A. K. Golovnin

An endogenous Su(Hw) insulator separates the yellow gene from the Achaete-scute gene complex in Drosophila

The best characterized chromatin insulator in Drosophila is the Suppressor of Hairy wing binding region contained within the gypsy retrotransposon. Although cellular functions have been suggested, no role has been found yet for the multitude of endogenous Suppressor of Hairy wing binding sites. Here we show that two Suppressor of Hairy wing binding sites in the intergenic region between the yellow gene and the Achaete-scute gene complex form a functional insulator. Genetic analysis shows that at least two proteins, Suppressor of Hairy wing and Modifier of MDG4, required for the activity of this insulator, are involved in the transcriptional regulation of Achaete-scute.

‘Insulator bodies’ are aggregates of proteins but not of insulators

Chromatin insulators are thought to restrict the action of enhancers and silencers. The best‐known insulators in Drosophila require proteins such as Suppressor of Hairy wing (Su(Hw)) and Modifier of mdg4 (Mod(mdg4)) to be functional. The insulator‐related proteins apparently colocalize as nuclear speckles in immunostained cells. It has been asserted that these speckles are ‘insulator bodies’ of many Su(Hw)–insulator DNA sites held together by associated proteins, including Mod(mdg4). As we show here using flies, larvae and S2 cells, a mutant Mod(mdg4) protein devoid of the Q‐rich domain supports the function of Su(Hw)‐dependent insulators and efficiently binds to correct insulator sites on the chromosome, but does not form or enter the Su(Hw)‐marked nuclear speckles; conversely, the latter accumulate another (C‐truncated) Mod(mdg4) mutant that cannot interact with Su(Hw) or with the genuine insulators. Hence, it is not the functional genomic insulators but rather aggregated proteins that make the so‐called ‘insulator bodies’.

SUMO conjugation is required for the assembly of Drosophila Su(Hw) and Mod(mdg4) into insulator bodies that facilitate insulator complex formation.

Chromatin insulators are special regulatory elements involved in modulation of enhancer–promoter interactions. The best studied insulators in Drosophila require Suppressor of Hairy Wing [Su(Hw)], Modifier of mdg4 [Mod(mdg4)] and centrosomal 190 kDa (CP190) proteins to be functional. These insulator proteins are colocalized in nuclear speckles named insulator bodies. Here, we demonstrate that post-translational modification of insulator proteins by small ubiquitin-like modifier (SUMO) and intact CP190 protein is crucial for insulator body formation. Inactivation of SUMO binding sites in Mod(mdg4)-67.2 leads to the inability of the mutant protein and Su(Hw) to be assembled into insulator bodies. In vivo functional tests show that a smaller amount of intact Mod(mdg4)-67.2, compared with the mutant protein, is required to restore the normal activity of the Su(Hw) insulator. However, high expression of mutant Mod(mdg4)-67.2 completely rescues the insulator activity, indicating that sumoylation is not necessary for enhancer blocking. These results suggest that insulator bodies function as a depot of sumoylated proteins that are involved in insulation and can facilitate insulator complex formation, but are nonessential for insulator action.

Enhancer-Promoter Communication Is Regulated by Insulator Pairing in a Drosophila Model Bigenic Locus

ABSTRACT The complexity of regulatory systems in higher eukaryotes, featuring many distantly located enhancers that nonetheless properly activate the target promoters, has prompted the hypothesis that the action of enhancers should be restricted by insulators. Continuing our research on the functional role of insulators and the consequences of their interaction in Drosophila, we studied the interplay of different Su(Hw)-dependent Drosophila insulators. The set of transgenic constructs comprised two consecutive genes (yellow and white) with their enhancers and insulator elements differently arranged in between and/or around the gene(s). All insulators were found to interact in twin or mixed tandems, demonstrating the bypass phenomenon. However, insulator pairing around a gene did not always improve its isolation from an outside enhancer. On the other hand, merely two insulator elements (identical or different) in appropriate positions can permit the expression of one gene but not the gene next to it or, conversely, largely block the transcription of the first gene, while allowing full enhancement of the second, or make them behave similarly. Thus, the results of this study support the model that loop formation by insulators is an essential component of insulator action on a positive and negative regulation of an enhancer-promoter communication.

Integrity of the Mod(mdg4)-67.2 BTB Domain Is Critical to Insulator Function in Drosophila melanogaster

ABSTRACT The Drosophila gypsy insulator contains binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Enhancer and silencer blocking require Su(Hw) recruitment of Mod(mdg4)-67.2, a BTB/POZ domain protein that interacts with Su(Hw) through a carboxyl-terminal acidic domain. Here we conducted mutational analyses of the Mod(mdg4)-67.2 BTB domain. We demonstrate that this domain is essential for insulator function, in part through direction of protein dimerization. Our studies revealed the presence of a second domain (DD) that contributes to Mod(mdg4)-67.2 dimerization when the function of the BTB domain is compromised. Additionally, we demonstrate that mutations in amino acids of the charged pocket in the BTB domain that retain dimerization of the mutated protein cause a loss of insulator function. In these cases, the mutant proteins failed to localize to chromosomes, suggesting a role for the BTB domain in chromosome association. Interestingly, replacement of the Mod(mdg4)-67.2 BTB domain with the GAF BTB domain produced a nonfunctional protein. Taken together, these data suggest that the Mod(mdg4)-67.2 BTB domain confers novel activities to gypsy insulator function.

Drosophila mini-white model system: new insights into positive position effects and the role of transcriptional terminators and gypsy insulator in transgene shielding

The white gene, which is responsible for eye pigmentation, is widely used to study position effects in Drosophila. As a result of insertion of P-element vectors containing mini-white without enhancers into random chromosomal sites, flies with different eye color phenotypes appear, which is usually explained by the influence of positive/negative regulatory elements located around the insertion site. We found that, in more than 70% of cases when mini-white expression was subject to positive position effects, deletion of the white promoter had no effect on eye pigmentation; in these cases, the transposon was inserted into the transcribed regions of genes. Therefore, transcription through the mini-white gene could be responsible for high levels of its expression in most of chromosomal sites. Consistently with this conclusion, transcriptional terminators proved to be efficient in protecting mini-white expression from positive position effects. On the other hand, the best characterized Drosophila gypsy insulator was poorly effective in terminating transcription and, as a consequence, only partially protected mini-white expression from these effects. Thus, to ensure maximum protection of a transgene from position effects, a perfect boundary/insulator element should combine three activities: to block enhancers, to provide a barrier between active and repressed chromatin, and to terminate transcription.

EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.

The su(Hw) Insulator Can Disrupt Enhancer-Promoter Interactions When Located More than 20 Kilobases Away from the Drosophila achaete-scute Complex

ABSTRACT Here we report that the su(Hw) insulator may not necessarily separate promoters from enhancers to allow inhibition of transcription by the su(Hw) protein. For this purpose we used the strains ofDrosophila melanogaster which carry inversion of the region containing the yellow gene and the achaete-scute complex (AS-C). Despite the reverse orientation of the region, the AS-C enhancers continue to activate achaete andscute gene expression. The su(Hw) insulator, located more than 20 kb away from the inversion, facilitates strong suppression ofachaete and scute gene expression, although is does not separate the promoters from the AS-C enhancers.

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.

The acid domain located at the C-terminus of the Su(Hw) protein represses transcription in the yeast two-hybrid system.

The determination of proteins that are responsible for the functioning of insulators has played a key role in clarifying the mechanisms of their action. One of the best studied insulators of Drosophila , which contains twelve binding sites for the Su(Hw) protein, interacts with two proteins—Su(Hw) and Mod(mdg4) [1, 2]. In the absence of the Mod(mdg4) protein, the Su (Hw) insulator is transformed into a repressor that inhibits transcription from some promoters (in particular, from the promoter of the yellow gene) [3, 4]. Genetic analysis with the use of Su(Hw) mutants showed that the C-terminal acid domain of the Su(Hw) protein is responsible for the repression of transcription of the yellow gene in the absence of the Mod(mdg4) protein [3]. Analysis of the interaction between the proteins Mod(mdg4) and Su(Hw) in yeast two-hybrid system showed that the Su(Hw) protein represses the transcription of the reporter genes in yeast. Deletion analysis showed that the C-terminal domain of the Su(Hw) protein is responsible for the repression of transcription in yeast. Therefore, this domain is a conservative negative regulator of transcription in different organisms, such as Drosophila and yeast.