suppressor of Hairy wing


Function of su(Hw) as an insulator protein (part 2/2)

The wing margin enhancer of cut is responsible for cut activity at the wing margin, the boundary between dorsal and ventral wing compartments. When the gypsy retrotransposon of Drosophila inserts between an enhancer and a proximal promoter it prevents the enhancer from activating transcription. Enhancers are blocked because Suppressor of Hairy-wing binds to gypsy. For example, gypsy insertions in an 85 kilobase region between a wing margin-specific enhancer and the promoter in the cut gene cause a cut wing phenotype that is suppressed by su(Hw) mutations. su(Hw) can cause a cut wing phenotype only if active around pupariation, when the wing margin enhancer is active. Enhancer-blocking by SU(HW) is reversible, and it occurs soon after binding of active SU(HW) to gypsy DNA. These results are consistent with models in which SU(HW) structurally interferes with enhancer-promoter interactions (Dorsett, 1993).

Polycomb response elements (PREs) can establish a silenced state that affects the expression of genes over considerable distances. The ability of insulator or boundary elements to block the repression of the miniwhite gene by the Ultrabithorax PRE has been tested. The gypsy element and the scs element interposed between PRE and the miniwhite gene protect miniwhite against silencing but the scs element is only weakly effective. Blocking the action of gypsy requires su(Hw). When the PRE-miniwhite gene construct is insulated from flanking chromosomal sequences by gypsy elements at both ends, the construct can still establish efficient silencing in some lines but not others. This silencing can be caused by interactions in trans with PREs at other sites. PRE-containing transposons inserted at different sites or even on different chromosomes can interact, resulting in enhanced silencing. These trans interactions are not blocked by the gypsy insulator and reveal the importance of nonhomologous associations between different regions of the genome for both silencing and activation of genes. The similarity between the behavior of PREs and enhancers suggests a model for their long-distance action. Thus blocking elements can prevent communication along a chromatin fiber, and enhance silencing of PREs in trans (Sigrist, 1997).

The suppressor of Hairy-wing [su(Hw)] binding region disrupts communication between a large number of enhancers and promoters and protects transgenes from chromosomal position effects. These properties classify the su(Hw) binding region as an insulator. While enhancers are blocked in a general manner, protection from repressors appears to be more variable. These studies investigate whether repression resulting from the Polycomb group genes (derived from a gypsy element) can be blocked by the su(Hw) binding region. The effects of this binding region on repression established by an Ultrabithorax Polycomb group response element were examined. A transposon carrying two reporter genes, the yellow and white genes, was used so that repression and insulation could be assayed simultaneously. The su(Hw) binding region is effective at preventing Polycomb group repression. These studies suggest that one role of the su(Hw) protein may be to restrict the range of action of repressors, such as the Polycomb group proteins, throughout the euchromatic regions of the genome (Mallina, 1998).

To gain further insights into the types of position effects that can be insulated by suppressor of Hairy-wing, the effects of the SU(HW)-binding region on dosage compensation of the X-linked mini-white gene were determined. Dosage compensation is the process that equalizes the unequal content of X-linked genes in males and females by increasing the X-linked transcription level twofold in males (See Sex lethal for more information). Transposition of X-linked genes to the autosomes commonly results in incomplete dosage compensation, indicating that the distinct male X chromatin environment is important for this process. Dosage compensation of autosomally integrated mini-white genes flanked by SU(HW)-binding regions was greatly improved, such that complete or nearly complete compensation is observed at the majority of insertion sites. The SU(HW) protein was essential for this enhanced dosage compensation because in a su(Hw) mutant background compensation is incomplete. These experiments provide evidence that the SU(HW)-binding region facilitates dosage compensation of the mini-white gene on the autosomes. This may result from protection of the mini-white gene from a negative autosomal chromatin environment (Roseman, 1995a).

Suppressor of Hairy wing interacts with a second protein, modifier of mdg4 [mod(mdg4)], that affects the ability of SU(HW) to act as a transcriptional silencer. su(Hw) protein inhibits the function of transcriptional enhancers located distally from the promoter with respect to the location of SU(HW)-binding sites. This polarity is due to the ability of the SU(HW)-binding region to form a chromatin insulator. Mutations in modifier of mdg4 enhance the effect of su(Hw) by inhibiting the function of enhancers located on both sides of the SU(HW)-binding region. This inhibition results in a variegated expression pattern, and mutations in mod(mdg4) act as classical enhancers of position-effect variegation. The MOD(MDG4) and SU(HW) proteins interact with each other. The MOD(MDG4) protein controls the nature of the repressive effect of SU(HW): in the absence of MOD(MDG4) protein, SU(HW) exerts a bidirectional silencing effect, whereas in the presence of MOD(MDG4), the silencing effect is transformed into unidirectional repression (Gerasimova, 1995).

Germ line transformation of white- Drosophila embryos with P-element vectors containing white expression cassettes results in flies with different eye color phenotypes due to position effects at the sites of transgene insertion. These position effects can be cured by specific DNA elements, such as the Drosophila scs and scs' and by gypsy elements, that have insulator activity in vivo. Matrix attachment regions (MARs) are DNA elements that are identified and defined by their ability to bind to DNA- and histone-depleted nuclei, which are generally termed nuclear matrices. MARs are typically AT-rich elements that contain consensus cleavage sites for topoisomerase II, and they may contain one or more loosely defined short sequence motifs, but, in general, their structures are not highly homologous. MARs are dispersed throughout eukaryotic genomes, having been found in centromeric DNA, within genes, and in intergenic regions. Especially interesting is the observation that the gypsy insulator of Drosophila has been identified as a MAR. This is a retroviral sequence that binds Suppressor of Hairy wing and the su(Hw) associated protein Mod(mdg4) (Nabirochkin,1998). The matrix-binding activities of MARs have been conserved throughout eukaryotic evolution. The functions of MARs in vivo are largely unknown, but one commonly held view is that MARs anchor individual chromatin loops to a proteinaceous matrix or scaffold in both interphase nuclei and mitotic chromosomes (Namciu, 1998 and references).

A test was performed of the ability of human MARS to insulate white from position effect variagation. Two different human MARs, from the apolipoprotein B and alpha1-antitrypsin loci, insulate white transgene expression from position effects in Drosophila. Both elements reduce variability in transgene expression without enhancing levels of white gene expression. In contrast, expression of white transgenes containing human DNA segments without matrix-binding activity is highly variable in Drosophila transformants. These data indicate that human MARs can function as insulator elements in vivo in Drosophila (Namciu, 1998).

Gypsy insulator blocks the interaction of even-skipped stripe enhancers when positioned between the enhancer and target promoter. The simultaneous use of two stripe enhancers (eve stripes 2 and 3) indicates that enhancers lying distal to the insulator are selectively blocked. The insertion of stripe-insulator-stripe sequences between two divergently transcribed promoters indicates that enhancers barred from acting on one basal promoter are fully accessable to appropriate regulatory factors for activating the other promoter. These results suggest that insulators do not propagate changes in chromatin structure (Cai, 1995).

A stripe expression assay was used in transgenic embryos to investigate the role of mod(mdg4) in gypsy insulator activity. The insulator was inserted between defined even-skipped stripe 2 enhancer and stripe 3 enhancer and placed among divergently transcribed reporter genes (white and lacZ) containing distinct core promoter sequences. These assays indicate that mod(mdg4) is essential for the enhancer-blocking activity of the insulator DNA. Reductions in mod(mdg4)+ activity cause the insulator to function as a promoter-specific silencer that selectively represses white, but not lacZ. It is thought that in the absence of mod(mdg4) the su(HW) protein can interact with an unknown repressor protein that selectively silences some promoters but not others. The repression of white does not affect the expression of the closely linked lacZ gene, suggesting that the insulator does not propagate changes in chromatin structure. These results provide an explanation for why mod(mdg4) exerts differential effects on different gypsy-induced mutations (Cai, 1997).

To determine whether the gypsy insulator can block silencer-promoter interactions, it was placed between the VRE of zen and the eve stripe 2 enhancer. The VRE contains both silencer elements and general activation The gypsy insulator does not substantially impede silencing from the VR600 element, so that stripe 2 expression is largely excluded from ventral regions. The insulator does block VR600 activation function, indicating that the gypsy element selectively blocks activator-promoter interactions compared with silencer-promoter interactions (Cai, 1995).

The gypsy insulator shows the same effect in the yolk protein (yp) gene as it shows in the eve gene. The effects of the su(Hw) binding region on yp gene expression have been determined. These genes are regulated by shared enhancers in the intergenic region, which provided a method to examine whether an enhancer blocked by the SU(HW) protein remains functional. A blocked enhancer is completely active on a second promoter that has no intervening SU(HW) binding site, supporting the proposal that the SU(HW) protein is an insulator protein that acts by forming a new boundary in a pre-existing chromatin domain, thereby preventing the interaction of regulatory elements located upstream of the insertion site with the promoter. yp promoter function is not diminished by sharing enhancers, suggesting that these enhancers are not rate limiting for transcriptional activation. In addition, yp promoter activity has been found to be interdependent, such that transcription from one promoter influences the level of activity of the linked promoter (Scott, 1995).

SU(HW) protein can repress transcription on a second chromosome when combined with mutations in modifer of mdg4. Mutations in mod(mdg4) result in the repression of a gene when the SU(HW) protein is bound to sequences in the copy of this gene located in a second homologous chromosome. This effect is dependent on the presence of the SU(HW) binding region from the gypsy retrotransposon in at least one of the chromosomes and is enhanced by the presence of additional gypsy sequences on the other homolog. This phenomenon is inhibited by chromosomal rearrangements that disrupt pairing, suggesting that close apposition between the two copies of the affected gene is important for trans repression of transcription. These results indicate that, in the absence of mod-(mdg4) product, the SU(HW) protein present in one chromosome can act in trans and inactivate enhancers located in the other homolog (Georgiev, 1995).

Flanking su(HW) binding sites [su(HW)BSs] can create a chromosomal domain, permitting activity of the chorion gene DNA replication origin. During Drosophila oogenesis the chorion (eggshell) gene loci are amplified about 80 fold through repeated initiation of DNA replication. The cis-acting amplification control element, on the third chromosome (ACE3), is required for high levels of amplification initiation at the nearby major origin of replication, Ori-ß. A transgenic chorion locus construct containing ACE3 and Ori-ß is able to amplify but is extremely sensitive to position effects: only 7 of 21 independent insertions amplify greater than 10-fold. The inclusion of flanking su(HW)BSs in the construct dramatically protects DNA replication from position effects: 31 of 31 insertions now amplify greater than ten-fold, and this protection is reduced in a su(HW) mutant background. Amplification is equal on both sides of the su(HW)BS, demonstrating that replication fork passage is not significantly impeded by these sites. Inclusion of only a single Su(HW)BS in the construct does not provide detectable protection for the chorion gene DNA replication origin from position effects (Lu, 1997).

The su(Hw) protein is responsible for the insulation mediated by the su(Hw)-binding region present in the gypsy retrotransposon. In the y2 mutant, su(Hw) protein partially inhibits yellow transcription by repressing the function of transcriptional enhancers located distally from the yellow promoter with respect to gypsy. y2 mutation derivatives have been induced by the insertion of two hobo copies on both sides of gypsy: into the yellow intron and into the 5' regulatory region upstream of the wing and body enhancers. The hobo elements have the same structure and orientation, opposite from the direction of yellow transcription. In the sequence context, where two copies of hobo are separated by the su(Hw)-binding region, hobo-dependent rearrangements are frequently associated with duplications of the region between the hobo elements. Duplication of the su(Hw)-binding region strongly inhibits the insulation of the yellow promoter separated from the body and wing enhancers by gypsy. These results provide a better insight into mechanisms by which the su(Hw)-binding region affects the enhancer function (Gause, 1998).

The prevailing model concerning the mechanism of insulator function proposes that insulators are chromatin boundaries. A domain assembled by boundaries prevents interactions between regulatory elements by promoting the folding of a higher-order chromatin structure in such a way as to increase the likelihood of interactions between regulatory elements within a domain, while decreasing these interactions between domains. The recent finding that blocked enhancers retain their full activity suggests that the effects of the su(Hw) protein on the enhancer function may be caused by the formation of such a domain boundary. In view of this, two su(Hw)-binding regions, resulting from a hobo-dependent rearrangement, may act as boundaries to define distinct chromosomal domains causing the suppression of insulation seen in ylh alleles. Distal enhancers under certain conditions may "bypass" the domain flanked from both sides by su(Hw)-binding regions and activate the proximal yellow promoter. However, this model fails to explain the activation of yellow promoter by enhancers flanked from both sides by a su(Hw)-binding region in the ymh and yrh alleles. Another type of model (termed the Decoy model) suggests that the su(Hw)-binding region functions as a flexible regulatory element, modulating enhancer-promoter interactions within complex genetic loci. It is proposed that insulators assemble complexes that might trap an enhancer in a nonproductive interaction, because the insulator lacks promoter function and no transcription occurs as a result. Other models postulate that an insulator binding protein interacts and interferes with higher eucaryotic proteins that facilitate interactions between the enhancer and promoter. The results obtained in the present work may be explained by either model. The ectopic intrachromosomal pairing between two gypsy elements or the interactions between su(Hw) proteins bound to two different su(Hw)-binding regions may prevent the organization of a nonproductive complex between su(Hw) protein and proteins, whose functions are either to activate transcription by enhancer binding or to facilitate the interaction between enhancer and promoter (Gause, 1998).

The mod(mdg4) gene encodes a protein that interacts with the su(Hw) protein and contributes to the insulating function of the su(Hw)-binding region. In the case of the y2 mutation, the hypomorph mod(mdg4)1u1 mutation changes the action of the su(Hw)-binding region in such a way that it inactivates yellow transcription driven by enhancers not separated by the su(Hw)-binding region from the yellow promoter. This observation may be explained by assuming that in the presence of the hypomorphic mod(mdg4)1u1 mutation, the su(Hw) protein directly inhibits the expression from the yellow promoter. An alternative explanation is that together the su(Hw) and mod(mdg4) proteins are able to affect chromatin structure. According to this hypothesis, binding of the su(Hw) protein to its target sequence creates a bidirectional repressive effect, similar to the silencing caused by heterochromatin. Subsequent interactions between the mod(mdg4) and su(Hw) proteins transform this nonspecific silencer into a polar insulator. The role of the chromatin structure in the action of mod(mdg4)1u1 is supported by the observation that y2, mod(mdg4)1u1 males have variegated yellow expression in the tip of the abdomen: dots of a darkly pigmented cuticle against the background of mutant-colored cuticle characteristic of y2. However, dots of a darkly pigmented cuticle were absent in males carrying a combination of mod(mdg4)1u1 with y alleles that had a deletion of enhancer elements. Therefore, variegated pigmentation on the tip of the abdomen may be interpreted as a result of the ability of enhancer elements to partially overcome su(Hw)-binding insulation in mod(mdg4)1u1 background. In this study, the duplication of gypsy in yrh and ylh alleles completely or partially suppressed the inhibitory effect of the mod(mdg4)1u1 mutation on yellow expression in the body and wings. Ectopic intrachromosomal pairing between gypsy elements could alter the properties of the su(Hw)-binding region as an insulator and suppress the effect of the mod(mdg4)1u1 mutation. However, it is difficult to explain this fact by assuming that the su(Hw) protein creates a bidirectional repressive effect in the absence of the mod(mdg4) protein (Gause, 1998).

Multimerization of sequences is thought to only enhance the possibility of formation of a higher order chromatin structure. The absence of the mod(mdg4)1u1 effect on yellow transcription in the yellow-containing construction, where the su(Hw)-binding region is inserted at position -1648, does not support the possibility that the mod(mdg4)1u1 mutation changes the chromatin structure. Although the su(Hw)-binding region in this construction is located between two enhancers of the yellow gene and blocks the wing enhancer, it does not repress yellow transcription in the presence of the mod(mdg4)1u1 mutation. This result can hardly be explained in terms of changes of the chromatin structure in the yellow gene by the su(Hw) protein. The role of the mod(mdg4)1u1 mutation with regard to the gypsy insulator has been previously studied in transgenic embryos. The su(Hw)-binding region was inserted between defined enhancers and placed among divergently transcribed reporter genes (white and lacZ) containing distinct core promoter sequences. The mod(mdg4)1u1 mutation causes the insulator to function as a promoter-specific silencer that selectively represses white, but not lacZ The repression of white does not affect the expression of the closely linked lacZ gene, suggesting that the insulator does not propagate changes in chromatin structure. Thus, the results presented in this work and some previous data support the possibility that the inhibiting action of the mod(mdg4)1u1 mutation is realized through a direct interaction of the su(Hw) protein with the yellow promoter, rather than through the action on chromatin structure (Gause, 1998).

Complex patterns of achete and scute expression are constructed by separable cis-controlling elements present within a large (ca. 90-kb) region. The yellow gene located 10 kb from ac has completely different expression patterns and is activated by different enhancers. Therefore, these genes may serve as a good model system for the analysis of proper enhancer-promoter recognition. Autologous recognition between genes and their respective promoters may depend on the existence of an interdomain boundary between AS-C and the yellow locus, or it may be determined by the specificity of the proteins assembled on a certain enhancer and promoter. An inversion is described that puts the yellow gene between the ac and sc genes and almost all of their cis-regulatory elements. This inversion shows only weak interference with the expression of the ac and sc genes. When the su(Hw)-binding region is deleted or inactivated by the su(Hw) mutation, the sc phenotype of the flies is practically indistinguishable from that of the wild type. The presence of the yellow gene between the AS-C enhancers and the promoters of the ac and sc genes does not interfere with ac and sc expression in most areas. This work shows, however, that it is not required that the su(Hw) insulator separate promoters from enhancers to allow inhibition of transcription by the su(Hw) protein. The presence of the su(Hw) insulator, located more than 20 kb away from the inversion, facilitates strong suppression of achaete and scute gene expression, although is does not separate the promoters from the AS-C enhancers (Golovnin, 1999).

The mechanism of direct interaction between AS-C enhancers and su(Hw) insulator is not yet clear. One possibility is that the pairing between the P elements located at the breakpoints of the inversion facilitates such interaction. However, deletions of the P elements on both sides of the inversion fail to influence the repression mediated by the su(Hw) insulator. Another possibility is that the inversion brings the su(Hw)-binding region into a close contact with the AS-C cis-regulatory elements due to changes in chromatin folding, which then leads to new long-range contacts between certain chromatin regions. As a result, the su(Hw)-mod(mdg4) complex formed on the su(Hw) insulator becomes capable of interacting directly with enhancer-bound transcription activators or with proteins responsible for enhancer-promoter interactions. The fact that the inactivation of AS-C control elements by the su(Hw)-binding region in the inversion is only partial may be explained by reversible interactions between the insulator and enhancers similar to the normal dynamic interactions observed between enhancers and promoters (Golovnin, 1999).

Chromatin insulators are regulatory elements that block the action of transcriptional enhancers when interposed between enhancer and promoter. The Drosophila Suppressor of Hairy wing [Su(Hw)] protein binds the Su(Hw) insulator and prevents enhancer-promoter interaction by a mechanism that is not understood. When two copies of the Su(Hw) insulator element, instead of a single copy, are inserted between enhancer and promoter, insulator activity is neutralized and the enhancer-promoter interaction may instead be facilitated. This paradoxical phenomenon could be explained by interactions between protein complexes bound at the insulators (Muravyova, 2000).

The Drosophila gypsy retrotransposon contains a chromatin insulator that consists of a cluster of 12 binding sites for the Su(Hw) zinc-finger protein. In the presence of Su(Hw) protein binding, the insulator blocks the activity of an enhancer separated from the promoter by an Su(Hw) binding region. However, this insulator action fails in certain genetic rearrangements that introduce more than one gypsy retrotransposon in the region of the yellow gene. The loss of insulator activity might result from intrachromosomal pairing between the two gypsy retrotransposons, causing chromatin to fold and allowing the enhancer to contact the promoter. Alternatively, interaction between the proteins bound to two Su(Hw) insulator elements might neutralize insulator action. Here, insulator activity was examined as affected by insulator element copy number and location (Muravyova, 2000).

The yellow gene is required for dark pigmentation of Drosophila larval and adult cuticle and its derivatives. Two upstream enhancers, En-b and En-w, activate expression in the body cuticle and wing blades, respectively. When a single Su(Hw) insulator is inserted at position -893 relative to the yellow transcription start, between the enhancers and the yellow promoter (ESY), enhancer action is blocked, resulting in yellow instead of dark pigmentation of body and wing cuticle. This block is relieved and pigmentation is restored when the construct is tested in a su(Hw)- background, confirming that the Su(Hw) protein is responsible. In the ESFSY construct, a fragment bearing two Su(Hw) insulators, separated by a 1.5-kilobase (kb) spacer fragment, was inserted at position -893. The spacer is derived from the second exon of the yellow gene and has no enhancer or insulator activity of its own. In seven ESFSY transgenic lines, yellow expression is higher than that in the control ESY lines, and in three lines it is at wild-type levels. When three of the less pigmented ESFSY were tested in a su(Hw)- background, wild-type pigmentation is restored. Thus, the second Su(Hw) insulator partially or completely neutralizes the effect of the first one. Similar results are obtained when the distance between the two Su(Hw) insulators is reduced to 200 base pairs (bp) (Muravyova, 2000).

The body and wing enhancers in these constructs are responsible for wild-type dark pigmentation because, when they are removed from ESFSY, yielding SFSY, body and wing pigmentation is yellow in all transgenic lines. Similarly, constructs containing the yellow gene alone never result in body or wing pigmentation. Increasing the distance between upstream enhancers and the yellow promoter does not weaken insulator activity because lines containing ESFY, bearing a single Su(Hw) insulator 2.4 kb from the yellow transcription start, all have yellow body and wing pigmentation, indicative of the block of wing and body enhancers (Muravyova, 2000).

If the loss of insulator activity is due to a steric constraint imposed by a physical interaction between the two insulators, flanking either the enhancers or the target gene with insulators might have the same effect. This was tested with the SFESY construct in which two Su(Hw) insulators frame the wing and body enhancers. Flies from nine SFESY transgenic lines exhibit yellow wing and body pigmentation. When two of these lines were crossed into a su(Hw)- background, wild-type levels of pigmentation were restored, confirming that the proximal Su(Hw) element retained insulator activity (Muravyova, 2000).

In the ES(-893)YS construct, the yellow gene is flanked by Su(Hw) insulators, one at position -893 and the other downstream of the yellow gene. In nine ES(-893)YS transgenic lines, yellow expression in the body and wings is blocked. When two of these lines were crossed into a su(Hw)- background, wild-type pigmentation of wings and body is restored. Thus, the second insulator, downstream of yellow, does not prevent the insulating function of the first (Muravyova, 2000).

Next, a different enhancer-promoter combination was tested. The white gene is required for eye pigmentation and is regulated by its eye-specific enhancer. Interposing the Su(Hw) insulator between the eye enhancer and white promoter completely blocks enhancer activity, whereas bracketing the mini-white gene between two Su(Hw) insulators protects white expression from position effects. In the EyeSYW construct, the eye enhancer was inserted between the yellow wing and body enhancers and was flanked by Flp recognition target (FRT) sites to permit its excision from transgenic flies. The three enhancers are separated from the yellow and white genes by a Su(Hw) insulator. Flies of 20 EyeSYW lines display eye pigmentation levels like those produced by an enhancerless white transposon, that is, ranging from pale yellow to red, depending on the insertion site. In two red-eyed EyeSYW lines, the deletion of the eye enhancer by Flp-dependent excision does not influence eye color, implying that in these two lines the white gene is activated by some genomic enhancer element. Thus, one Su(Hw) insulator interposed between eye enhancer and white gene blocks enhancer-promoter communication. The body and wing enhancers of the yellow gene are also blocked in these lines, indicating that the insulator functions normally. Similarly, if the insulator is placed in front of the white gene, to give EyeYSW, the 14 transgenic lines obtained have eye colors in the range expected in the absence of eye enhancer. Deletion of the eye enhancer in five dark orange-eyed lines does not change eye pigmentation. Thus, the eye enhancer is blocked by one copy of the Su(Hw) element inserted either near or far from the white promoter (Muravyova, 2000).

The EyeSYSW construct uses the same enhancer configuration described above and contains one Su(Hw) insulator at position -893 relative to the yellow transcription start and another inserted between the yellow gene and the mini-white promoter. Therefore, just one insulator intervenes between the enhancers and yellow but two insulators between the enhancers and white. In 19 of 21 transgenic EyeSYSW lines, wing and body pigmentation is yellow, indicating that the yellow enhancers are blocked, whereas white expression is stronger than in lines bearing the mini-white gene without eye enhancer. To demonstrate that the eye enhancer stimulates white expression in these lines, the enhancer was excised by Flp-induced recombination between FRT sites. In nine DeltaEyeSYSW lines tested, the deletion of the eye enhancer strongly diminished eye pigmentation, indicating that the enhancer can activate the white gene despite the two intervening insulators. Therefore, also in this case, two insulators between enhancer and promoter neutralize one another. However, interaction between the two insulators does not simply inactivate them, because the upstream insulator can still block the activation of the yellow gene (Muravyova, 2000).

In the same EyeSYSW lines, white expression was studied in a su(Hw)- background. In five lines, the absence of Su(Hw) protein reduces white expression, implying that the Su(Hw) protein actually has a positive role, facilitating enhancer-promoter interactions. In four other lines, the absence of Su(Hw) protein had no effect. Thus, the stimulating effect of the two Su(Hw) insulators may depend on genomic context and/or local chromatin structure. To show that the Su(Hw) protein does not by itself activate white expression, five lines bearing the EyeSYSW transposon with deleted eye enhancer (DeltaEyeSYSW) were crossed into a su(Hw)- background. The absence of Su(Hw) protein did not influence white expression (Muravyova, 2000).

To determine what configuration of two insulators neutralizes insulator activity, EyeSYWS, in which the two Su(Hw) insulators frame the yellow and white genes, was constructed. Fourteen EyeSYWS lines display weak expression of both white and yellow, indicating that all three enhancers upstream of the interposed Su(Hw) insulator are blocked. However, when the mini-white gene flanked by two Su(Hw) insulators is inserted at position -893 relative to the yellow transcription start site (EyeSWFSY), the yellow gene is expressed in the body and wings. In a su(Hw)- background, yellow expression decreased in three lines and did not change in one line, showing that the activation of the yellow promoter by distant yellow enhancers is improved by an interposed insulator pair. Thus, for both white and yellow, the insertion of two Su(Hw) insulators between the respective enhancers and promoters may facilitate their interaction instead of blocking it. When the Su(Hw) insulator between white and yellow genes is removed, yielding EyeSFWY, yellow expression in the body and wings is suppressed, showing again that a single insulator blocks the wing and body enhancers. Two copies of Su(Hw) do not simply neutralize one another by an exclusive binary interaction. In the EyeSFSYSW construct, three insulator copies intervene between eye enhancer and white gene and two copies are between the yellow enhancers and the yellow gene. In 12 of 16 lines carrying this transposon, both yellow and white are activated, producing flies with strongly pigmented eyes and wing and body cuticle (Muravyova, 2000).

In summary then, when two or more Su(Hw) insulators are introduced between enhancer and promoter, their enhancer-blocking effect is neutralized in most cases and enhancer-promoter communication is often improved. The implication is that two insulators interact, probably through the protein complexes bound to them. This interaction by itself does not neutralize the blocking action, because when the insulators frame the enhancers or the target gene, the block still occurs. A possible explanation is that the 'looping out' of the sequences separating enhancer and promoter displaces the insulators out of the way and, by bringing the enhancer and promoter closer, may even stimulate expression. This may explain why the stimulating effect increases with the distance between enhancers and promoter (Muravyova, 2000).

These effects may have a bearing on the mechanism of insulator action. A possible way to envision how the insulator interferes with the access of the enhancer to the promoter is by associating with the nearest Su(Hw)-related complexes in the nucleus. The effect of this association would be to tether loops containing members of an enhancer-promoter pair, thereby interfering with the interaction of the enhancer on one loop with the promoter on another loop. When two Su(Hw) elements are placed between enhancer and promoter, the loop would form preferentially between the two neighboring Su(Hw) elements, thereby shortening the distance between enhancer and promoter rather than inhibiting their interaction. This type of mechanism may also help to explain the role of boundary elements in the Drosophila bithorax complex. In the Abd-B regulatory region, boundary elements like Fab-7 and Fab-8 flank the iab enhancer regions, insulating them from the silencing or activating effects of adjacent regulatory regions. However, as insulators, the boundary elements would also block activation of the Abd-B promoter by more distant iab enhancers, thus defeating the purpose of these enhancers. Although other explanations are possible, these results with insulator pairs may account for this discrepancy. Interaction between boundary elements flanking each enhancer may effectively protect the iab enhancers from outside repressing effects and facilitate, instead of blocking, enhancer-promoter communication. It is possible, in fact, that one role of certain kinds of insulator is to promote the interaction between distant enhancers and promoters (Muravyova, 2000).

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. Two Suppressor of Hairy wing binding sites in the intergenic region between the yellow gene and the Achaete-scute gene complex are shown to 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 (Golovnin, 2003).

To explain how the long-range activation potential of eukaryotic enhancers are restricted to the relevant target promoter, it has been proposed that eukaryotic chromatin is organized into functionally independent domains that prevent illegitimate enhancer-promoter communication. Recent publications suggest a model in which distant chromosomal binding sites of Su(Hw) are brought together by Mod(mdg4) into a small number of insulator bodies located at the nuclear periphery. In this way Su(Hw) marks the base of topologically independent looped chromatin domains. However, despite the presence of many endogenous Su(Hw) binding sites in polytene chromosomes, no specific function has been attributed to any site in a particular gene (Golovnin, 2003).

Using in vivo and in vitro assays, it has been shown that there exists a functional Su(Hw) insulator between a P-element inserted yellow gene and AS-C. At least four Su(Hw) binding sites have been shown to be required for effective enhancer blocking. It has been shown that a 125 bp fragment including only two Su(Hw) binding sites can partially block the strong yellow enhancer, while a larger 454 bp fragment including the same Su(Hw) sites completely blocks yellow enhancers. Thus, additional proteins binding to neighboring sequences are required for strong insulator action of the element between yellow and AS-C. The sequencing of the Drosophila genome shows the absence of large clusters of endogenous Su(Hw) binding sites, such as are found in the gypsy retrotransposon. It seems possible that in endogenous insulators, Su(Hw) cooperates with additional DNA-binding proteins to produce insulator activity. This assumption may also explain the absence of lethal phenotypes in the su(Hw)- background since other proteins would partly compensate for the loss of Su(Hw) function (Golovnin, 2003).

The results further confirm the initial observation of the interaction between two gypsy insulators. The two Su(Hw) binding sites in the 125 bp fragment and the gypsy insulator mutually neutralize each other's enhancer-blocking activity. Thus, the difference in the number of Su(Hw) binding sites between interacting insulators is not critical for the effective neutralization of the enhancer blocking activity (Golovnin, 2003).

Increasing the number of Su(Hw) binding sites increases insulator strength, and three copies of the 125 bp insulator block better than a single copy. How can this be reconciled with the observation that two Su(Hw) insulators neutralize one another? It is supposed that the neutralization requires the pairing between two insulators. Interaction between neighboring insulators would pre-empt their interaction with larger assemblies of Su(Hw) binding sites that have been proposed to associate together at the nuclear periphery through the Mod(mdg4) protein. Thus, for neutralization, it is supposed that the Su(Hw) binding sites must adopt a paired configuration, therefore requiring a sufficient distance between them for DNA to form a loop. In contrast, putting more Su(Hw) binding sites very close together merely ensures that enough Su(Hw) protein will be bound at any one time to produce insulator action (Golovnin, 2003).

The role of the Su(Hw) and Mod(mdg4) proteins in the expression of ASC genes becomes obvious when the normal architecture of the ASC regulatory region is altered by chromosome rearrangements. Many previously described inversions with breakpoints in the AS-C regulatory region and centric heterochromatin have weak mutant phenotypes, suggesting the presence of sequences that effectively impede the spread of heterochromatic silencing. The appearance of strong variegating repression of the ac and sc genes when the inversions are combined with loss of su(Hw) or mod(mdg4) function suggests that the Su(Hw) and Mod(mdg4) proteins are involved in the stability of the ac and sc expression (Golovnin, 2003).

In the In(1)y3p mutation, a heterochromatic breakpoint in the upstream regulatory region does not effect yellow expression suggesting that the yellow promoter is relatively resistant to heterochromatin proximity at this breakpoint. At the same time, ac and sc expression is strongly affected by su(Hw) or mod(mdg4) mutations, supporting the idea that Su(Hw) binding sites between yellow and ac block heterochromatin spreading (Golovnin, 2003).

The In(1)sc8 and In(1)scv2 inversions separate the ac and sc genes. The requirement of the Su(Hw) and Mod(mdg4) proteins for normal sc expression suggests the existence of additional Su(Hw) binding sites in the AS-C regulatory region. The strong genetic interaction between sc2 and mutations in mod(mdg4) or su(Hw) also supports the presence of additional Su(Hw) binding sites in ASC. The expression of ASC genes is regulated by a large number of enhancer-like elements. It seems reasonable that these ASC enhancers should be separated by boundary elements as was found for the 3' cis-regulatory region of Abdominal B (Abd-B), which is subdivided into a series of iab domains. Boundary elements like MCP, Fab-7 and Fab-8 separate the iab domains and protect each against positive and negative chromatin modifications induced by neighboring iab domains. The genetic results might be explained by the assumption that the Su(Hw)-Mod(mdg4) protein complex participates in formation of boundary elements between certain AS-C enhancers. The absence of noticeable changes in the wild-type AS-C gene expression on the su(Hw) or mod(mdg4) mutant background might be the consequence of the functional redundancy of the Su(Hw)-Mod(mdg4) protein complex. No clusters of potential endogenous Su(Hw) binding sites are found inside the AS-C sequence. Thus, it seems possible that Su(Hw)-Mod(mdg4) cooperates with other non-identified proteins in formation of the functional boundaries in the regulatory region of AS-C. The identification and characterization of new Su(Hw) binding sites may help in understanding the role of Su(Hw)/Mod(mdg4) in transcriptional regulation of AS-C genes and provide new insights into the mechanisms of the insulator action (Golovnin, 2003).

Study of long-distance functional interactions between Su(Hw) insulators that can regulate enhancer-promoter communication

The Su(Hw) insulator found in the gypsy retrotransposon is the most potent enhancer blocker in Drosophila melanogaster. However, two such insulators in tandem do not prevent enhancer-promoter communication, apparently because of their pairing interaction that results in mutual neutralization. Furthering studies of the role of insulators in the control of gene expression, this study presents a functional analysis of a large set of transgenic constructs with various arrangements of regulatory elements, including two or three insulators. Their interplay can have quite different outcomes depending on the order of and distance between elements. Thus, insulators can interact with each other over considerable distances, across interposed enhancers or promoters and coding sequences, whereby enhancer blocking may be attenuated, cancelled, or restored. Some inferences concerning the possible modes of insulator action are made from collating the new data and the relevant literature, with tentative schemes illustrating the regulatory situations in particular model constructs (Savitskaya, 2006).

A single Su(Hw) insulator at any position between the yellow enhancer and promoter completely precludes gene activation in cis, whereas tandem pairing of the insulators results in 'mutual neutralization' or 'cancellation.' The insulators can also pair across interposed regulatory elements (enhancers or promoter); likewise, two Su(Hw) insulators interact across a strong scs insulator, which thereby is also rendered incapable of enhancer blocking (Savitskaya, 2006).

Previously, it was have shown that a pair of closely spaced insulators does not in itself stimulate transcription of yellow in wings and body cuticle. The very act of putting together a couple of insulators and an enhancer (or other element) does not create an artificial enhancer-like region that could have smeared the results (Savitskaya, 2006).

Further, downstream gene activation is completely blocked when two insulators surround the enhancer quite closely (within 4 kb of each other) but only partly blocked when the insulators are 10 kb apart. In a system with a 'cancelled' tandem pair of insulators, enhancer blocking can be almost completely restored by a third downstream insulator (Savitskaya, 2006).

It is not yet known how the insulator disrupts enhancer-promoter communication, but some educated guesses can be made. Though the 'promoter decoy' model is most common, there is no actual evidence that an insulator complex binds to an enhancer complex to neutralize it (as in insulator pairing) or traps its vital component(s), as is inherent in the model; on the contrary, the definitive position dependence of insulator action implies that insulators do not inactivate enhancers, silencers, or promoters. Furthermore, enhancer blocking by an intervening Su(Hw) insulator in cis does not prevent the enhancer action in trans; this makes insulator-enhancer pairing quite unlikely, because such neutralization should have affected both modes of gene activation. In the aggregate, this gives grounds to the supposition that the insulator interacts with the enhancer only when and inasmuch as the enhancer tries to negotiate the insulator. One can immediately see that the decoy model as amended for transient interaction represents a particular case of this general issue. It is concluded that insulators are not just barriers to interaction between other elements but rather versatile agents that may be widely involved in regulation of complex genetic loci (Savitskaya, 2006).

PRE-mediated bypass of Two Su(Hw) insulators targets PcG proteins to a downstream promoter

Drosophila Polycomb group response elements (PRE) silence neighboring genes, but silencing can be blocked by one copy of the Su(Hw) insulator element. Polycomb group (PcG) proteins can spread from a PRE in the flanking chromatin region and PRE blocking depends on a physical barrier established by the insulator to PcG protein spreading. In contrast, PRE-mediated silencing can bypass two Su(Hw) insulators to repress a downstream reporter gene. Strikingly, insulator bypass involves targeting of PcG proteins to the downstream promoter, while they are completely excluded from the intervening insulated domain. This shows that PRE-dependent silencing is compatible with looping of the PRE in order to bring PcG proteins in contact with the promoter and does not require the coating of the whole chromatin domain between PRE and promoter (Comet, 2006).

The present work suggests two complementary mechanisms for promoter silencing by PcG proteins. (1) The data show directly that PcG proteins recruited at a PRE can spread over several kilobases along the flanking chromatin. Therefore, promoters located within short distances from PREs might be silenced by PcG spreading and interference with the transcription machinery. However, PcG spreading induced by the Ubx PRE did not extend beyond few kilobases in these experiments, and ChIP on chip also showed limited extension of PcG protein binding from known PREs. This limited spreading might depend on genomic sequences or proteins bound to them that might attenuate chromatin association of PcG proteins. Thus, spreading alone might not be sufficient for silencing promoters located several tens of kilobases away, as in the case of the Ubx gene, suggesting that additional mechanisms allow PcG proteins to gain access at distant promoters. It was found that pairing of two Su(Hw) insulators can induce promoter association of PcG complexes without PcG-mediated coating of the insulated domain. (2) This suggests an additional mechanism of PRE-dependent promoter silencing, whereby PREs located at large distances from their promoters might contact them via looping of intervening domains. This looping might be favored by natural regulatory elements present at these loci, which might play a role similar to the pair of Su(Hw) insulators used in this study (Comet, 2006).

The endogenous distribution of PcG proteins might reflect spreading from a PRE into the flanking genomic region as well as their ability to bypass insulators. At the two endogenous target loci en and ph, where PREs are located in the promoter region, the distribution of PC and PH suggests spreading from the PREs. The distribution of PC and PH was characterized at Ubx, a locus where the PRE is over 20 kb upstream from the Ubx promoter. In addition to Ubx, this region contains the bxd locus, driving the production of noncoding transcripts. PC and PH binding shows a peak at the bxd transcription start site downstream to the PRE, in addition to the previously described peaks corresponding to the PRE and the Ubx promoter. Furthermore, binding of PH and PC drops between the bxd peak and the Ubx promoter and rises again at the promoter. This distribution is consistent with spreading from the PRE for short-distance chromatin silencing, and direct targeting of PRE bound PcG complexes to the downstream promoter to drive silencing over larger distances (Comet, 2006).

The sharp transitions in PcG protein binding detected at insulators are surprising, especially considering that the PRC1 complex is larger than 1 MDa, a size equivalent to several nucleosomes. The block in PcG spreading might depend on a physical barrier imposed by protein complexes tightly bound to the insulator. The bypass of the insulated domain might be explained by topological features imposed by insulators on three-dimensional chromatin folding. The Su(Hw) and Mod(mdg4) proteins that regulate the Su(Hw) insulator are organized into discrete “insulator bodies” in the cell nucleus. PcG proteins are also organized into “PcG bodies” that might be the sites of PRE-mediated silencing. A single Su(Hw) insulator located near a PRE might thus exclude the downstream domain from the PcG body associated to the PRE. A second insulator paired with the first one in the insulator body might bring the downstream promoter at the PRE-associated PcG body, while excluding from it the intervening chromatin domain. This type of regulation of three-dimensional chromatin folding by insulator elements might modulate gene expression at a number of loci in Drosophila and other species (Comet, 2006).

Enhancer blocking and transvection at the Drosophila apterous locus

Intra- and interchromosomal interactions have been implicated in a number of genetic phenomena in diverse organisms, suggesting that the higher-order structural organization of chromosomes in the nucleus can have a profound impact on gene regulation. In Drosophila, homologous chromosomes remain paired in somatic tissues, allowing for trans interactions between genes and regulatory elements on the two homologs. One consequence of homolog pairing is the phenomenon of transvection, in which regulatory elements on one homolog can affect the expression of a gene in trans. This paper reports a new instance of transvection at the Drosophila apterous (ap) locus. Two different insertions of boundary elements in the ap regulatory region were identified. The boundaries are inserted between the ap wing enhancer and the ap promoter and have highly penetrant wing defects typical of mutants in ap. When crossed to an ap promoter deletion, both boundary inserts exhibit the interallelic complementation characteristic of transvection. To confirm that transvection occurs at ap, a deletion of the ap wing enhancer was generated by FRT-mediated recombination. When the wing-enhancer deletion is crossed to the ap promoter deletion, strong transvection is observed. Interestingly, the two boundary elements, which are inserted ~10 kb apart, fail to block enhancer action when they are present in trans to one another. This study demonstrates that this is unlikely to be due to insulator bypass. The transvection effects described here may provide insight into the role that boundary element pairing plays in enhancer blocking both in cis and in trans (Gohl, 2008).

This study presents evidence for transvection at the Drosophila apterous locus. While interallelic complementation at ap has been previously reported, the ap alleles were not molecularly characterized. Consequently, it was not clear whether the complementation between these alleles involved trans-regulatory interactions or occurred at the level of the mutant ap gene products. This study has observed trans-regulatory interactions with several different classes of ap mutations (Gohl, 2008).

The first type is the transvection seen in trans combinations between mutations that disrupt enhancers and mutations that disrupt the promoter. At the ap locus, this is illustrated by the apDG/apUGO35 combination. Interestingly, the transvection observed between apDG and apUGO35 is sufficient to express ap at or near wild-type levels, as >90% of the wings are completely wild type. ap mutants are recessive, so there is likely a range of ap activity that is sufficient to produce wild-type wings (Gohl, 2008).

It is unknown to what extent Dipterans have learned to exploit this interesting feature of their genomes for normal gene regulation. For example, it is unlikely that trans regulation occurs at the endogenous y locus in wild-type flies, since the enhancers appear to be strongly tethered in cis by the promoter. Instead, trans regulation is observed only at y when the enhancers are freed by deletion of the cis promoter. ap is clearly different from y in this respect, since relatively strong trans regulation is also observed when the enhancer deletion, apDG, is combined with presumed ap-coding region mutations that are likely to retain an intact promoter. Since the suppression of these coding region mutants by apDG is not as strong as that observed with the promoter deletion apUGO35, cis interactions between the upstream wing enhancer and the promoter of the mutant gene must compete with the apDG promoter in trans (Gohl, 2008).

The second type of trans-regulatory interaction observed at ap is the transvection effects observed with boundary elements. Two different boundary insertions were observed in the ap regulatory region. apMM-Mcp is an experimentally generated insertion of the Mcp-containing Flipper 2 transposon 403 bp upstream of the ap transcriptional start site between the wing enhancer and the ap promoter. MCP is a polycomb response element from the Drosophila bithorax complex. Although the Mcp element in this transgene contains both a boundary element and a PRE, the results indicate that the wing defects seen in homozygous or hemizygous apMM-Mcp flies are due to the enhancer-blocking activity of the boundary and not due to silencing by the Mcp PRE. In the absence of an Mcp boundary insertion that lacks the PRE, the possibility remains that the Mcp PRE contributes to the ap wing phenotype. However, if this is the case, it is likely that the role of the PRE is a modulatory one, as the bxd PRE alone is not sufficient to cause wing defects. apf00451 is a su(Hw)-containing piggyBac element and was also experimentally inserted between ap enhancer elements and the ap promoter (Gohl, 2008).

One version of this boundary-element-induced transvection is that seen in the interallelic complementation between the boundary insertions and the ap promoter deletion, apUGO35. This trans-regulatory interaction is observed with both the Mcp and su(Hw) elements. The Mcp insert, apMM-Mcp, has a strong ap wing phenotype, but when it is combined with the promoter deletion, apUGO35, the wing defects are partially suppressed. The fact that full suppression is not observed in this combination, while it is observed when the enhancer deletion is combined with the promoter deletion, indicates that the Mcp element must be capable of partially blocking trans interactions between the apUGO35 wing enhancers and the apMM-Mcp promoter. This suggestion is substantiated by a comparison of the wing phenotypes in combinations between apUG035 and the enhancer deletion with (apDG-Mcp) and without (apDG) the Mcp element. While nearly full suppression is observed in the latter case, the suppression of the wing defects in apDG-Mcp/apUGO35 flies is comparatively modest. This difference can be attributed to the ability of the Mcp element to block the ap enhancers in trans from activating the ap promoter in cis to the boundary. In contrast, a comparison of the wing phenotype of the apDG-Mcp/apUGO35 trans combination with flies that are either hemizygous or homozygous for the Mcp insertion, apMM-Mcp, reveals that the enhancer-blocking activity of this boundary element is stronger when the enhancer and promoter are in cis than when they are in a trans configuration (Gohl, 2008).

The other version of boundary-element-induced transvection that was observed is the trans combination between the boundary insertions and the ap wing-enhancer deletion, apDG. This combination was tested for the Mcp and su(Hw) inserts and in both cases the wing phenotype of the enhancer deletion was suppressed. Since the extent of suppression in both cases is considerably less than seen when the enhancer deletion apDG is combined with the promoter deletion apUGO35, it would appear that the boundary in cis to the enhancer is able to partially block its interactions with the ap promoter in trans. As noted above, the converse is also true: boundary elements in trans to the enhancer are able to partially block interactions with the ap promoter in cis (Gohl, 2008).

Since these results demonstrate that the Mcp and su(Hw) boundaries can act not only in cis but also in trans, one might predict either that no interallelic complementation would be observed when two different boundary inserts are combined or that the phenotype would actually become even stronger because of the ability of boundaries to inhibit regulatory interactions in trans. Surprisingly, however, neither of these expectations holds. Instead, flies trans-heterozygous for the Mcp insert apMM-Mcp, and the su(Hw) insert apf00451 have completely wild-type wings. One mechanism that could account for this unexpected result is insulator bypass. Studies on the su(Hw) insulator have shown that enhancer-blocking activity is neutralized when there are two copies of this element in tandem between the enhancer and the promoter. While bypass is thought to involve su(Hw)-pairing interactions, other insulators, including Mcp, can be substituted for one of the two su(Hw) elements. A strong prediction of the insulator bypass model is that interallelic complementation should also be observed when the su(Hw) element in apf00451 is in trans to the enhancer deletion that retains an intact Mcp element, apDG-Mcp. However, this is not the case as the wing phenotype of apDG-Mcp/apf00451 trans-heterozygotes is the same as that of apf00451 alone. This result indicates that the Mcp element is able to prevent trans activation of the ap promoter in cis by the wing enhancers on the apf00451 chromosome. The ability to block enhancers on the trans chromosome from contacting the promoter in cis to a boundary element was also observed when apMM-Mcp is combined with the Mcp-containing enhancer deletion apDG-Mcp (Gohl, 2008).

Thus, the interallelic complementation observed in apMM-Mcp/apf00451 flies is not likely to be an instance of insulator bypass. Instead, it seems that the additive effects of the unblocked, ap proximal portion of the apf00451 enhancer and trans activation by the enhancer on the apMM-Mcp chromosome can account for the wild-type wings of apMM-Mcp/apf00451 flies (Gohl, 2008).

Including the studies reported in this study on boundary insertions in the ap locus, there are now several examples in which the blocking activity of a boundary element can be partially bypassed by interactions between enhancers on one chromosome and the target gene/promoter on the other chromosome. These findings raise the question of why boundary elements are more permissive for regulatory interactions in trans than they are for interactions in cis (Gohl, 2008).

Answering this question depends upon how enhancers communicate with promoters and how boundaries block this communication. Two general models have been proposed to explain how enhancers interact with their target promoters. In the first model, the enhancer (or an activator molecule recruited by the enhancer) processively tracks along the chromosome (perhaps modifying the intervening chromatin) until it encounters the promoter. In this model, boundary elements function as roadblocks (or 'promoter decoys'), stopping the tracking activator and/or the spread of active chromatin. As this model requires the enhancer to act in cis, it is difficult to reconcile it with the phenomenon of transvection, which depends upon regulatory interactions occurring in trans. In addition, if transvection is explained in this model by postulating that the tracking activator skips from one paired chromosome to the other, then it is hard to understand how a boundary element would ever be able to prevent an enhancer from activating a promoter since an activator molecule that can skip freely in trans should also be able to skip over a boundary in cis (Gohl, 2008).

The second model, which is strongly supported by recent studies, hypothesizes that the sliding of the chromatin fiber against itself within a higher-order chromatin domain brings the enhancer and promoter into contact while looping out the intervening DNA. This is more easily reconciled with transvection since the enhancer could interact with a promoter in trans by a similar sliding-looping mechanism as long as the chromatin fibers of the two chromosomes are paired. Indeed, chromosomal rearrangements that disrupt pairing also tend to disrupt transvection. In this model, boundary elements prevent enhancer-promoter contact by isolating the enhancer and the promoter from each other in topologically independent looped domains. It is thought that boundaries generate topologically independent looped domains through pairing interactions with the neighboring boundaries (or by interacting with some fixed structure such as the nuclear matrix). This mechanism is supported by studies on su(Hw), scs/scs', and several boundaries from the Drosophila BX-C. For example, pairing between tandem su(Hw) insulators neutralizes their boundary function, enabling an upstream enhancer to activate a downstream promoter. According to this model for enhancer blocking, the Mcp [or su(Hw)] boundary would isolate the ap wing enhancer from the ap promoter in cis through interactions with the hypothetical upstream and downstream boundaries that define the ap domain (Gohl, 2008).

This mechanism for boundary function in cis still leaves open the question of why boundaries can be partially bypassed in trans. One possibility is that pairing interactions between boundaries occur not only in cis but also in trans. In this model, the arrangement of loop domains would be the same on each chromosome when they both contain the Mcp or su(Hw) boundary insert-there would be two loops, one containing the ap enhancer and the other containing the ap promoter. These loops would be generated by interactions between Mcp and the neighboring proximal and/or distal boundaries. The situation would be more complicated when one chromosome has the boundary element insertion and the other does not. In this case, the wild-type chromosome should have a single ap loop containing both the enhancer and the promoter, while the chromosome containing Mcp should have two loops, one containing the enhancer and the other the promoter. However, this arrangement of loops on the two chromosomes might be dynamically unstable if trans-boundary interactions also tend to stabilize cis contacts between the boundary elements that flank the ap locus. This dynamic instability could disrupt or weaken cis interactions between Mcp and the boundaries flanking the ap locus. In this case, the arrangement of loops on the Mcp-containing chromosome might switch back and forth from two to one, permitting a partial bypass of Mcp through trans-regulatory interactions (Gohl, 2008).

While both the Mcp and su(Hw) boundary elements can be partially bypassed by interactions between the ap enhancer and promoter in trans, trans interactions do not occur when the same boundary insertion is present on both homologs. In contrast, when the Mcp and su(Hw) boundary insertions are present in trans on the two chromosomes (apMM-Mcp/apf00451), this seems to abrogate their blocking activity. One explanation for this effect is that Mcp and su(Hw) are unable to interact with each other; however, it was previously demonstrated that su(Hw) and Mcp can pair with one another, possibly through the interaction of GAGA factor and Mod(mdg4). Since the Mcp and su(Hw) boundary insertions are located at distant sites within the ap locus, another possibility is that the pairing of the two structurally dissimilar alleles in this arrangement results in conformational stress that precludes the formation of stable Mcp/su(Hw) interactions either with each other or with the hypothetical flanking ap boundaries. In this model for enhancer bypass of the Mcp and su(Hw) boundaries in trans, homologous pairing between sequences in the ap locus would loop out the transposons containing the Mcp and su(Hw) boundary elements, preventing them from blocking enhancer-promoter contacts. An alternative possibility is that boundary interactions occur only in pairwise combinations. Thus, instead of interacting simultaneously with the boundaries that flank the ap locus, Mcp and su(Hw) might be paired only with either the upstream or the downstream ap boundary at a given time. If the pairing of Mcp and su(Hw) with the flanking boundaries occurs independently [or if Mcp and su(Hw) differ in their pairing preferences], either of these distinct domains might be predicted to confer enhancer blocking to both homozygous or hemizygous flies. However, when these two alleles are crossed together, the domains in effect would be complementary, with one unblocked enhancer and one unblocked ap gene. It may be possible to distinguish between these different models by generating new insertions into the ap locus in which the Mcp and su(Hw) boundaries are brought closer together and by substituting other boundary elements for Mcp or su(Hw) (Gohl, 2008).

Context differences reveal insulator and activator functions of a Su(Hw) binding region

Insulators are DNA elements that divide chromosomes into independent transcriptional domains. The Drosophila genome contains hundreds of binding sites for the Suppressor of Hairy-wing [Su(Hw)] insulator protein, corresponding to locations of the retroviral gypsy insulator and non-gypsy binding regions (BRs). The first non-gypsy BR identified, 1A-2, resides in cytological region 1A. Using a quantitative transgene system, this study showed that 1A-2 is a composite insulator containing enhancer blocking and facilitator elements. 1A-2 separates the yellow (y) gene from a previously unannotated, non-coding RNA gene, named yar for y-achaete (ac) intergenic RNA. The role of 1A-2 was elucidated using homologous recombination to excise these sequences from the natural location, representing the first deletion of any Su(Hw) BR in the genome. Loss of 1A-2 reduced yar RNA accumulation, without affecting mRNA levels from the neighboring y and ac genes. These data indicate that within the 1A region, 1A-2 acts an activator of yar transcription. Taken together, these studies reveal that the properties of 1A-2 are context-dependent, as this element has both insulator and enhancer activities. These findings imply that the function of non-gypsy Su(Hw) BRs depends on the genomic environment, predicting that Su(Hw) BRs represent a diverse collection of genomic regulatory elements (Soshnev, 2008).

Prevailing models of gypsy insulator function predict that the gypsy insulator establishes independent transcriptional domains through cooperation with genomic insulators defined by non-gypsy Su(Hw) BRs. Recent findings indicate that the sequence and organization of non-gypsy BSs differ from the Su(Hw) BR in the gypsy retrovirus. These observations imply that properties of non-gypsy BRs may be distinct from those of the gypsy insulator. The properties of 1A-2 were defined in order to gain insights into mechanisms of Su(Hw) insulator action (Soshnev, 2008).

The quantitative FBE1-yp2-LacZ reporter system was used to define the sequence requirements for enhancer blocking by 1A-2(520). Prior application of this system demonstrated that at least four gypsy Su(Hw) sites were needed for robust blocking. This study showed that 1A-2(157) provided as strong an enhancer block as the gypsy insulator. A fragment containing only the Su(Hw) BRs [1A-2(79)] reconstituted a weaker enhancer block than 1A-2(157), but had a greater blocking capacity than the synthetic insulators made from reiterated copies of BS3 of the gypsy insulator. While the reason for the more robust blocking is not known, it is noted that these regions differ in sequence and distance of separation from Su(Hw) sites. Blocking effectiveness does not appear to be due to differences in DNA recognition, as the in vitro binding constants for Su(Hw) for the 1A-2 and gypsy BSs are similar. These experiments revealed that 1A-2 contains a second regulatory element located in 1A-2(78). When these sequences were positioned next to the inactive, synthetic Su(Hw) BR (3R:3), a functional insulator was reconstituted. These data are consistent with previous findings that Su(Hw) chromosome association is limited. Taken together, it is proposed that 1A-2 is a composite insulator that contains an enhancer blocking and a facilitator function that may improve Su(Hw) chromosome association. Further, it is predicted that in vivo effectiveness of enhancer blocking by the Su(Hw) protein is related to the accessibility of Su(Hw) BSs. If single or small clusters of Su(Hw) BSs are located in genomic regions of open chromatin, then these regions will demonstrate enhancer blocking, as defined in transgene assays. This proposal implies that genomic context greatly influences the properties of non-gypsy Su(Hw) BRs (Soshnev, 2008).

1A-2 is located between the independently regulated y and ac genes. Chromatin immunoprecipitation studies demonstrated that 1A-2 is associated with Su(Hw), Mod67.2 and E(y)2 in vivo, suggesting that this element binds a complex competent for establishing a genomic insulator. Based on these properties, it is postulated that 1A-2 was responsible for the regulatory independence of the y and ac genes in the 1A locus. As a first step in testing this proposal, transcription was investigated in the y-ac region to evaluate the current accuracy of the genomic annotation of this region. These studies identified a previously unannotated gene, yar, located ~1.2 kb downstream of the y gene and ~3.0 kb upstream of ac. Multiple, differentially spliced, polyA- RNAs are encoded by yar, with the largest translation product predicted to be 75 amino acids, indicating that this is a non-coding RNA gene. Emerging data suggest that non-coding RNAs are abundant in eukaryotes and have a wide repertoire of biological functions, ranging from structural components in protein complexes to regulatory molecules involved in transcription and translation. It is unknown whether yar has a function. Since flies carrying a large genomic deletion that removes sequences upstream of y and extends downstream of ac are viable and fertile, yar is a non-essential gene (Soshnev, 2008).

Having re-defined the transcriptional profile in the 1A locus, the function was tested of 1A-2 and a second, weaker Su(Hw) BR, 1A-2' on gene regulation, using gene targeting to delete these elements. These studies represent the first deletional analysis of any non-gypsy Su(Hw) BR in the Drosophila genome. Two targeted deletion lines, yδ1A-2 and yδ1A-2/δ1A-2'; were established. Levels of y, ac, sc and yar RNA accumulation during development were studied using quantitative PCR. Loss of 1A-2 and 1A2′ was found to have no effect on the timing and level of y, ac or sc RNAs relative to the wild type control, but strongly reduced yar RNA. These data suggest that the effects of loss of 1A-2 are limited to local changes of gene expression, implying that these sequences are not a chromatin insulator at the endogenous location. Instead, these data indicate that 1A-2 may be an activator of yar expression, consistent with other studies that have suggested a role for Su(Hw) in gene activation. These data, coupled with genetic studies on the effects of the loss of Su(Hw) on expression of genes adjacent to Su(Hw) BRs , demonstrate that Su(Hw) BRs have diverse functions in the genome (Soshnev, 2008).

The complexity of the transcriptional effects associated with Su(Hw) BRs is reminiscent of regions in mammalian genomes that bind the versatile regulatory protein CTCF. High throughput genomic analyses have identified hundreds of CTCF binding sites within the mouse and human genomes. Although many of these sequences possess enhancer blocking activity, CTCF has been implicated in transcriptional activation, repression, and chromosome pairing. These observations suggest that, similar to the non-gypsy Su(Hw) BRs, genomic context will have an important influence on the properties of CTCF BSs within a given region (Soshnev, 2008).

The mechanism(s) used to maintain transcriptional autonomy in the 1A locus are unclear. The discovery of yar provides an alternative explanation to the need for a chromatin insulator. Based on the developmental timing displayed by the 1A genes, it is postulated that activation of yar transcription may cause inactivation of ac through transcriptional interference. Similarly, activation of y may repress yar transcription. Although yδ1A-2 and yδ1A-2/δ1A-2' flies show reduced yar expression, transcription is not abolished, suggesting that the remaining yar activity may be sufficient to turn off ac. Alternatively, other mechanisms can be considered that might influence enhancer preference, including selectivity of enhancers for certain classes of promoters, the presence of promoter targeting sequences that direct enhancer action, or promoter tethering elements that capture enhancers. Further experiments to define the properties of DNA elements within the 1A locus will resolve how transcriptional independence is achieved (Soshnev, 2008).

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, it was 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, 2011).

Insulators regulate gene activity in a variety of organisms. The defining feature of insulators as a class of regulatory elements is their ability to block enhancer-promoter interactions only when positioned between them (Erokhin, 2011).

Two mutually non-exclusive but rather complementary mechanisms can account for the ability of insulators to block enhancers and support long-distance interactions. Experiments with transgenic lines suggest that the interaction between insulators can result in the formation of chromatin loops that either block or facilitate long-distance enhancer-promoter communication depending on the nature of the interacting insulators as well as on the distances between all the elements involved (enhancers, insulators and promoters) and their relative 'strength'. Alternatively, insulator action can be explained by the ability of insulators to form direct contacts with either an enhancer (the decoy model) or a promoter, thereby inactivating them. For example, the insulator protein CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and blocks enhancer-promoter communication by directly interacting with Igf2 promoters. Insulators of the Drosophila Abd-B gene can establish contact with a region upstream of the promoter that is required for proper enhancer-promoter communication. Several Drosophila insulators [scs, scs', IdefixU3 and Faswb] have been shown to contain promoters, which, according to the decoy model, may tether enhancers in nonproductive interactions. The stalled promoters of the bithorax complex display insulator activity in embryos. Many insulator proteins, such as CTCF, CP190, Mod(mdg4)-67.2 [Mod(mdg4) - FlyBase] and BEAF (BEAF-32), are frequently found bound to the promoters (Erokhin, 2011 and references therein).

Previously, two well-studied tissue-specific Drosophila genes, yellow and white, were shown to contain insulators immediately downstream of their coding regions. The yellow gene is responsible for dark pigmentation of the larval and adult cuticle and its derivatives, whereas the white locus determines eye pigmentation. The 1A2 insulator located on the 3' side of the yellow gene contains two binding sites for the Su(Hw) protein. Additional proteins, Mod(mdg4)-67.2, CP190 and E(y)2, interact with Su(Hw) and are required for the activity of Su(Hw)-dependent insulators. None of the known DNA-binding insulator proteins binds to the Wari insulator located on the 3' side of the white gene. However, stage-specific binding of CP190 and E(y)2 to the Wari insulator has been observed (Erokhin, 2010), which was indicative of its relationship to Su(Hw) insulators (Erokhin, 2011).

This study presents evidence that the 1A2 and Wari insulators interact with their target promoters and that this facilitates the formation of a gene loop between the promoter and terminator regions (Erokhin, 2011).

These insulators can support a gene loop that brings together a promoter and a terminator. The results obtained by ChIP assay suggest that insulator-promoter interactions are transcription dependent. To date, transcription-dependent gene looping has been demonstrated in yeast and HIV provirus. In yeast, loop formation was reported to be organized by TFIIB and the Ssu72 and Pta1 components of the 3'-end processing machinery. It is possible that this mechanism is conserved between eukaryotes and that the interaction between an insulator and a promoter is required to facilitate the formation of a gene loop and/or its stabilization (Erokhin, 2011).

It has been suggested that gene loop formation might be a common feature of gene activation that serves to promote efficient transcriptional elongation and transcription reinitiation by facilitating RNAP II recycling from the terminator to the promoter, reinforcing the coupling of transcription with mRNA export and enhancing terminator function. This study has found that the interaction of insulators with promoters is required for the basal activity of the white and yellow promoters in the eye. In addition to the possible role of a gene loop in the enhancement of RNAP II recycling and mRNA export, insulators might serve to bring to the promoter the remodeling and histone modification complexes that improve the binding and stabilization of the TFIID complex (Erokhin, 2011).

Recently, Chopra (2009) have found that the enhancer-blocking activity of several promoters and insulators depends on general transcription factors that inhibit RNAP II elongation. That study suggests that insulators interact with components of the RNAP II complex at stalled promoters and that the resulting chromatin loops can prevent the inappropriate activation of stalled genes by enhancers associated with the neighboring locus. This study found that the upstream promoter regions required for interactions with enhancers are not necessary for insulator-promoter interactions, which provides evidence that insulator proteins can interact with general transcription factors or proteins involved in the organization of promoter architecture. Certain types of insulators [the Su(Hw)-dependent 1A2, the Zw5-dependent scs, and Wari] can effectively interact with the yellow promoter, whereas others appear not to (the GAF-dependent Fab-7 and CTCF-dependent Mcp). GAF and CTCF are frequently found bound to promoter regions (Smith, 2009; Bartkuhn, 2009; Bushey, 2009; Nègre, 2010), which indicates that insulators that utilize these proteins are also involved in long-distance interactions with some promoters. For example, it is speculated that the Fab-7 insulator can interact with stalled promoters, such as the Abd-B promoter (Erokhin, 2011).

This study has shown that the GAL4 activator is unable to stimulate the promoter when GAL4 binding sites are placed downstream of the insulator. It appears likely that the loop is also formed between the insulator and promoter in this case, but that GAL4 is rendered outside the loop and blocked by the insulator. Thus, a chromatin loop formed by the promoter and insulator can prevent undesirable interactions with downstream regulatory elements. This provides evidence that the promoter-binding capacity of at least some insulators might contribute to their enhancer-blocking activity (Erokhin, 2011).

The genome-wide analysis of binding sites for insulator proteins has shown that they are present at the 3' and 5' UTRs of many Drosophila genes (Nègre, 2010). The 1A2 and Wari insulators at the 3' end of the yellow and white genes were identified only as a result of the extensive use of these genes in insulator assays. Thus, it appears that insulators are likely to be located at the 3' UTRs of many genes. Further experiments are required to resolve this issue and to elucidate the mechanisms and functional role of insulator-promoter interactions in transcriptional regulation (Erokhin, 2011).

A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber

Regulation of gene expression involves long-distance communication between regulatory elements and target promoters, but how this is achieved remains unknown. Insulator elements have been proposed to modulate the communication between regulatory elements and promoters due to their ability to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, this study shows that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Even at a distance of 8 kb from the PRE, the distal insulator imposes a chromatin constraint that is removed upon mutation of the su(Hw) gene. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3 histone mark reflect this insulator-dependent chromatin conformation, suggesting that Polycomb action at a distance can be organized by local chromatin topology (Comet, 2011).

Long-distance communication between cis regulatory elements and their promoters may involve either chromatin looping or the ability of factors recruited to the regulatory element to scan the surrounding chromatin by mechanisms of linear spreading or tracking along the fiber until they reach the target promoter. 3C technology can distinguish between these mechanisms by detailed analysis of the interaction profile of a chromatin fragment with the surrounding chromatin fiber. This interaction profile reflects contact probabilities, thus giving a perception of the conformational constraints imposed on the chromatin fiber in a given locus (Comet, 2011).

Using this technology, preferential contacts between regulatory elements and their target promoters have been shown to correlate with their regulatory function, suggesting that chromatin looping can be involved in long-distance regulatory phenomena. The data demonstrate that the gypsy insulator functions via chromatin looping. However, it should be noted that these results cannot distinguish between mechanisms whereby homotypic interactions between the gypsy elements directly establish chromatin loops or whether transient contacts between elements tracking along the chromatin fiber are stabilized by such interactions (Comet, 2011).

A second feature of this element is that one copy of the insulator is able to prohibit contacts between adjacent chromatin fragments. Due to the remarkable resolution of H3C, inhibition of contacts between fragments spaced by <1 kb could be detected or by greater distances (up to 8kb was detected). This topological constraint delimits the range of action of a PRE on its surrounding regions independently of the relative distances between the insulator and the PRE. How does the insulator function to prohibit interactions? A model, called enhancer decoy, suggests that an insulator would mimic a promoter architecture without actually being able to drive transcription. This outcome would predict that a regulatory element such as an enhancer or a PRE may interact with it but this interaction would not be functional. Whereas strong homotypic interactions were seen between insulators, the data did not show high contact levels between the PRE and the insulator. Instead, contact levels reduced just adjacent to it and were low at the insulator and on a 5-kb region downstream of it. This result suggests that insulator complexes are repulsive with respect to the PRE. Because the configuration in which the PRE can bypass two insulators induced clearly detectable contacts between PRE and promoter, it is believed that the insulator is not mimicking a promoter with respect to long-range chromatin interactions. Instead, the insulator seems to topologically separate upstream chromatin regions from those downstream. This separation may depend either on the organization of a specific 3D chromatin fiber architecture or on the formation of large insulator bodies that would be repulsive to chromatin regions not bound by insulator proteins. It should be noted, however, that an endogenous Su(Hw) binding site was previously mapped ~4 kb upstream of the transgenic PRE site in the (P)(S)YSW-22E line, in which there are not interactions with either of the transgenic insulators. Moreover, this binding site does not affect local topological constraints or the profiles of PC binding or H3K27me3 deposition on the local chromatin fiber, suggesting that not all endogenous Su(Hw) binding sites are insulators or belong to the same class of insulator (Comet, 2011).

How PcG proteins are recruited to chromatin by PREs to repress distal gene promoters is still unknown. ChIP analysis showed that the PC protein is detectable in large domains flanking the position of a PRE. This could be explained by spreading or by looping models. This study shows that a PRE-containing chromatin fragment can establish many contacts with its surrounding chromatin region, with a probability decreasing with linear distance. Moreover, the ChIP profiles of PC and H3K27me3 are very similar to the PRE-chromatin interaction profile. In particular, when apparent PC 'spreading' can be detected several kilobases away from the insulator, this process is reflected in an almost identical contact profile between the PRE and the 'region of spreading.' Polycomb group protein complexes have been shown to compact nucleosomal arrays in vitro, which may be expected to enhance physical contacts between the PRE and surrounding chromatin. However, removal of PC due to PRE excision or its physical block due to insulator function does not affect the transgene chromatin conformations. These data suggest that in vivo PC targeting to downstream chromatin is not the simple result of chromatin compaction. The most parsimonious scenario thus posits that PRE-bound PcG proteins of the PRC2 histone methyltransferase complex loop out from the PRE to contact and deposit the H3K27me3 mark on PRE-flanking chromatin. The PC distribution surrounding the PRE may thus be due to multiple transient contacts between the PRE and its flanking regions, followed by H3K27me3 binding of PC via its chromodomain. Finally, in the presence of two insulators, PC is found at the PRE and at the distally located target promoter but not in the region located between the two insulators, in perfect agreement with the chromatin contact data. As a corollary it is important to note that PC is not bound to the promoter in the absence of a neighboring PRE. This result shows that the mini-white promoter itself is not able to recruit PC. Therefore, the ChIP signal observed at this promoter strictly reflects 3D chromatin fiber interactions rather than DNA sequence-autonomous protein binding to their target chromatin sites. Because Su(Hw), as well as many other insulator proteins, binds more than a thousand genomic sites, this kind of 3D looped chromatin organization may represent an important component modulating PRE-mediated silencing of gene expression (Comet, 2011).

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. This study demonstrates 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 (Golovnin, 2012).

Posttranslational modification by SUMO has been shown to regulate subcellular localization of many targets, including RanGAP, PML, SATB2 and others. This study presents data that SUMO is necessary for co-localizing the Su(Hw), Mod(mdg4)-67.2, and CP190 proteins in nuclear speckles, named insulator bodies. Previously an opposite model has been proposed according to which sumoylation of Mod(mdg4)-67.2 and CP190 leads to disruption of insulator bodies. This model was mainly based on the observation that, in diploid cells from the larval brain, mutations in the gene encoding Ubc9 restored aggregation of the CP190 protein in the mod(mdg4)u1 background. This study found that inactivation of Mod(mdg4)-67.2 did not affect the ability of CP190 to form insulator bodies in S2 cells (Golovnin, 2012).

mod(mdg4)u1 mutation also did not affect CP190 incorporation into the insulator bodies in diploid cells of wing and eye imaginal discs. Thus, the significance of Mod(mdg4)- 67.2 for CP190 recruitment to the insulator bodies is confined to diploid cells of the larval brain. To test the role of sumoylation in the formation of insulator bodies, the lwr5 mutation, generated by a single amino acid substitution in the Ubc9 region (R104H) located on the loop between strand 7 and helix B, has been used. This region of Ubc9 is required for the interaction of its active site with the substrate. Although untested, it appears that R104H makes the surface of the mutant enzyme (Ubc95) more hydrophobic, thereby strengthening binding interactions for certain enzyme-substrate pairs. Thus, lwr5 is not a null-mutation in the gene, and Ubc95 can either increase or decrease sumoylation, depending on the protein substrate. Therefore, additional studies are required to demonstrate role of Ubc95 in the formation of insulator bodies in the imaginal disks of larvae (Golovnin, 2012).

The data provide evidence for a critical role of CP190 and a passive role of Su(Hw), a DNA-binding protein, in the formation of insulator bodies. In addition to Su(Hw), CP190 forms complexes with dCTCF that is also co-localized in the insulator bodies. Thus, it is likely that Mod(mdg4)-67.2 and CP190 proteins recruit DNA-binding dCTCF and Su(Hw) proteins to the insulator bodies (Golovnin, 2012).

As shown previously, SUMO is necessary for the formation of PML nuclear bodies (PML-NBs). These bodies are formed due primarily to the self-assembly ability of the PML N-terminal domain. Moreover, SUMO-1 modification of PML was shown to target the protein from the nucleoplasm to the NBs. The occurrence of both sumoylation sites and SUMO-interacting motifs (SIMs) in the PML protein provides a basis for the network of interactions that constitute the nucleation event for subsequent recruitment of sumoylated proteins and SIM-containing proteins (Golovnin, 2012).

Cells that lack PML are unable to form NBs, with other NB components remaining diffusely distributed in the nucleus. While analysis of the CP190 sequence suggests the presence of two SIMs, no direct interaction was observed between CP190 and SUMO in vitro. At the same time, CP190 and Mod(mdg4)-67.2 contain several protein-protein interaction domains, including BTB/POZ that might be involved in direct interaction with many DNA-binding transcription factors, such as Su(Hw) and dCTCF, to facilitate their assembly into the insulator bodies. It is noteworthy that heat shock has proved to induce redistribution of CP190 to the nuclear periphery, in complex with SUMO. This is evidence that the formation of insulator bodies requires interactions with additional proteins, which are disrupted as a result of heat shock treatment (Golovnin, 2012).

Sumoylation is essential for the functional activity of proteins in transcriptional repression, activation, and recruitment of modifying complexes. This study has demonstrated that inactivation of sumoylation sites in the Mod(mdg4)-67.2 protein does not affect its functional activity in the insulator complex. This finding is in accordance with the previous observation that only 10% of Su(Hw) binding sites coincide with SUMO on polytene chromosomes (Golovnin, 2012).

This study confirms the role of Mod(mdg4)-67.2 in recruiting the Su(Hw) protein to the insulator bodies and insulators. When the mutant Mod(mdg4)-67.2 protein was expressed at a low level, Su(Hw) binding was reduced, whereas low expression of the wild-type Mod(mdg4)-67.2 protein was sufficient for completely restoring Su(Hw) binding to insulators. Therefore, the assembly of the Su(Hw) and Mod(mdg4)-67.2 proteins in insulator bodies is essential for subsequent recruitment of insulator complexes to DNA. A higher level of the mutant Mod(mdg4)-67.2 protein increases the probability of formation of the Su(Hw)/Mod(mdg4)-67.2 complex out of insulator bodies, thereby providing for more effective binding of the Su(Hw) and mutant Mod(mdg4)-67.2 proteins to the insulators (Golovnin, 2012).

Taken together, these results support the model of insulator bodies as a depot of proteins involved in transcription regulation and insulation. According to these results, the insulator proteins can interact and form complexes without SUMO. However, partial sumoylation of the Mod(mdg4)-67.2 and CP190 proteins lead to further aggregation of the protein complexes in insulator bodies. The sumoylated Mod(mdg4)-67.2 and CP190 proteins interact with Su(Hw) and recruit it to the insulator bodies. The insulator bodies possibly protect the insulator complex from degradation and facilitate the formation of complexes between Su(Hw)/Mod(mdg4)-67.2/CP190 and other transcription factors. 'Mature' insulator complexes may then transiently interact with the chromatin fibril and detach from the insulator bodies by means of desumoylation. As was suggested for PML bodies, proteins deposited in the insulator bodies may be used during cell stress. For example, it was found that heat shock treatment induced relocation of CP190 from the insulator bodies to the nuclear periphery but did not affect the insulator complexes bound to DNA. Such an unusual relocation of the CP190 protein resulted in a diffuse distribution of the Su(Hw) and Mod(mdg4)-67.2 proteins. Thus, it appears that insulator proteins may have an as yet unknown yet role in cell response to heat shock stress. During DNA replication, a large amount of insulator proteins is required for newly synthesized chromosomes. It is possible that desumoylation of insulator bodies during DNA replication results in the release of protein complexes that form functional insulators on the newly synthesized DNA.Further studies are required to verify this model (Golovnin, 2012).

Nature and function of insulator protein binding sites in the Drosophila genome

Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. This study tested this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. Distinct combinatorial binding was found of insulator proteins to different classes of sites, and a novel type of insulator element was uncovered that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. Insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes (Schwartz, 2012).

The binding sites of insulator proteins are often taken to represent elements that partition the genome into independent regulatory domains and demarcate chromosomes into regions of 'active' and 'repressed' chromatin. The results presented in this study give little support to this view as a general principle of genome organization although it may be true in certain regions. Instead it is argued that: 1) insulator proteins bind to genomic sites in specific combinatorial patterns, 2) the properties of sites bound by key insulator proteins SU(HW) and CTCF are markedly different depending on whether the two co-bind with CP190, 3) many of the known insulator proteins sites do not function as robust enhancer blockers, and 4) at least in cultured cells the depletion of insulator proteins has a limited impact on genome-wide gene expression (Schwartz, 2012).

Classifications of combinatorial binding of insulator proteins have been described previously. These classifications relied on the overlapping of bound regions defined according to arbitrary statistical thresholds and the position of these regions relative to TSSs. Because they did not take into account the relative strengths of binding, such classifications grouped together binding sites with very different biochemical and functional properties (Schwartz, 2012).

In contrast, this study defines the persistent co-binding patterns based on the strength of binding of the associated proteins, treating regions strongly bound by a combination of proteins differently from regions at which the same proteins are detected according to a statistical threshold but where the extent of their binding is disproportional. It is argued that this approach retains the information on biochemical interrelations between the co-bound proteins and separates the sites with different functional properties. The strongest support for this argument comes from RNAi knock-down experiments which demonstrate that the effect of the loss of one insulator protein on the binding of another insulator protein is constrained to a specific class of co-bound regions. For example, the knock-down of SU(HW) results in the loss of CP190 from class 3 (gypsy-like) sites but not from class 9 (CTCF+CP190) or class 5 (BEAF-32+CP190) sites (Schwartz, 2012).

The approach to select the sites representative of each co-binding class is conservative and inevitably excluded a fraction of binding sites from downstream analyses. For example, strong SU(HW) binding sites assigned to class 14 by initial overlap comparison were not analyzed further due the uncertainty of their co-binding by CP190. It is therefore cautioned that selection of representative binding sites is not a complete genomic catalogue and readers are advised to use the ChIP-chip binding profiles, deposited to GEO and modMINE, to gauge whether their locus of interest has a strong insulator protein binding site (Schwartz, 2012).

The prevailing model in the field suggests that CP190 is recruited to different insulator elements by DNA binding proteins where it serves as a universal adapter that mediates interactions between different insulator elements. The current results present a more complex picture. First, RNAi knock-down experiments demonstrate that the binding of SU(HW) protein to class 3 (gypsy-like) sites is dependent on CP190, indicating that CP190 is not passively tethered to common sites by SU(HW) and instead plays an active role in recruitment and/or stabilization of the bound complex. Second, the sequence analysis of class 9 (CTCF+CP190) sites suggests that the binding of both proteins to these sites is likely due to the coincidence of cognate recognition sequences. Third, RNAi knock-down experiments indicate that BEAF-32 is dispensable for CP190 binding at shared sites. Clearly CP190 plays an active role in the selection of sites shared with SU(HW), CTCF or BEAF-32. It is still possible that once it co-binds, or binds sufficiently close to another insulator protein, it may mediate the trans-interactions of the bound sites. However, such interactions would have to be rather transient, at least in cultured cells, as they are not easily detected in the ChIP-chip data (Schwartz, 2012).

A variably occupied CTCF binding site in the Ultrabithorax gene in the Drosophila Bithorax Complex

Although the majority of genomic binding sites for the insulator protein CTCF are constitutively occupied, a subset show variably occupancy. Such variable sites provide an opportunity to assess context-specific CTCF functions in gene regulation. This study has identified a variably occupied CTCF site in the Drosophila Ultrabithorax (Ubx) gene. This site is occupied in tissues where Ubx is active (third thoracic leg imaginal disc) but is not bound in tissues where the Ubx gene is repressed (first thoracic leg imaginal disc). Using chromatin conformation capture this site was shown to preferentially interact with the Ubx promoter region in the active state. The site lies close to Ubx enhancer elements and is also close to the locations of several gypsy transposon insertions that disrupt Ubx expression, leading to the bx mutant phenotype. Gypsy insertions carry the Su(Hw)-dependent gypsy insulator and were found to affect both CTCF binding at the variable site and the chromatin topology. This suggests that insertion of the gypsy insulator in this region interferes with CTCF function and supports a model for the normal function of the variable CTCF site as a chromatin loop facilitator, promoting interaction between Ubx enhancers and the Ubx transcription start site (Magbanua, 2014).

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. This study has 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 (Golovnin, 2015).

Insulators belong to the class of regulatory elements that organize the architecture of chromatin compartments. Insulators, or chromatin boundaries, are characterized by two properties: they interfere with enhancer-promoter interactions when located between them and buffer transgenes from chromosomal positions effects. To date, chromatin insulators have been characterized in a variety of species, indicative of their involvement in the global regulation of gene expression (Golovnin, 2015).

The well-studied Drosophila insulator proteins, dCTCF (homolog of vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. The Su(Hw) protein, encoded by the suppressor of Hairy wing [su(Hw)] gene, was one of the first insulator proteins identified in Drosophila. The best-studied Drosophila insulator found within the 5'-untranslated region of the gypsy retrovirus consists of 12 directly repeated copies of Su(Hw) binding sites. Genetic and molecular approaches have led to the identification and characterization of three proteins recruited by Su(Hw) to chromatin-Mod(mdg4)-67.2, CP190, and E(y)2/Sus1-that are required for the activity of the Su(Hw)-dependent insulators. The mod(mdg4) gene, also known as E(var)3-93D, encodes a large set of BTB/POZ protein isoforms. One of these isoforms, Mod(mdg4)-67.2, by its specific C-terminal domain interacts with the enhancer-blocking domain of the Su(Hw) protein. The BTB domain is located at the N-terminus of Mod(mdg4)-67.2 and mediates homo-multimerization (Golovnin, 2015).

Su(Hw), dCTCF, and most of other identified insulator proteins interact with Centrosomal Protein 190 kD (CP190). This protein (1096 amino acids) contains an N-terminal BTB/POZ domain, an aspartic-acid-rich D-region, four C2H2 zinc finger motifs, and a C-terminal E-rich domain. The BTB domain of CP190 forms stable homodimers that may be involved in protein-protein interactions. In addition to these motifs, CP190 also contains a centrosomal targeting domain (M) responsible for its localization to centrosomes during mitosis. It has been shown that CP190 is recruited to chromatin via its interaction with the DNA insulator proteins in interphase nucleus (Golovnin, 2015).

The Su(Hw), dCTCF, Mod(mdg4)-67.2, and CP190 proteins colocalize in discrete foci, named insulator bodies, in the Drosophila interphase cell nucleus. Contradictory reports have been published in which the insulator bodies are described either as protein-based bodies in the interchromatin compartment or as chromatin domains. As shown recently, insulator proteins rapidly coalesce from diffusely distributed speckles into large punctate insulator bodies in response to osmotic stress (Golovnin, 2015).

Cell exposure to hypertonic treatment, which enhances molecular crowding, makes it possible to discriminate between nucleoplasmic bodies formed mainly of RNA and proteins (such as PML bodies) and chromatin compartments such as Polycomb bodies formed due to the interaction of distantly located chromatin regions bound by Polycomb proteins. Nucleoplasmic bodies disappear under less crowded conditions and reassemble under normally crowded conditions, which can be interpreted as a consequence of increased intermolecular interactions between components of nucleoplasmic bodies. Similar to PML bodies, insulator bodies are preserved under hypertonic treatment, in contrast to chromatin-based structures that disappear as proteins dissociate from chromatin. The CP190 protein is suggested to be critical for the activity of insulators and to regulate the entry of other insulator proteins into the speckles. At the same time, CP190 associates with centrosomes throughout the nuclear division cycle in syncytial Drosophila embryos. Nuclear localization of CP190 is also sensitive to various kinds of stress, suggesting that this process is highly regulated. However, the mechanisms and proteins responsible for localization of CP190 in different nucleus compartments are unknown. This study has shown that the nuclear distribution of CP190 depends on the level of EAST, which is located mainly in the interchromatin compartment of the nucleus. EAST is a nuclear protein of 2362 amino acids which, except for 9 potential nuclear localization sequences and 12 potential PEST sites, contains no previously characterized motifs or functional domains. Together with Skeletor, Chromator, and Megator proteins, EAST forms the spindle matrix during mitosis. In the interphase nuclei, EAST localizes to the extrachromosomal compartment of the nucleus and is essential for the spatial organization of chromosomes (Golovnin, 2015).

Despite that the bulk of interphase EAST resides in the interchromosomal domain, the current model assumes that EAST can transiently interact with chromosomes. EAST physically interacts with Megator, a 260-kDa protein with a large N-terminal coiled-coil domain capable of self-assembly. It has been speculated that Megator can form polymers that, together with EAST, may serve as a structural basis for the nuclear extrachromosomal compartment. The results show that EAST interacts with CP190 and Mod(mdg4)-67.2 proteins and modulates their aggregation into the nuclear speckles. In case of EAST overexpression, CP190 binding to chromatin is reduced; consequently, the binding of Mod(mdg4)-67.2 and Su(Hw) is reduced as well, since CP190 is essential for it. On the basis of these results, it is hypothesized that EAST regulates localization of CP190 and insulator protein complexes in the interchromatin compartment, with these complexes subsequently determining organization of chromatin insulators (Golovnin, 2015).

The results suggest that insulator bodies are sensitive to the concentration of EAST in interphase cells. The properties of insulator bodies described previously and in this study suggest that they are formed by multiple interactions between proteins and resemble nuclear bodies composed of aggregated proteins and RNAs. As shown previously, the CP190 and Mod(mdg4) proteins interact with Su(Hw) and dCTCF and help the latter to enter the insulator bodies (Golovnin, 2015).

Taking into account the high level of dCTCF and Mod(mdg4) co-binding to chromosomes, it appears that dCTCF interacts with an as yet unidentified Mod(mdg4) isoform. Mod(mdg4)-67.2 and CP190 conjugate to the small ubiquitin-like modifier protein (SUMO). Specific interactions mediated by SUMO, the ability of Mod(mdg4) BTB to form oligomers, and the interaction between the BTB domain of Mod(mdg4)-67.2 and CP190 contribute to specific aggregation of the Su(Hw)/Mod(mdg4)-67.2/CP190 and dCTCF/CP190 complexes into the insulator bodies (Golovnin, 2015).

According to current views, the Megator protein can form polymers that, together with EAST, may serve as a structural basis for the nuclear extrachromosomal compartment. The overexpression of EAST leads to an extension of the EAST-Megator compartment, with consequent reduction in the effective volume available for the insulator proteins in the cell. As a result, the concentration of the insulator proteins increases, contributing to stabilization of the compact protein conformations visualized as insulator bodies. By interacting with Mod(mdg4)-67.2 and CP190, EAST may also be directly involved in nucleation of insulator bodies. It is possible that the truncated version of EAST (from 933 to 2362 aa) can more easily interact with the insulator proteins, which leads to noticeable enlargement of insulator bodies in S2 cell expressing EAST933-2362. The overexpression of EAST leads to segregation of the CP190 protein in independent speckles. The results suggest that EAST interacts with the CP190 region that includes BTB, D, and M domains. These domains are also required for CP190 interactions with other insulator proteins (Golovnin et al., in preparation). Thus, an increase in the EAST concentration may lead to displacement of the insulator proteins from the complex with CP190 (Golovnin, 2015).

The results do not exclude the possibility that EAST overexpression directly leads to dissociation of CP190 from chromatin. During mitosis, CP190 colocalizes with EAST in the spindle matrix, and the increase in the amount of EAST may well be responsible for dissociation of CP190 prior to chromosome condensation (Golovnin, 2015).

According to the current model, the insulator bodies help to form protein complexes that subsequently bind to regulatory elements such as insulators and promoters. In view of this hypothesis, it is likely that disturbances in the insulator bodies caused by EAST overexpression are responsible for the decrease in CP190 binding to the regulatory regions such as dCTCF- and Su(Hw)-dependent insulators and promoters. As shown recently, CP190 is required for recruiting Su(Hw) and Mod(mdg4)-67.2, but not dCTCF, to chromatin. Accordingly, it was observed that EAST overexpression affects the chromosomal binding of Su(Hw), but not of dCTCF. CP190 specifically interacts with the Mod(mdg4)-67.2 isoform, and Mod(mdg4)-67.2 at all Su(Hw) binding sites is colocalized with CP190. Thus, CP190 may be essential for recruiting the specific Mod(mdg4)-67.2 isoform to the Su(Hw) binding sites, with subsequent decrease in the amount of CP190 at the Su(Hw) binding sites, which leads to the substitution of Mod(mdg4)-67.2 by other Mod(mdg4) isoforms, as has been observed in this study (Golovnin, 2015).

Strong inactivation of EAST in S2 cells reduces the entry of the Mod(mdg4)-67.2/ Su(Hw) complex, but not of CP190, into the nucleus. It appears that EAST is involved in the regulation of nuclear localization of Mod(mdg4)-67.2, whose BTB domain can form multimeric complexes. Further study is required to elucidate this issue (Golovnin, 2015).

back to suppressor of Hairy wing Regulation part 1/2

suppressor of Hairy wing: Biological Overview | Protein Interactions | Developmental Biology | Effects of Mutation | References

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