing molecular evidence of pairing between the GAF sites and the Su(Hw) insulator. Finally, we show that placing the Mcp boundary together with the Su(Hw) insulator between an enhancer and a promoter leads to bypass, again in a GAF- and Mod(mdg4)-dependent manner. Our data provide direct evidence that heterologous insulators can be bypassed by distal enhancers and identify the interaction between GAF and Mod(mdg4) as a possible means to regulate insulator activity.

Melnikova L ，Juge F ，Gruzdeva N ，Mazur A ，Cavalli G ，Georgiev P ... -  《-》

Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs.

Much of the research on insulators in Drosophila has been done with transgenic constructs using the white gene (mini-white) as reporter. Hereby we report that the sequence between the white and CG32795 genes in Drosophila melanogaster contains an insulator of a novel kind. Its functional core is within a 368 bp segment almost contiguous to the white 3'UTR, hence we name it as Wari (white-abutting resident insulator). Though Wari contains no binding sites for known insulator proteins and does not require Su(Hw) or Mod(mdg4) for its activity, it can equally well interact with another copy of Wari and with unrelated Su(Hw)-dependent insulators, gypsy or 1A2. In its natural downstream position, Wari reinforces enhancer blocking by any of the three insulators placed between the enhancer and the promoter; again, Wari-Wari, Wari-gypsy or 1A2-Wari pairing results in mutual neutralization (insulator bypass) when they precede the promoter. The distressing issue is that this element hides in all mini-white constructs employed worldwide to study various insulators and other regulatory elements as well as long-range genomic interactions, and its versatile effects could have seriously influenced the results and conclusions of many works.

Chetverina D ，Savitskaya E ，Maksimenko O ，Melnikova L ，Zaytseva O ，Parshikov A ，Galkin AV ，Georgiev P ... -  《-》

Insulators form gene loops by interacting with promoters in Drosophila.

Chromatin insulators are regulatory elements involved in the modulation of enhancer-promoter communication. The 1A2 and Wari insulators are located immediately downstream of the Drosophila yellow and white genes, respectively. Using an assay based on the yeast GAL4 activator, we have found that both insulators are able to interact with their target promoters in transgenic lines, forming gene loops. The existence of an insulator-promoter loop is confirmed by the fact that insulator proteins could be detected on the promoter only in the presence of an insulator in the transgene. The upstream promoter regions, which are required for long-distance stimulation by enhancers, are not essential for promoter-insulator interactions. Both insulators support basal activity of the yellow and white promoters in eyes. Thus, the ability of insulators to interact with promoters might play an important role in the regulation of basal gene transcription.

Erokhin M ，Davydova A ，Kyrchanova O ，Parshikov A ，Georgiev P ，Chetverina D ... -  《-》