suppressor of Hairy wing

DEVELOPMENTAL BIOLOGY

Embryonic

SU(HW) protein was detected in the nuclei of cells from all stages of development. Staining was detected in males as well as females, contrary to what might be expected from the ovary-specific phenotype of su(Hw) mutations (Harrison, 1993).

Nuclear distribution of su(Hw) protein

Chromatin insulators might regulate gene expression by controlling the subnuclear organization of DNA. A DNA sequence normally located inside of the nucleus moves to the periphery when the gypsy insulator is placed within the sequence. The presence of the gypsy insulator also causes two sequences, normally found in different regions of the nucleus, to come together at a single location. Alterations in this subnuclear organization imposed by the gypsy insulator correlate with changes in gene expression that take place during the heat-shock response. These global changes in transcription are accompanied by dramatic alterations in the distribution of insulator proteins and DNA. The results suggest that the nuclear organization imposed by the gypsy insulator on the chromatin fiber is important for gene expression (Gerasimova, 2000).

To test the functional significance of the aggregation of large clusters of individual gypsy insulator sites at specific nuclear locations, their precise arrangement was determined using antibodies against the su(Hw) and mod(Mdg4) proteins. Both proteins colocalize in approximately 21 sites to form an equivalent number of insulator bodies. All gypsy insulator bodies, with the exception of five located in the center of the nucleus, are located in the nuclear periphery. These interior bodies tend to be smaller and less intense than those located in the periphery. Because the total number of individual sites for the mod(Mdg4) protein is approximately 500, these results suggest that, on average, approximately 25 individual gypsy insulator sites come together to form one insulator body; and these structures are present mostly in the nuclear periphery (Gerasimova, 2000).

The distribution of su(Hw) protein was examined in live cells. Transgenic flies were generated, expressing a green fluorescent protein (GFP)-su(Hw) protein under the control of the normal su(Hw) promoter; in addition, these flies carry a null mutation in the su(Hw) gene. Analysis of nuclei from imaginal disc cells shows the same pattern of su(Hw) distribution previously observed in fixed cells. These results support the idea that the large insulator bodies are present in live cells and might be the result of the aggregation of multiple individual insulator sites at specific single nuclear locations. This aggregation might be mediated through interactions between the su(Hw) and mod(mdg4) proteins or other putative, and as yet unknown, components of the gypsy insulator. The final outcome of these interactions might be to physically attach the chromatin fiber to a nuclear peripheral substrate -- possibly the nuclear lamina -- and in the process, organize the chromatin fiber into distinct domains (Gerasimova, 2000).

To obtain further insight into the significance of the organization imposed by the presence of gypsy insulator sites on the structure of a specific chromosome during interphase, the number of gypsy insulator bodies formed in a single X chromosome was determined. To this end, diploid interphase cells from imaginal discs of a wild-type Drosophila male were stained with antibodies against the Male-specific lethal 2 (msl-2) and mod(Mdg4) proteins; Msl-2 is present specifically in the X chromosome of males. It is apparent that the territory occupied by the X chromosome contains five large aggregates of individual gypsy insulator sites. This number varies between four and six in different nuclei, and is also the average of ten different nuclei examined in the same fashion. Each large aggregate represents several individual gypsy insulator sites, suggesting that the X chromosome of Drosophila is organized in approximately five different rosettes. Because the X chromosome contains approximately 21,000 kb of DNA, each of these rosettes would be expected to contain approximately 4,000 kb of DNA, and the sequences from 4B and 7D should be present within the same rosette. The X chromosome contains approximately 80 individual gypsy insulator sites, and, therefore, each rosette should be composed, on average, of 16 individual sites. Two individual sites coalescing into a gypsy insulator body form a loop or domain of higher-order chromatin organization. The length of the DNA contained within each loop should be approximately 250 kb (Gerasimova, 2000).

If the role of the gypsy insulator is to organize the chromatin fiber within the nucleus, this organization might be static and serve only a structural role or, alternatively, it might have a functional significance in a manner that is relevant to gene expression. In the latter case, there should be a correlation between the organization imposed by the gypsy insulator and the ability of genes to be transcribed. To address this issue, the possible effects of heat shock on the subnuclear arrangement of gypsy insulator bodies was determined. During the heat-shock response in Drosophila, there are dramatic changes in the transcriptional state of the cell. A small number of genes, the heat-shock genes, are turned on, while transcription of the rest of the genome is turned off. One would then expect that if the organization of gypsy insulator bodies were important for transcription, these global changes in gene expression would be accompanied by alterations in their nuclear arrangement. Results from the analysis of the distribution of GFP-su(Hw) protein in live nuclei suggest that this is the case. Cells from larval imaginal discs show the typical punctated pattern of gypsy insulator bodies. After a 20-min heat shock at 37°C, the su(Hw) protein appears to be distributed throughout the nucleus in a uniform pattern; the punctated distribution has disappeared, with only some cells showing one faint dot of su(Hw) protein. To test whether this alteration is also true for the mod(Mdg4) protein, its distribution was determined in fixed cells using antibodies. As was the case for su(Hw), the mod(Mdg4) protein is distributed in a diffuse pattern after a brief heat shock, with some cells still displaying one gypsy insulator body (Gerasimova, 2000).

The alteration in the distribution of gypsy insulator bodies observed after temperature elevation could be caused by a decrease in the cellular levels of the su(Hw) and mod(Mdg4) proteins; Western analyses show that this is not the case and that the levels of the two proteins are the same before and after heat shock. A second possibility is that the su(Hw) protein does not bind to its target sequence after heat shock and, as a consequence, both protein components of the gypsy insulator fall off the chromosomes in cells subjected to temperature elevation. To test this possibility, the distribution of su(Hw) and mod(Mdg4) proteins were examined on polytene chromosomes from salivary glands. The number and intensity of bands is the same for both proteins before and after heat shock, suggesting that the alterations observed in diploid nuclei are not caused by disruption of protein-DNA or su(Hw)-mod(Mdg4) interactions at individual insulator sites. The changes observed in the localization of su(Hw) and mod(Mdg4) proteins are accompanied by changes in the localization of the DNA to which these proteins are bound. Because the proteins stay bound to the DNA after heat shock, the only explanation for this observation is that the rosette structure formed by the gypsy insulator is disrupted as a consequence or pre-requisite for the heat-shock response (Gerasimova, 2000).

Models to explain the molecular mechanisms of insulator action are based on their idiosyncratic effects on gene expression. Some of these effects can be explained by assuming that insulators interfere with the propagation of a directional signal from the enhancer to the promoter. This signal could be (1) the looping of intervening DNA; (2) the tracking of a transcription factor along the DNA toward the promoter; (3) the topological state of the DNA or (4) some form of chromatin alteration that affects the primary structure of the chromatin fiber, such as histone modifications or changes in nucleosome condensation or spacing. Other properties of insulators point to an involvement of these sequences in the establishment or maintenance of higher-order chromatin organization. For example, the ability of insulators to buffer the expression of transgenes against chromosomal position effects and their location at the boundaries between active and inactive chromatin are both indicative of a role in the establishment of higher-order chromatin domains. In addition, the observed interactions between proteins involved in chromatin dynamics, such as enhancers/suppressors of position-effect variegation and trxG/PcG proteins, and the protein components of some insulators, are also suggestive of a role for these sequences in higher-order chromatin organization. The results presented here support the involvement of insulators in nuclear organization and suggest that this organization is the primary cause of their effects on gene expression by subsequently affecting the transmission of signals from the enhancer to the promoter (Gerasimova, 2000).

The su(Hw) and mod(Mdg4) proteins are components of the gypsy insulator and localize at approximately 500 sites on polytene chromosomes from salivary glands of third instar larvae. The observed distribution of insulator proteins in polytene chromosomes suggests that they should bind at more or less regular intervals in the chromosomes of diploid cells in interphase. Given the large number of sites and their regular distribution, one would expect to observe a diffuse homogeneous scattering of insulator sites in the nuclei of interphase diploid cells. Surprisingly, this is not the case and, instead, gypsy insulator proteins accumulate at a small number of nuclear locations. This has led to the suggestion that each of the locations where su(Hw) and mod(Mdg4) proteins accumulate in the nucleus is made up of several individual sites that come together, perhaps through interactions among protein components of the insulator. Interestingly, the locations in the nucleus where individual insulator sites appear to aggregate are not random. Analysis of the distribution of gypsy insulator bodies in 3D reconstructions of nuclei from diploid cells in interphase indicates that, although not all the aggregate sites are present in the nuclear periphery, approximately 75% of them are present immediately adjacent to the location of the nuclear lamina. This finding suggests that the formation of gypsy insulator bodies perhaps requires a substrate for attachment, and the physical attachment might play a role in the mechanism by which insulators affect enhancer-promoter interactions. The nuclear lamina itself might serve as a substrate for attachment, perhaps through interactions between lamin and protein components of the insulator. The nature of the substrate involved in the attachment of the aggregate sites found in the interior of the nucleus is unknown, but it is interesting that a lamin network has also been detected in the inside of the nucleus. This attachment might impose a topological or physical constraint on the DNA that interferes with the transmission of a signal from an enhancer located in one domain to a promoter located in an adjacent one. The preferential aggregation of insulator sites at the nuclear periphery and the possibility that this targeting might take place through interactions with the nuclear lamina led to the idea that the gypsy insulator might be equivalent to matrix attachment regions/scaffold attachment regions (MARs/SARs). This hypothesis is directly supported by the finding of MAR activity within the DNA sequences containing the gypsy insulator (Gerasimova, 2000).

The nonuniform distribution of gypsy insulator bodies inside of the nucleus allowed for a test of the hypothesis that these sites indeed correspond to several individual insulators coming together in a single nuclear location. The model was tested with insulator sequences carried by the gypsy retrovirus, but it is likely that the conclusions are also applicable to putative endogenous insulators. As predicted by this hypothesis, a DNA sequence usually located randomly with respect to the periphery versus interior compartments in the nucleus becomes preferentially located in the periphery when a copy of the gypsy insulator is present in this sequence. The correspondence between the presence of the insulator and the peripheral localization is not 100%, as one would expect from the location of 25% of gypsy insulator bodies in the interior of the nucleus. The idea that insulator bodies result from the coalescence of individual gypsy insulators is also supported by the overlap in the nucleus between a DNA sequence containing the gypsy insulator and sites in the nucleus where insulator bodies are present. The strongest evidence in support of a role for the gypsy insulator in the organization of the chromatin fiber within the nucleus through the aggregation of individual insulator sites comes from the analysis of the localization of two specific DNA sequences. The two sequences utilized in the experiments described in this work are located at 4D and 7B in the X chromosome. Because the X chromosome contains approximately 21,600 kb of DNA, each chromosomal division would contain approximately 1,000 kb of DNA. Therefore, these two sequences are separated by approximately 3,000 kb of DNA, and they appear as two distinct hybridization signals in nuclei of diploid cells. Nevertheless, the presence of insulator sites in these two sequences in cells containing copies of gypsy at 4D and 7B causes them to associate into a single nuclear location (Gerasimova, 2000).

The organization imposed by the gypsy insulators on the chromatin fiber might explain the effect of these sequences on the silencing effects of Polycomb response elements (PREs). The gypsy element is able to block silencing caused by interactions among PREs located on the same chromosome, but is not able to block silencing due to PREs located in different chromosomes. These results could be explained if the rosette organization imposed by gypsy insulators on a specific chromosome interfers with other types of intrachromosomal interactions mediated by PREs, whereas the gypsy-induced organization still allows interchromosomal interactions between these elements. An important question arising from the role of insulators in nuclear organization is whether this organization is static and has a mostly structural role, or is dynamic and has a direct functional significance. If the latter were the case, one would expect a correlation between the pattern of nuclear organization of insulator bodies and the transcriptional state of the cell. Changes in transcription that take place during the heat-shock response in Drosophila represent an ideal situation to address this question because the alterations in gene expression induced by temperature elevation are global, affecting the whole genome. If the gypsy insulator plays a role in establishing higher-order chromatin domains, and this organization of the chromatin fiber is important for transcription, it should then be possible to detect alterations in the pattern of insulator bodies within the nucleus concomitant with changes in gene expression induced by heat shock. Indeed a dramatic modification in the distribution of su(Hw) and mod(Mdg4) proteins can be observed in the nuclei of heat-shocked cells. The normal punctated pattern disappears and these two proteins are present diffusely throughout the nucleus. The observed changes are not due to effects of heat shock on su(Hw) and mod(Mdg4) levels, or their ability to interact with DNA or with each other, because both proteins are still present in polytene chromosomes at normal levels after temperature elevation. The most likely explanation for the observed changes is a reorganization in the higher-order chromatin structure imposed by the gypsy insulator. This conclusion is strongly supported by the changes observed in the localization of gypsy-containing DNA. Sequences containing the gypsy insulator and located in the nuclear periphery before heat shock move toward the center of the nucleus after temperature elevation. Similarly, two insulator-containing DNA sequences, normally located in close proximity, move apart when cells are subjected to a brief heat shock. These results suggest a correlation between the transcriptional changes taking place during the heat-shock response and alterations in the nuclear arrangement of the DNA determined by the gypsy insulator, supporting a role for insulators in the organization of the chromatin fiber in a manner that has functional significance in the control of gene expression (Gerasimova, 2000).

Visualization of chromatin domains created by the gypsy insulator of Drosophila

Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, this study shows that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops (Byrd, 2003).

The existence and exact composition of the nuclear matrix has been a subject of intense debate. Lamin, which is the main component of the nuclear lamina, is present in the nuclear matrix fraction. It is possible that protein components of the gypsy insulator interact with the nuclear lamina or with other components of the nuclear matrix. In the current experiments, two different biochemical nuclear matrix purification procedures were used. In both cases, it is clear that the Su(Hw) and Mod(mdg4)2.2 proteins associate with the nuclear matrix fraction, whereas other proteins, such as histones and Ubx, are extracted by high salt. Also an in situ cell extraction procedure followed by visualization of the extracted nuclei using light microscopy was used as a means of confirming the association of insulator proteins with the nuclear matrix. Using this approach, gypsy insulator proteins also appear to be associated with the nuclear residue that is resistant to salt extraction. Whether this resistant fraction is a filamentous network of defined composition or a matrix formed by interactions among different proteins and nucleic acids is not known, but it is clear that insulator proteins are not extractable by 2 M NaCl and are not present in the chromosomal regions that extrude from the nucleus after high salt extraction. The interaction of Su(Hw) and Mod(mdg4)2.2 with nuclear matrix components supports observations indicating that gypsy insulator proteins are preferentially present in the nuclear periphery of the interphase nucleus (Byrd, 2003).

Although the insulator DNA and associated proteins remain in the nuclear matrix fraction, the intervening DNA is extruded from the nucleus by high salt extraction and is found in the form of a large loop. The DNA contained within this loop appears only as a small dot after FISH analysis of unextracted nuclei. These two observations suggest that the chromatin fiber present in the loop formed by two insulators is not completely decondensed during interphase and only becomes extended after extraction of histones and other associated proteins. This suggests that the loop formed by two insulators might represent a domain of higher-order chromatin structure. This higher-order structure might be established and/or maintained by specific covalent modifications of histone tails. For example, it has been found that the chicken ß-globin locus, which is flanked by two CTC-binding factor insulators, contains histones H3 and H4, acetylated in various lysine residues. Covalent modification of histone tails might modulate internucleosome interactions, which, in turn, determine the degree of higher-order chromatin structure (Byrd, 2003).

The existence of chromatin insulator-induced domains would explain their unusual gene regulatory property of preventing an enhancer from activating a promoter in a different domain while not preventing the same enhancer from activating a promoter located in its own domain. The ability of the insulator, when flanking a transgene, to provide position-independent expression of the transgene is also consistent with the formation of a loop domain. This domain appears to be created by the interaction of flanking insulators with each other and the nuclear matrix. In fact, recent experiments have shown that boundary function in yeast can be elicited by tethering boundary-associated proteins to the nuclear pore complex (NPC). This tethering would presumably result in the formation of a loop, similar to the ones observed in this study, by the DNA located between the two boundary elements, which would attach the base of the loop to the NPC. In the case of Drosophila, the requirement for interactions between individual Su(Hw) binding sites for the formation of the loops is underscored by the observation that a brief heat shock interferes both with the formation of insulator bodies and with the ability of the gypsy retrotransposon to form a new loop when inserted in the ct locus (Byrd, 2003).

The organization of the chromatin fiber into loops has also been shown for the Drosophila specialized chromatin structures (scs) and scs' boundary sequences. The proteins that interact with these elements have been shown to interact with each other both in vitro and in vivo. Consistent with the idea that interaction between the two proteins might facilitate pairing of boundary elements and formation of chromatin loops, sequences corresponding to the scs and scs' elements can be found in close proximity to each other in Drosophila nuclei. The formation of similar loops by the gypsy insulator could also explain results that demonstrated that two gypsy insulators inserted between an enhancer and promoter have no enhancer-blocking effect. These results could be explained in the context of the loop organization observed in this study by assuming that two closely linked insulators, due to their proximity, may preferentially interact with each other. This interaction would take place at the expense of interactions with other insulators, and it would result in the formation of a minidomain within a larger domain. Enhancers located within the larger domain would then be free to activate transcription from promoters in the same domain. The ability of the gypsy insulator to establish these chromatin loops raises the question of whether insulators/boundary elements are functionally equivalent to MARs/SARs. These sequences have been defined biochemically, based on their ability to attach to the nuclear matrix protein fraction in vitro. In some cases, MARs/SARs have also been shown to possess boundary activity using in vivo assays, although most MARs lack this activity. It is possible that insulators and MARs have similar properties but play very different roles in the cell. MARs might have a fixed structural function in establishing chromatin organization within the nucleus. MARs might only be functional during mitosis, when the interphase to metaphase and back to interphase transition requires orderly changes in chromosome condensation and organization. Alternatively, MARs might create a scaffold of proteins and DNA that is more or less permanent during cell differentiation and among various cell types. Insulators, on the other hand, might act at a different level by creating an organization superimposed to that of MARs. Contrary to MARs, insulator activity might be regulatable, allowing this organization to change during development as cells differentiate and different patterns of gene expression are established (Byrd, 2003).

Functional analyses of genome-wide expression patterns in yeast, Drosophila, and mammals also support the idea of the compartmentalization of the chromatin fiber into domains of gene expression. Studies in this diverse group of organisms have shown that genes located together in the same chromosomal region are transcriptionally coregulated. Although coregulation has not been shown to depend on insulator function, the existence of these transcriptional domains could have a structural basis in the formation of chromatin loops in which gene expression is globally regulated (Byrd, 2003).

Effects of Mutation or Deletion

The only phenotypic effect of null mutations in su(Hw) is female sterility. Mutant females are unable to lay eggs because the egg chambers degenerate before completion of oogenesis. Nurse cell nuclei of stage 4 egg chambers have a bulbous chromosome morphology. Ovaries of su(Hw) mutants show an irregular bulbous structure of nurse cell chromatin visible at all stages of oogenesis beyond stage 3 (Harrison, 1993).

An acidic domain located in the C-terminal end of D. melanogaster SU(HW) is not present in the protein from other Drosophila species, suggesting a nonessential role for this part of the protein. A second acidic domain located in the N-terminal region is present in all species analyzed. This domain is dispensable in the D. melanogaster protein when the C-terminal acidic domain is present, but the protein is nonfunctional when both regions are simultaneously deleted. Mutations in the zinc fingers result in an inability to bind to DNA. The leucine zipper motif is necessary for the negative effect of SU(HW) on enhancer function, suggesting an interaction, either direct or indirect, with transcription factors bound to enhancers, or an interaction of SU(HW) with chromatin components (Harrison, 1993).

The roles played by each of the 12 zinc fingers has been examined in binding gypsy DNA. The zinc fingers can be classified into four groups: essential (fingers 6 through 10); beneficial but nonessential (fingers 1, 2, 3, and 11); unimportant (fingers 5 and 12); and inhibitory (finger 4). Because finger 10 is not required for female fertility but is essential for binding gypsy, these results imply that the SUHW-binding sites required for oogenesis differ in sequence from the gypsy-binding sites. The functions of the N- and C-terminal domains of SUHW were examined by determining the ability of various deletion mutants to support female fertility and to alter expression of gypsy insertion alleles of the yellow, cut, forked, and Ultrabithorax genes. No individual segment of the N- and C-terminal domains of SUHW is absolutely required to alter expression of gypsy insertion alleles. However, the most important domain lies between residues 737 and 880 in the C-terminal domain. This region also contains the residues required for female fertility, and the fertility domain may be congruent with the enhancer-blocking domain. These results imply that SUHW blocks different enhancers and supports oogenesis by the same or closely related molecular mechanisms (Kim, 1996).

Chromatin insulators are thought to regulate gene expression by establishing higher-order domains of chromatin organization, although the specific mechanisms by which these sequences affect enhancer-promoter interactions are not well understood. The gypsy insulator of Drosophila can affect chromatin structure. The insulator itself contains several DNase I hypersensitive sites whose occurrence is dependent on the binding of the Suppressor of Hairy-wing [Su(Hw)] protein. The presence of the insulator in the 5' region of the yellow gene increases the accessibility of the DNA to nucleases in the promoter-proximal, but not the promoter-distal, region. This increase in accessibility is not due to alterations in the primary chromatin fiber, because the number and position of the nucleosomes appears to be the same in the presence or absence of the insulator. Binding of the Su(Hw) protein to insulator DNA is not sufficient to induce changes in chromatin accessibility, and two domains of this protein, presumed to be involved in interactions with other insulator components, are essential for this effect. The presence of Modifier of mdg4 [Mod(mdg4)] protein, a second component of the gypsy insulator, is required to induce these alterations in chromatin accessibility. The results suggest that the gypsy insulator affects chromatin structure and offer insights into the mechanisms by which insulators affect enhancer-promoter interactions (Chen, 2001).

The gypsy insulator contains at least two protein components characterized to date -- Su(Hw) and Mod(mdg4) -- but additional proteins not yet identified could also be important for the function of this insulator. The effects of the gypsy insulator on chromatin structure could be due simply to the binding of Su(Hw) to DNA or to the assembly of a functional insulator. To distinguish between these two possibilities, the effect was examined of mutations in Su(Hw) known to affect its interactions with other proteins and insulator function, while not affecting its ability to bind DNA. The leucine zipper domain of the Su(Hw) protein and the adjacent sequences known as region B have been shown to be essential for the ability of the gypsy insulator to block enhancer function but not required for binding of Su(Hw) to DNA; these two regions of Su(Hw) have been shown to be necessary and sufficient for its interaction with Mod(mdg4). The su(Hw)^L775K allele encodes a nonfunctional protein that can bind DNA but is unable to block the function of upstream enhancers; it is caused by a point mutation in the leucine zipper region that results in a change of the last leucine in the zipper to a lysine. The su(Hw)^D765N allele has a point mutation in an aspartic acid residue not conserved among Su(Hw) proteins of different Drosophila species; this alteration has no effect on the function of the Su(Hw) protein or the gypsy insulator. The su(Hw)^DeltaB allele carries a deletion of the B region and has a similar phenotype to su(Hw)^L775K. To investigate whether the leucine zipper and B domains of Su(Hw) are important for the effects of the gypsy insulator on chromatin structure, nuclease accessibility to the insulator region in flies carrying these su(Hw) mutations was examined; in this case, the yellow gene present in the y² allele, which contains a complete copy of the gypsy element instead of just insulator sequences, was examined. Purified nuclei from third instar larvae of different strains were incubated with increasing amounts of DNase I for a period of time. Purified genomic DNA was digested with BamHI and NcoI; the latter restriction enzyme has a recognition site adjacent to the Su(Hw) binding region in the gypsy element. Four new DNase I hypersensitive sites map to gypsy sequences located between the insulator and the yellow gene in the y² allele. The accessibility of both the insulator region and promoter-proximal sequences decreases in flies carrying mutations in the leucine zipper [su(Hw)^L775K] or B region [su(Hw)^DeltaB] of Su(Hw) with respect to wild-type flies. The Asp to Asn change in the su(Hw)^D765N allele, which has no effect on the function of the Su(Hw) protein, also has no effect on the accessibility of the insulator or adjacent sequences to DNase I. Thus, interactions of Su(Hw) with other proteins through the leucine zipper and B regions are required for normal insulator function and for the observed changes in chromatin organization (Chen, 2001).

Since domains of the Su(Hw) protein thought to interact with Mod(mdg4), and perhaps with other proteins, are important for changes in chromatin structure induced by the gypsy insulator, it was asked whether mod(mdg4) mutations affect chromatin accessibility to DNase I. A combination of two mod(mdg4) alleles were used that allow survival to early pupal stages. The combination of both alleles in y²; mod(mdg4)^T16/ mod(mdg4)^E(var)3-93D flies causes early pupal lethality and allows the selection of third instar larvae for DNase I sensitivity experiments. The hypersensitive sites present in the promoter-proximal region disappear in flies carrying mutations in the mod(mdg4) gene. These results suggest that the observed changes in chromatin structure induced by the gypsy insulator in the promoter-proximal region require the binding of Su(Hw) to insulator sequences as well as the presence of Mod(mdg4) protein. The observed effects of mod(mdg4) mutations on the chromatin structure of sequences located upstream and downstream of the insulator correlate with the observed effect of these mutations on enhancers located proximal and distal to the insulator with respect to the promoter (Chen, 2001).

An important conclusion from the results presented here is that changes in chromatin structure are not simply the result of the binding of Su(Hw) protein to insulator DNA. Further interactions between Mod(mdg4) and Su(Hw) are needed to induce changes in chromatin structure, a conclusion supported both by the requirement of the Mod(mdg4) protein and of domains of Su(Hw) involved in mediating interactions between both proteins. Since interactions between Su(Hw) and Mod(mdg4) lead to the establishment of loops or domains of higher-order chromatin organization, this observation further supports the role of the gypsy insulator in the regulation of chromatin structure at this level of organization. The establishment of these domains by insulators might be a first step and a prerequisite to allow further steps necessary for the activation of transcription. The differences in the effect of the gypsy insulator on promoter-proximal vs. promoter-distal sequences could be due to the ability of the insulator to discern between either side with respect to its own location. This property would require the insulator to orient itself with respect to the promoter and should be based on interactions between insulator proteins and the transcription complex. Alternatively, insulators might just affect chromatin structure at a global level in a manner that allows subsequent interactions of the chromatin fiber with components of the transcriptional machinery. These transcription factors could then further affect chromatin structure in a promoter-specific manner, resulting in the observed promoter-proximal specific effects (Chen, 2001).

Combining gypsy with a P element results in novel mutagenic properties. A P element was modified so that it carried two copies of the Suppressor of Hairy-wing binding regions isolated from the gypsy transposable element. This transposon was mobilized, and the genetic consequences of its insertion were analyzed. The composite transposon (gypsy+P element) combines the mutagenic efficacy of the gypsy element with the controllable transposition of P elements. Compared to standard P elements, this composite transposon causes an expanded repertoire of mutations and produces alleles that are suppressed by su(Hw) mutations. The large number of heterochromatic insertions obtained is unusual compared to other insertional mutagenesis procedures (Roseman, 1995b).

Chip may encode an enhancer-facilitator, acting to facilitate the activity of distal enhancers. The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein [su(Hw)] that binds to gypsy retrovirus insertions between enhancers and promoters. su(Hw) bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. Chip is needed for the wing margin enhancer of cut. The Chip mutation dominantly enhances the mutant phenotypes displayed by partially suppressed gypsy insertions in both cut and Ultrabithorax and is a homozygous larval lethal, indicating that Chip regulates multiple genes. Chip is normally required for wing margin enhancer function of cut because Chip mutations also enhance the cut wing phenotype of a cut mutation and heterozygotes for Chip display cut wing phenotypes when either scalloped or mastermind (mam) are also heterozygous mutant. Both Sc and Mam are known to regulate the cut distal enhancer, but in contrast to sd and mam mutants, Chip mutants display stronger genetic interactions with gypsy insertions than with wing margin enhancer deletions. Thus, in a heterozygous Chip mutant, a heterozygous gypsy insertion in cut displays a cut wing phenotype, whereas a heterozygous enhancer deletion does not. Dependence on the nature of the heterozygous lesion in the regulatory region strongly suggests that Chip directly regulates cut. More strikingly, it indicates that in a Chip heterozygote, a gypsy insertion is more deleterious to enhancer function than deletion of the enhancer. The simplest explanation is that su(Hw) bound to gypsy in one cut allele acts in a transvection-like manner (interchromosomally) to block the wing enhancer in the wild-type cut allele on a second chromosome. This implicates Chip in enhancer-promoter communication (Morcillo, 1997 and references).

Chip was cloned and found to encode a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins, which bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. It is suggested that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer-promoter interactions (Morcillo, 1997).

Pairing between gypsy insulators facilitates the enhancer action in trans throughout the Drosophila genome

The Suppressor of the Hairy wing [Su(Hw)] binding region within the gypsy retrotransposon is the best known chromatin insulator in Drosophila. Two copies of the gypsy insulator inserted between an enhancer and a promoter neutralize each other's actions, indicative of an interaction between the protein complexes bound to the insulators. The role was investigated of pairing between the gypsy insulators located on homologous chromosomes in trans-interaction between yellow enhancers and a promoter. trans activation of the yellow promoter strongly depends on the site of the transposon insertion; this provides evidence for a role of surrounding chromatin in homologous pairing. The presence of the gypsy insulators in both homologous chromosomes even at a distance of 9 kb downstream from the promoter dramatically improves the trans-activation of yellow. Moreover, the gypsy insulators have proven to stabilize trans activation between distantly located enhancers and a promoter. These data suggest that gypsy insulator pairing is involved in communication between loci in the Drosophila genome (Kravchenko, 2005).

To confirm the role of the gypsy insulator in trans-activation between nonhomologous enhancerless and promoterless derivatives, trans-activated allele combinations were tested in su(Hw)^- or mod(mdg4)^u1 backgrounds. Both mutants have a strongly reduced level of trans activation. Surprisingly, the mod(mdg4)^u1 mutation affects transvection between nonhomologous insertions much more strongly than transvection between homologous insertions; apparently, the long-range trans activation is more sensitive to the gypsy insulator's components. In contrast, its effect on both homologous and nonhomologous trans activation is much less severe than that of the su(Hw)^- mutation. This agrees with the extent of instability of insulator bodies on the su(Hw)^- and mod(mdg4)^u1 backgrounds: they are completely destroyed in the former case but only partially affected in the latter case. Thus, the gypsy insulator supports transvection between nonhomologous loci, and the efficiency of trans activation mainly depends on the relative arrangement of the loci in the nuclear architecture rather than on the linear distances between them on the chromosomes (Kravchenko, 2005).

The level of trans activation by the wing and body enhancers strongly depends on the site of insertion and does not correlate with the level of the yellow promoter activation in cis. Thus, the genomic regions do not provide identical yellow activation in trans that might be explained by their pairing strength between the homologs. Hence, there might be some specific elements facilitating the pairing between homologous chromosomes. The gypsy insulator inserted either 5 kb or 9 kb downstream from the yellow promoter improves its trans activation by the enhancers located on the homologous chromosome. The interaction between the gypsy insulators can improve the local pairing between homologous chromosomes. Cytological data have indicated that the gypsy insulators create chromatin loop domains by associating with the nuclear matrix. Two homologous chromosomes form only one loop, suggesting that the proteins present in the gypsy insulator and the nuclear matrix could maintain homologous chromosome pairing during the interphase (Kravchenko, 2005).

It is noteworthy that the reported yellow sequences significant for efficient transvection between enhancerless and promoterless y alleles include the 1A-2 insulator located on the 3' side of the yellow gene. The insulator containing two binding sites for the Su(Hw) protein was not present in the constructs used in this study. Thus, reliable and efficient trans activation observed previously upon the transgene insertion at all seven genomic sites can be explained by the presence of the endogenous Su(Hw) insulator improving local homologous pairing. Because the Su(Hw) protein binds to approximately 200 sites in the Drosophila genome, the Su(Hw) binding sites appear to play a role in the pairing between homologous chromosomes. Recent studies of the scs and scs' insulators confirm that they interact with each other and, therefore, may also be involved in this process (Kravchenko, 2005).

Although the examples of transvection are many, the nature of chromosome pairing during the interphase is still obscure. Today, only the Zeste protein is known to be involved in some transvection effects. The zeste gene encodes the sequence-specific DNA-binding protein with the binding sites distributed throughout genome. Inactivation of Zeste disrupts allelic pairing, thus enhancing the heteroallelic mutant phenotype. Zeste also supports transvection-like effects in the decapentaplegic, white, eyes absent, and Ubx genes (Kravchenko, 2005).

Polycomb response elements (PREs) are another class of regulatory elements that may facilitate pairing between homologous chromosomes. PREs are short DNA segments initiating the assembly of silencing complexes composed of the Polycomb group (PcG) proteins. The silencing of a PRE-containing transposon construct is often dramatically enhanced in flies homozygous for the transposon insertion. The interaction between two copies of PREs on the homologous chromosomes is supposed to improve the stability and silencing power of the PcG complex. At the same time, the interaction between the PcG complexes may support homologous chromosome pairing. The combination of the binding sites for the proteins like Su(Hw), Zeste, and PcG may generate a unique code for making this process more efficient (Kravchenko, 2005).

The Zeste, Su(Hw), and PcG proteins, along with having the ability to strengthen homologous effects, are involved in the formation of higher-order nuclear structures. Thus, Zeste can form high-order aggregates, which suggests that it can hold together certain DNA regions. Likewise, PcG proteins are organized into discrete nuclear bodies that may be the sites of the PRE-mediated silencing. The well-defined Fab-7 cellular memory module also leads to association of transgenes even when inserted into different chromosomes. The same has been shown for the Mcp element, which contains an insulator and PRE. Such long-distance interactions depend on the PcG proteins, at least partially. This study has shown that the gypsy insulator provides for trans activation between selected genomic loci at distances exceeding 13 Mb. Together with previous data on the punctuated distribution of gypsy insulator proteins in the nucleus, the results of this study suggest their involvement in the arrangement of the chromatin fiber within the nucleus and in organization of communication between distant loci in the Drosophila genome (Kravchenko, 2005).

Thus, the interaction between the gypsy insulators facilitates trans activation of the yellow promoter, which is evidence for the involvement of gypsy insulators in the regulation of homologous chromosome pairing and communication between distant loci. Further investigations are required to find out whether the transvection stabilization by gypsy insulator in homologous and distant locations relies on the same mechanism. The model system utilizing the effects of transvection between yellow transgenes provides a powerful tool for the analysis or identification of the proteins supporting interactions between loci (Kravchenko, 2005).

Analysis of chromatin boundary activity in Drosophila cells.

Chromatin boundaries, also known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. However, the mechanisms of boundary action are poorly understood, in part due to limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared. This paper reports the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. The activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are shown to be supported in these cells. It was further shown that double stranded RNA (dsRNA)-mediated knockdown of SuHw and dCTCF factors disrupts the enhancer-blocking function of suHw and Fab8, respectively, thereby establishing the effectiveness of using RNA interference in this cell-based assay for probing insulator function. It is concluded that the novel boundary assay provides a quantitative and efficient method for analyzing insulator mechanism and can be further exploited in genome-wide RNAi screens for insulator components. It provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements (Li, 2009).

Despite their diverse genomic origins and distinct cis- and trans- components, the Drosophila suHw, SF1, Fab7 and Fab8 elements function as potent enhancer-blockers in the Drosophila cells. This finding suggests that chromatin boundary represents a basic cell function that is shared by diverse tissues. The cell-based insulator assay was combined with RNAi-mediated gene knockdown to systematically test the requirement of SuHw and dCTCF in the function of several Drosophila insulators. RNAi-mediated knockdown of SuHw and dCTCF specifically disrupted the function of the suHw and Fab8 boundaries, respectively, thereby validating the functional specificity of the assay. The results suggest that multiple independent pathways in Drosophila mediate insulator function. This is in contrast with the pivotal role the CTCF protein plays in the enhancer-blocking activities in vertebrates (Li, 2009).

Cell culture assays have several important advantages that complement studies using in vivo system. The homogeneous cell populations in these assays can be used in biochemical and cell biological analyses. They allow more efficient and quantitative assessment of reporter readout from a large number of individual cells. Insulator activity has previously been demonstrated in Drosophila cells; this system has improved the assay with several novel features. First is the use of P-element-based transgene vector, which is known to mediate single to low copy number, non-tandem genomic integration of the assay transgenes. This would provide more native genomic and regulatory environment for studying chromatin boundary function. Large numbers of stably transfected cells with randomly integrated transgenes also provide a broader sampling of the genomic environment, a feature that can be exploited to examine boundary activity in blocking chromosomal position effect. The second improvement is the use of divergently transcribed dual reporters, which provides a linked readout to control for the 'off-targets' effects on the non-insulator components in the assay system, such as enhancers, promoters, reporters, the state of general transcription or other cellular functions that impact the reporter readout. It should also provide an important control for the chromosomal position effect near the transgene integration site in stably transfected cells. The use of fluorescent protein reporters further allows rapid and quantitative FACS assessment of the enhancer-blocking activity, a feature particular important in high-throughput applications. The activity of multiple Drosophila insulators has been established, along with the efficiency of RNAi-mediated gene knockdown; this should facilitate biochemical dissection of insulator function and genome-wide high throughput RNAi screens for novel boundary components (Li, 2009).

As most cell-based systems, the enhancer-blocking assay is limited in its application by potential tissue or developmental stage incompatibilities of the insulator and the cell. Studies have suggested that certain chromatin boundaries, such as Fab7 and SF1, are composed of distinct insulator activities that function in different tissues and/or developmental stages. Although this study has documented the functionality of several Drosophila insulators in S2 and Kc cells, both derived from embryonic cell lineages, other insulators may not function in these two cell lines. In addition, cultured cells may have, over the course of many passages, lost the physiological stoichiometry of relevant DNA or protein components, resulting in impaired function of certain insulators. Furthermore, the dynamic regulation of insulator activity in response to developmental and physiological cues would depend on the context of the whole animal. Therefore, the cell-based insulator assay presented in this study provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements (Li, 2009).

Genome-wide studies of the multi-zinc finger Drosophila Suppressor of Hairy-wing protein in the ovary

The Drosophila Suppressor of Hairy-wing [Su(Hw)] protein is a globally expressed, multi-zinc finger (ZnF) DNA-binding protein. Su(Hw) forms a classic insulator when bound to the gypsy retrotransposon and is essential for female germline development. These functions are genetically separable, as exemplified by Su(Hw)(f) that carries a defective ZnF10, causing a loss of insulator but not germline function. This sutyd, completed the first genome-wide analysis of Su(Hw)-binding sites (SBSs) in the ovary, showing that tissue-specific binding is not responsible for the restricted developmental requirements for Su(Hw). Mapping of ovary Su(Hw)(f) SBSs revealed that female fertility requires binding to only one third of the wild-type sites. It was demonstrate that Su(Hw)(f) retention correlates with binding site affinity and partnership with Modifier of (mdg4) 67.2 protein. Finally, clusters of co-regulated ovary genes flanked by Su(Hw)(f) bound sites were identifed, and it was shown that loss of Su(Hw) has limited effects on transcription of these genes. These data imply that the fertility function of Su(Hw) may not depend upon the demarcation of transcriptional domains. These studies establish a framework for understanding the germline Su(Hw) function and provide insights into how chromatin occupancy is achieved by multi-ZnF proteins, the most common transcription factor class in metazoans (Soshnev, 2012).

Su(Hw) is a broadly expressed transcription factor that is required for oogenesis. Much of the understanding of Su(Hw) function has been obtained through investigation of the gypsy insulator. These studies have led to the concept that Su(Hw) is an architectural protein involved in establishing higher order chromosomal structure critical for regulation of gene expression. However, emerging evidence suggests that the function of Su(Hw) extends beyond that of an insulator protein, including the recent demonstration that 1A-2, a cluster of two SBSs, is required for activation of yar, a non-coding RNA gene (Soshnev, 2008). These data suggest that Su(Hw) has multiple functions in the genome (Soshnev, 2012).

Previous studies estimate that between five to eighteen percent of SBSs are cell type specific, with evidence that 1%-3% of SBSs are developmentally regulated. This study used ChIP-seq coupled with extensive ChIP-qPCR to show that Su(Hw) chromosome occupancy is largely constitutive throughout development. While a small set of 'ovary-specific' SBSs were identified among the low fold enrichment SBSs, it was shown that these sites are occupied in non-ovary tissues. The data are consistent with the previous analysis of SBSs in the three megabase alcohol dehydrogenase region, in which Su(Hw) binding was conserved between different tissues. These studies provide a cautionary note for investigations relying solely on computational evaluation of high-throughput genomic datasets, as it was found that extensive validation is required to establish confident binding thresholds needed for data interpretation (Soshnev, 2012).

The ovary-specific developmental requirement for Su(Hw) may be explained based on its function at the gypsy insulator. The insulator properties of Su(Hw) suggest that oogenesis may require establishment of domain boundaries that permit appropriate gene expression in the ovary. To test this postulate, genome-wide binding sites were defined for Su(Hw)^f, a mutant isoform that lacks insulator activity, but retains fertility. These studies revealed that Su(Hw)^f was retained at only one third of wild-type sites. Ostensibly, these observations are surprising for an architectural protein, as two-thirds of SBSs can be lost without effects on essential functions needed for fertility. These global analyses were extended through direct studies of co-regulated gene clusters delimited by f-retained SBSs. Loss of Su(Hw) was shown to have limited, if any, effects on expression of these genes in the ovary. Based on these observations, it is suggested that the essential ovary function of Su(Hw) may not be related to establishment of boundaries of transcriptional domains, a conclusion supported by recent findings that null and nearly null alleles of mod(mdg4) and Cp190 do not affect oogenesis. It is suggested that Su(Hw) may act locally to change gene expression. Recent studies demonstrate that Su(Hw) is associated with repressed chromatin domains and is enriched in lamin-associated domains. These observations, together with findings that enhancer blocking activity of the gypsy insulator is disrupted by a lamin mutation, suggest that Su(Hw)-dependent regulation may involve gene silencing that requires Su(Hw) targeting to the nuclear periphery (Soshnev, 2012).

The availability of a high-quality dataset of SBSs provided the opportunity to investigate the genome-wide association of Su(Hw) with its partner proteins, Mod67.2 and CP190. These analyses showed that SBS-O sites represented the largest class. Further, it was found that SBS-O and SBS-C sites displayed sequence conservation that extended beyond the Su(Hw)-binding motif, which was not observed for the SBS-CM class. These data suggest that Mod67.2 confers greater flexibility to Su(Hw) association, a postulate supported by the demonstration that Mod67.2 facilitates Su(Hw) occupancy. These findings imply that the structurally related BTB-domain protein CP190 cannot replace the function of Mod67.2 in facilitating Su(Hw) occupancy of SBSs. Although SBSs collectively display no enrichment with genic features, a skewed localization of SBS-CM sites to the 5'- and 3'-end of genes and coding exons was found. Taken together, these data indicate that different classes of SBSs may have distinct regulatory contributions in the genome (Soshnev, 2012).

Su(Hw) has 12 ZnFs, with ten corresponding to C₂H₂ fingers and two corresponding to C₂HC. Previous studies suggest that the major mode for Su(Hw) chromosome association is DNA binding, as loss of ZnF7 causes complete loss of in vivo localization to chromosomes that correlates with defective in vitro binding. This study has demonstrated that loss of ZnF10 eliminates Su(Hw)^f occupancy at two-thirds of SBSs, with binding site selection of Su(Hw)^f showing greater constraints than Su(Hw)⁺. While Su(Hw)^f is lost at many genomic sites, this protein binds f-lost SBSs in vitro, although with reduced affinity relative to Su(Hw)⁺. Yet, this reduced Su(Hw)^f-binding affinity cannot account for all f-lost sites, as there is an absence of a strict correlation between in vitro DNA binding and in vivo chromosome Su(Hw)^f occupancy. Further investigation revealed that some SBSs showed tissue-specific Su(Hw)^f retention and that Su(Hw)^f retention was optimal at SBSs that associate with Mod67.2, a protein partner associated with enhanced occupancy of Su(Hw). Taken together, these data suggest that Su(Hw)^f retention is affected by multiple factors, including DNA sequence, tissue-specific effects that may depend on local chromatin structure and a protein partner of the gypsy insulator complex (Soshnev, 2012).

Multi-ZnF domains are the most common DNA-binding motif among transcription factors in metazoan genomes. The data are relevant to understanding how mutation of a single ZnF within a large ZnF-binding domain impacts chromatin occupancy of this class of transcription factors. It was shown that individual fingers may make distinct contributions to chromosome association, without altering the recognition sequence of the binding site. Interestingly, a second well-characterized vertebrate insulator protein CCCTC-binding factor (CTCF) is an eleven ZnF DNA-binding protein. Mutations in the gene encoding CTCF have been found in several human tumor samples, including breast, prostate and kidney. These tumor-associated alleles carried missense mutations that changed specific CTCF ZnFs, with none producing a truncated protein. Interestingly, in vitro studies demonstrated that these CTCF ZnF mutants had altered in vitro DNA-binding properties, reminiscent of Su(Hw)^f. However, no in vivo binding studies were completed. Data obtained from analysis of Su(Hw)^f predict that the cancer-associated CTCF mutations may alter the in vivo landscape of CTCF occupancy genome-wide. As a result, these effects may lead to complex changes in gene expression that may promote tumorigenesis (Soshnev, 2012).

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date revised: 26 December 2015 

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