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 ... -  《-》

Interaction between a pair of gypsy insulators or between heterologous gypsy and Wari insulators modulates Flp site-specific recombination in Drosophila melanogaster.

Chromatin insulators block the action of transcriptional enhancers when interposed between an enhancer and a promoter. An Flp technology was used to examine interactions between Drosophila gypsy and Wari insulators in somatic and germ cells. The gypsy insulator consists of 12 binding sites for the Su(Hw) protein, while the endogenous Wari insulator, located on the 3' side of the white gene, is independent from the Su(Hw) protein. Insertion of the gypsy but not Wari insulator between FRT sites strongly blocks recombination between Flp dimers bound to FRT sites located on the same chromatid (recombination in cis) or in sister chromatids (unequal recombination in trans). At the same time, the interaction between Wari and gypsy insulators regulates the efficiency of Flp-mediated recombination. Thus, insulators may have a role in controlling interactions between distantly located protein complexes (not only those involved in transcriptional gene regulation) on the same chromosome or on sister chromatids in somatic and germ cells. We have also found that the frequency of Flp-mediated recombination between FRT sites is strongly dependent on the relative orientation of gypsy insulators. Taken together, our results indicate that the interactions between insulators can be visualized by Flp technology and that insulators may be involved in blocking undesirable interactions between proteins at the two-chromatid phase of the cell cycle.

Krivega M ，Savitskaya E ，Krivega I ，Karakozova M ，Parshikov A ，Golovnin A ，Georgiev P ... -  《-》

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》

Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements.

Insulators are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. According to previous data, an interaction (pairing) between some Drosophila insulators can support distant activation of a promoter by an enhancer. Here, we have demonstrated that pairs of well-studied insulators such as scs-scs, scs'-scs', 1A2-1A2 and Wari-Wari support distant activation of the white promoter by the yeast GAL4 activator in an orientation-dependent manner. The same is true for the efficiency of the enhancer that stimulates white expression in the eyes. In all insulator pairs tested, stimulation of the white gene was stronger when insulators were inserted between the eye enhancer or GAL4 and the white promoter in opposite orientations relative to each other. As shown previously, Zw5, Su(Hw) and dCTCF proteins are required for the functioning of different insulators that do not interact with each other. Here, strong functional interactions have been revealed between DNA fragments containing binding sites for either Zw5 or Su(Hw) or dCTCF protein but not between heterologous binding sites [Zw5-Su(Hw), dCTCF-Su(Hw), or dCTCF-Zw5]. These results suggest that insulator proteins can support selective interactions between distant regulatory elements.

Kyrchanova O ，Chetverina D ，Maksimenko O ，Kullyev A ，Georgiev P ... -  《-》

E(y)2/Sus1 is required for blocking PRE silencing by the Wari insulator in Drosophila melanogaster.

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers to the spread of repressive chromatin. Recently, we have found an insulator, named Wari, located on the 3' side of the white gene. Here, we show that the previously identified 368-bp core of this insulator is sufficient for blocking Polycomb response element-mediated silencing. Although Wari does not contain binding sites for known insulator proteins, the E(y)2 and CP190 proteins bind to Wari as well as to the Su(Hw)-containing insulators in vivo. It may well be that these proteins are recruited to the insulator by as yet unidentified DNA-binding protein. Partial inactivation of E(y)2 in a weak e(y)2 ( u1 ) mutation impairs only the anti-silencing but not the enhancer-blocking activity of the Wari insulator. Thus, the E(y)2 protein in different Drosophila insulators serves to protect gene expression from silencing.

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