modifier of mdg4


The nuclear distribution of mod(Mdg4) 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).

The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements

In Drosophila, defects in asymmetric cell division often result in the formation of stem cell derived tumors. This study shows that very similar terminal brain tumor phenotypes arise through a fundamentally different mechanism. Brain tumors in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by de-repression of target genes in the Salvador-Warts-Hippo (SWH) pathway. ChIP-seq was used to identify L(3)mbt binding sites, and it was shown that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting expression of the insulator protein gene mod(mdg4) results in upregulation of SWH-pathway reporters. As l(3)mbt tumors are rescued by mutations in bantam or yorkie or by overexpression of expanded the deregulation of SWH pathway target genes is an essential step in brain tumor formation. Therefore, very different primary defects result in the formation of brain tumors, which behave quite similarly in their advanced stages (Richter, 2011).

Drosophila nervous system recapitulates many steps in mammalian neurogenesis. Neurons in the adult fly brain arise from stem cells called neuroblasts which undergo repeated rounds of asymmetric cell division during larval stages. After division, one daughter cell remains a neuroblast while the other is called the ganglion mother cell (GMC) and divides just once more into two differentiating neurons. Most larval neuroblasts are inherited from the embryo but the so-called optic lobe neuroblasts (NB) located laterally on each brain lobe pass through a neuroepithelial (NE) stage and are therefore a particularly suitable model for mammalian neurogenesis. During early larval stages, the NE cells of the optic lobes (OL) proliferate and separate into the inner (IOA) and outer (OOA) optic anlagen. During late larval stages, NE cells switch to a neurogenic mode. On the medial side, they generate optic lobe neuroblasts (OL NBs), which generate the neurons of the medulla, the second optic ganglion. OL neurogenesis is controlled by a wave of lethal of scute (l(1)sc) expression passing through the neuroepithelium from medial to lateral. The activity of the Jak/STAT pathway inhibits neural wave progression and thereby controls neuroblast number. Differentiation of neuroepithelial cells also involves the Notch, Epidermal Growth Factor (EGF) and Salvador-Warts-Hippo (SWH) pathways (Richter, 2011).

Characterization of Drosophila genes identified in brain tumor suppressor screens has demonstrated that defects in neuroblast asymmetric cell division result in the formation of stem cell derived tumors that metastasize and become aneuploid upon transplantation. These screens also identified lethal (3) malignant brain tumor (l(3)mbt), a conserved transcriptional regulator that is also required for germ-cell formation in Drosophila. L(3)mbt binds to the cell cycle regulators E2F and Rb but the relevance of these interactions is unclear. This study shows that in Drosophila, L(3)mbt regulates target genes of the Salvador-Warts-Hippo (SWH) pathway that are important in proliferation and organ size control. The SWH-pathway is regulated by the membrane proteins Expanded (Ex) and Fat, which activate a protein complex containing the kinases Hippo and Warts to phosphorylate the transcriptional co-activator Yorkie. Yorkie acts together with the transcription factors Scalloped and Homothorax to activate proliferative genes like Cyclin E and the microRNA bantam (ban) and Drosophila inhibitor of apoptosis 1 (diap1: thread). Upon phosphorylation, Yorkie is retained in the cytoplasm and its target genes are not activated. In Drosophila the main role of the SWH-pathway is to limit proliferation in imaginal discs and its absence leads to tumorous overgrowth. In vertebrates, many homologs of key pathway members are tumor suppressors indicating that this function is conserved (Richter, 2011).

L(3)mbt contains three MBT domains which bind mono- or dimethylated histone tails. Biochemical experiments in vertebrates have suggested a role in chromatin compaction but whether this role is conserved is not known. Results published while this paper was under review have shown that germline genes are upregulated in l(3)mbt mutant brains and are necessary for tumor formation. The current data indicate that L(3)mbt is bound to insulator sequences, which affect promoter-enhancer interactions and influence transcription. In Drosophila, the proteins CTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4) and GAF are found at insulator sequences but how these factors act is largely unknown (Richter, 2011).

The data presented in this study show that tumor formation in l(3)mbt mutants is initiated by the uncontrolled overproliferation of neuroepithelial cells in the optic lobes due to the upregulation of proliferation control genes normally repressed by the SWH-pathway. L(3)mbt is located at DNA sequences bound by chromatin insulators and we propose that the function of L(3)mbt as a chromatin insulator is essential for repressing SWH target genes and preventing brain tumor formation (Richter, 2011).

brat, lgl and dlg were previously identified as Drosophila brain tumor suppressors. In all cases, defects in asymmetric cell division cause a huge expansion of the neuroblast pool. In l(3)mbt mutants, however, the neuroblast pool is expanded because an upregulation of SWH target genes results in a massive expansion of neuroepithelial tissue. Why those neuroblasts proliferate indefinitely upon transplantation is currently not understood for any of those mutants (Richter, 2011).

While the SWH-pathway is essential for tumorigenesis in l(3)mbt mutants, its overactivation can not recapitulate the neuroblast tumor phenotype seen in l(3)mbt mutants (this study and Reddy, 2010). Similar to the multifactorial origin of mammalian tumors, therefore, the combined deregulation of several signaling pathways could be required. The Notch pathway could be involved as it regulates the formation of OL neuroblasts from neuroepithelia and Notch pathway gene insulator sequences are bound by L(3)mbt. Increased activity of the Jak/STAT pathway, a major regulator of OL development, was also observed. Finally, the deregulation of germline genes in l(3)mbt mutants that has been described while this manuscript was under review (Janic, 2010) could provide another exciting explanation (Richter, 2011).

The results indicate that L(3)mbt acts on insulator elements, which isolate promoters from the activity of nearby enhancers acting on other genes. the analysis showed that L(3)mbt binding sites overlap with CP190, CTCF and BEAF-32, placing the protein into what has been called the class I of chromatin insulators (Negre, 2010; Richter, 2011 and references therein).

The identification of a DNA consensus motif for a histone binding protein like L(3)mbt is highly unexpected as insulators are typically nucleosome free. Currently, the activity of these important transcriptional regulators could be explained in several ways. Either, they form physical barriers blocking the interaction between enhancers and promoters. Alternatively, they mimic promoters and compete with endogenous promoters for enhancer interaction. Finally, they could interact with each other or nuclear structures to form loop domains that regulate transcriptional activity. The data suggests another model in which insulators interact with histones on nearby nucleosomes and influence the structure of higher order chromatin. Importantly, in the regions flanking CTCF binding sites nucleosomes are enriched for histones that are mono- and di-methylated on H3K4 or mono-methylated on H3K9 or H4K20, the variants to which MBT domains can bind in vitro. As the human L(3)mbt homolog L3MBTL1 was shown to compact nucleosome arrays in vitro, a model becomes feasible in which simultaneous binding to insulators and the surrounding nucleosomes reduces flexibility and thereby restricts the ability of nearby enhancers to interact with promoters on the other side of the insulator. However, the data could equally well be worked into the other prevalent models for insulator activity. Since L(3)mbt is currently the only chromatin insulator besides CTCF that is conserved in vertebrates, analysis of its homologs will certainly allow to distinguish between those possibilities (Richter, 2011).

OL development resembles vertebrate neurogenesis. Both processes consist of an initial epithelial expansion phase followed by neurogenesis through a series of asymmetric divisions. Together with previous findings, these data demonstrate that l(3)mbt and the SWH-pathway are crucial regulators of the initial neuroepithelial proliferation phase. Interestingly, the SWH-pathway has been implicated in regulating neural progenitors in the chicken embryo and it will be exciting to test the role of mammalian L(3)mbt in this process. It is remarkable that YAP is upregulated and L3MBTL3 is deleted in a subset of human medulloblastomas. Medulloblastoma is the leading cause of childhood cancer death and investigating the role of the SWH-pathway might contribute to the progress in fighting this disastrous disease (Richter, 2011).

Effects of Mutation or Deletion

mod(mdg4) interacts with a number of chromatin genes that suppress position-effect variegation. mod(mdg4) suppresses the strong dominant suppressor effect of the mutations Su(var)2-11, Su(var)2-54, Su(var)2-55, Su(var)3-310 and Su(var)3-96, which indicates that the products of these genes interact. Many of the known suppressor loci encode chromatin proteins. Su(var)2-11 affects histone H4 deacetylation; mutations of this locus display a recessive lethal interaction with the heterochromatic Y chromosome. Su(var)2-5 encodes HP1, a structural component of heterochromatin, and the Suppressor of variegation 3-9 protein may belong to the same class of proteins. The observation that mod(mdg4) mutations interact with all suppressor mutations tested suggests that the products of mod(mdg4) are also chromatin proteins. Mutations in mod(mdg4) exhibit an imprinting-like effect on the Y chromosome. This effect is transmitted paternally over several generations. Homeotic transformations in mod(mdg4) mutants indicate an involvement of the gene products in regulation of homeotic gene complexes. mod(mdg4), like trithorax, is a positive regulator of the Bithorax complex. An antiserum raised against Mod(mdg4) protein detects this chromosomal protein in a large subset of sites in polytene chromosomes. Genetic and molecular data suggest that the protein coded by mod(mdg4) are generally involved in establishing and/or maintaining an open chromatin conformation (Dorn, 1993).

The suppressor of Hairy wing (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 (mod[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; 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), su(Hw) exerts a bidirectional silencing effect, whereas in the presence of mod(mdg4), the silencing effect is transformed into unidirectional repression (Gerasimova, 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 within 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 homologous chromosome (Georgiev, 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).

The suppressor of Hairy-wing [su(Hw)] protein inhibits the function of transcriptional enhancers located distal to the promoter with respect to the location of su(Hw)-binding sites, thus functioning to insulate enhancers from promoters. Mutations in the modifier of mdg4 [mod(mdg4)] interfere with insulation and enhance the effect of the su(Hw)-binding region inserted in the y2 mutation by inhibiting the function of regulatory elements located on both sides of the su(Hw)-binding region. From P-M hybrid dysgenic crosses, 21 mutations that suppress the negative effect of the mod(mdg4)1u1 mutation on the y2 allele were obtained among 47,000 flies scored. These Su(mg) mutations have a dominant suppressor effect and map to at least 13 different loci. Some of Su(mg) mutations also suppress the effect of mod(mdg4)1u1 on two other gypsy-induced mutations, scD1 and ct6. Most of Su(mg) mutations do not affect the viability or fertility of homozygous flies. It is speculated that the Su(mg) genes represent a new family of redundant regulatory genes in Drosophila melanogaster (Bezborodova, 1997).

Polycomb and trithorax group proteins mediate the function of a chromatin insulator

Chromatin boundaries or insulator elements affect the interaction between enhancers and promoters. The gypsy insulator contains two proteins, su(Hw) and Mod(mdg4). Both proteins colocalize on several hundred sites on polytene chromosomes and are distributed in a punctated pattern in the nuclear matrix. Mutations in mod(mdg4) have properties characteristic of a trxG gene. In addition, mutations in trxG genes enhance insulator effects on adjacent enhancers, whereas mutations in Pc have the opposite result. These alterations correlate with changes in the pattern of nuclear localization of insulator components. The results suggest a model in which PcG and trxG proteins regulate insulator function by establishing higher order domains of chromatin organization required for the assembly of functional insulators at the nuclear matrix (Gerasimova, 1998).

Escaper males carrying the mod(mdg4)E(var)3-93D allele display homeotic transformations of the fifth to fourth abdominal segments (Dorn, 1993). This observation is surprising in view of the finding that Mod(mdg4) protein is ubiquitously distributed in embryos and imaginal discs, suggesting that mod(mdg4) is not a homeotic gene. One possible explanation for these results is that the Mod(mdg4) protein regulates the expression of homeotic genes, as is the case for members of the trxG and PcG families. If mod(mdg4) is a trxG gene, mutations in mod(mdg4) should satisfy three criteria: enhance the phenotype of trxG mutations, suppress the dominant Pc phenotype, and decrease homeotic gene expression. To test this possibility, the phenotype of doubly heterozygous mutant combinations carrying the mod(mdg4)u1 and mod(mdg4)T6 alleles and mutations in various trxG and PcG genes were examined for strong transformations of haltere to wing, transformations of first and third leg to second, and formation of partial seventh tergite. An adult fly of the genotype trx+ mod(mdg4)T16/trxB11 mod(mdg4)+ displays a dramatic transformation of the haltere into wing. Flies of the genotype mod(mdg4)T16/+ or trxB11/+ do not show this phenotype, but in combination, 1.4% of the adults show this strong homeotic transformation. A similar result was obtained with other mod(mdg4) alleles. Mutations in the Trithorax-like (Trl) gene also cause a similar increase in the number of homeotic transformations observed when in heterozygous combination with mod(mdg4) alleles, resulting in 3.7% of individuals displaying homeotic transformations in Trl+ mod(mdg4)T16/Trl62 mod(mdg4)+ compared to 0.3% observed in Trl62/+. Mutations in mod(mdg4) do not significantly increase the number or severity of homeotic transformations in two other trxG genes: absent, small or homeotic discs1 (ash1) and brahma (brm). When mutations in these two genes are combined with mutations in mod(mdg4), the frequency of transformations increases dramatically. For example, 77.7% of flies of the genotype trx+ ash1+ mod(mdg4)T16/trxB11 ash1VF101 mod(mdg4)+ display homeotic transformations, whereas only 14.1% of the trxB11 ash1VF101/++ show a mutant phenotype. These results suggest that mod(mdg4) might be a member of the trxG family, since mutations in this gene enhance the phenotype of mutations in trxG family members (Gerasimova, 1998).

To explore this possibility further, genetic interactions between mutations in the mod(mdg4) and Polycomb genes were examined. Flies heterozygous for a deficiency of the Pc gene show a transformation of the second and third legs into first leg; this transformation is easily visualized by the presence in the second and third legs of males of ectopic sex combs characteristic of the first leg. When Pc/+ flies are also heterozygous for a mutation in mod(mdg4), both the frequency and severity of this transformation are reversed, and the sex combs are visible only in the first leg. This result indicates that mutations in mod(mdg4) suppress the dominant phenotype of Pc, supporting the idea that mod(mdg4) might be a member of the trxG family (Gerasimova, 1998).

If mod(mdg4) functions as a trxG gene, it should play a positive role in controlling the expression of homeotic genes, both during embryonic and later stages of development. To determine whether mod(mdg4) mutations affect homeotic gene expression, their effects were analyzed on the expression of homeotic genes during larval development. The effect of mod(mdg4) mutations were examined on the expression of the Antennapedia (Ant) gene. To this end, a combination of two mod(mdg4) alleles were used, mod(mdg4)16/mod(mdg4)E(var)3-93D, resulting in lethality at the early pupa stages. Tissues were taken from live individuals during late larval stages of development. At this time, the Antp protein is expressed in the ventral ganglion in three bands of cells that correspond to the three thoracic segments. In the mod(mdg4)16/mod(mdg4)E(var)3-93D mutant individuals examined, the brain lobes are small, the ventral ganglion is malformed, and expression of the Antp protein is undetectable. A second homeotic member of the Antp complex, Sex combs reduced (Scr), is expressed in a stripe of cells located in the most anterior region of the ventral ganglion in wild-type third-instar larvae. This band is not observed in mod(mdg4)16/mod(mdg4)E(var)3-93D mutants. Mod(mdg4) also regulates homeotic genes involved in the development of posterior body segments. Ubx is expressed in a band of cells in the ventral ganglion located posterior to the domain of Antp expression. This stripe of Ubx expression is not detectable in the ventral ganglion of mod(mdg4)16/mod(mdg4)E(var)3-93D larvae, suggesting that the Mod(mdg4) protein positively regulates Ubx expression. Mutations in mod(mdg4) also affect the expression of the Abdominal B (Abd B) gene, which is expressed in the most posterior region of the ventral ganglion during larval development but is lacking in mod(mdg4)16/mod(mdg4)E(var)3-93D mutants. A similar effect is observed for the expression of homeotic proteins in the wing and leg imaginal discs; in mod(mdg4) mutants, these structures often appear malformed, and there is no detectable accumulation of Antp, Scr, Ubx, or Abd-B proteins. These results indicate that several homeotic genes of the Antennapedia and bithorax complexes are not properly expressed in mod(mdg4) mutants, suggesting that the Mod(mdg4) product plays a positive role in regulating their expression, in agreement with its putative role as a trxG gene (Gerasimova, 1998).

hobo induced rearrangements in the yellow locus influence the insulation effect of the gypsy su(Hw)-binding region in Drosophila melanogaster

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).

The lawc gene is a new member of the trithorax-Group that affects the function of the gypsy insulator of Drosophila

Mutations in the lawc gene result in a pleiotropic phenotype that includes homeotic transformation of arista into leg. lawc mutations enhance the phenotype of trx-G mutations and suppress the phenotype of Pc mutations. Mutations in lawc affect homeotic gene transcription, causing ectopic expression of Antennapedia in the eye-antenna imaginal disc. These results suggest that lawc is a new member of the trithorax family. The lawc gene behaves as an enhancer of position-effect variegation and interacts genetically with mod(mdg4), which is a component of the gypsy insulator. In addition, mutations in the lawc gene cause alterations in the punctated distribution of Mod(mdg4) protein within the nucleus. These results suggest that the Lawc protein is involved in regulating the higher-order organization of chromatin (Zorin, 1999).

As expected from its properties as a trx-G gene, mutations in lawc affect the expression of homeotic genes. The observation that the lawc/Df background leads to the reduction of some homeotic gene products (Ubx, Lab, Dfd) and not others (Scr, Antp) is not exceptional. In the case of ash-2, the level of Antp is not reduced in the first leg disc, and there is no change in the level of Ubx expression in the CNS, although there is patterned loss in the haltere and third leg disc. ash-2 also causes a reduction of Scr in the first leg disc. ash-1 mutations lead to a reduction of Antp in the first leg disc and lower levels of Ubx in the CNS, but only variable loss of Scr in the first leg disc. Because the trx-G gene products appear to form a complex, it is possible that different trx-G gene proteins interact with different homeotic genes. In forming this complex, some trx-G products such as Trl might bind to DNA, whereas others bind to each other. trx-G members are diverse and range from transcription factors, such as the Trl GAGA factor, to putative nucleosome displacement factors, such as Brahma. trx-G proteins could exert their effects on gene expression at various levels in the process of regulating transcription. Some trx-G products must have a general role in transcription because they bind to many sites on polytene chromosomes, other than the sites of homeotic genes (Zorin, 1999 and references).

The gypsy insulator of Drosophila affects chromatin structure in a directional manner

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).

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

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).

The Mod(mdg4) component of the Su(Hw) insulator inserted in the P transposon can repress its mobility in Drosophila melanogaster

Transposable element P of Drosophila melanogaster is one of the best-characterized eukaryotic transposons. Successful transposition requires the interaction between transposase complexes at both termini of the P element. Full-length P elements are 2.9 kb and encode an 87-kD transposase protein. P-element transposition requires ~150 bp of sequence at each end of the P element. These sequences include 31-bp terminal inverted repeats, internal transposase-binding sites, and internal 11-bp inverted repeats. Insertion of one or two copies of the Su(Hw) insulator in the P transposon reduces the frequency of its transposition. Inactivation of a Mod(mdg4) component of the Su(Hw) insulator suppresses the insulator effect. Thus, the Su(Hw) insulator can modulate interactions between transposase complexes bound to the ends of the P transposon in germ cells (Karakozova, 2004).

The results show that the Su(Hw) insulator affects the P transpositions in germ cells. It is most likely that the Su(Hw) insulator interferes with the interaction between protein complexes bound to the ends of the P transposon. Previous studies have shown that the loss of isform Mod(mdg4)-67.2, which interacts with the enhancer-blocking domain of the Su(Hw) protein, attenuates enhancer blocking by the Su(Hw) insulator at some genes but not at others. In several cases, the absence of Mod(mdg4)-67.2 converts the Su(Hw) insulator into a repressor. These data suggest that the Mod(mdg4)-67.2 isoform is involved in only some Su(Hw) insulator functions. In contrast, Mod(mdg4)-67.2 fulfills the main role in the repression of P transpositions. According to the accepted structural models, the putative interaction between BTB domains of Mod(mdg4)-67.2 is required for generation by the Su(Hw) insulators of looped chromatin domains that preclude interactions between regulatory elements residing in distinct domains. From this viewpoint, a plausible explanation of the inability of paired, closely spaced Su(Hw) insulators to block enhancer-promoter communication is that they would preferentially interact with each other. This local interaction precludes the paired Su(Hw) insulators from interacting with other Su(Hw) insulators, which is necessary to separate the enhancer from the promoter (Karakozova, 2004).

This study shows that, in contrast to the neutralization of the enhancer blocking, pairing between two Su(Hw) insulators located between the ends of the P transposon does not significantly neutralize the repression of P transpositions. Thus, it is most likely that Mod(mdg4)-67.2 directly blocks the interaction between protein complexes bound to the ends of the P element. As shown for the BTB-containing PZLF and Bcl6 proteins, a charge pocket, formed by apposition of the two monomers, represents a molecular structure involved in recruitment of transcriptional repression complexes. It is possible that the Mod(mdg4)-67.2 dimers either directly interact with transposase complexes or recruit other proteins that interfere with the formation of the transposase complexes at the ends of the P transposon. Alternatively, Mod(mdg4)-67.2 might interact with proteins bound to the promoter region of the P transposon. Since the transposase binds to the site overlapping the promoter, the assumed interaction between the promoter complex and Mod(mdg4)-67.2 might interfere with the transposase binding to the P transposon. Further molecular study is required for understanding the molecular basis of the described phenomenon (Karakozova, 2004).

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).

Modifier of Mdg4 is required in male meiosis for the maintenance of homolog conjunction

In Drosophila males, homologous chromosomes segregate by an unusual process involving physical connections not dependent on recombination. Two meiotic proteins specifically required for this process have been identified. Stromalin in Meiosis (SNM) is a divergent member of the SCC3/SA/STAG family of cohesin proteins, and Modifier of Mdg4 in Meiosis (MNM) is one of many BTB-domain proteins expressed from the mod(mdg4) locus. SNM and MNM colocalize along with a repetitive rDNA sequence known to function as an X-Y pairing site to nucleolar foci during meiotic prophase and to a compact structure associated with the X-Y bivalent during prometaphase I and metaphase I. Additionally, MNM localizes to autosomal foci throughout meiosis I. These proteins are mutually dependent for their colocalization, and at least MNM requires the function of teflon, another meiotic gene. SNM and MNM do not colocalize with SMC1, suggesting that the homolog conjunction mechanism is independent of cohesin (Thomas, 2005).

The two mnm alleles Z3-3298 and Z3-5578 mutations proved to be allelic to modifier of mdg4 (mod[mdg4]), a complex locus that encodes more than 30 different proteins by alternative splicing. The proteins share a 402-residue N terminus containing a BTB domain but have different, albeit similar, C termini, each encoded by one or two specific exons in the variable region of the gene. Most C termini encode a conserved C2H2 motif. Mutations in mod(mdg4) exhibit a variety of recessive and dominant phenotypes, including recessive lethality, disorganized neuromuscular synapses, modification of gypsy-induced mutations, enhancement of PEV, and others, but no meiotic phenotypes have been previously described (Thomas, 2005).

Mod(mdg4)67.2, the only functionally characterized isoform, binds along with the zinc-finger protein Su(Hw) to chromatin insulator elements in the gypsy retrotransposon and is required for insulator function. It localizes to hundreds of sites in polytene chromosomes, most of which are not within gypsy elements. In diploid cells, these coalesce into 15-25 perinuclear foci that anchor multiple chromatin loops that have been suggested to constitute functionally independent chromatin domains. Other Mod(mdg4) isoforms exhibit different chromosome localization patterns, suggesting that the variable C termini function to determine chromosome localization (Thomas, 2005).

Sequencing of genomic DNA revealed that Z3-5578 and Z3-3298 contain single mutations within 100 base pairs of each other in the C-terminal coding exon of the DOOM/Mod(mdg4)56.3/MNM isoform. Both mutations disrupt the C2H2 motif within the partially conserved C-terminal FLYWCH domain present in most Mod(mdg4) isoforms. Z3-3298 changes the first histidine residue into a tyrosine, and Z3-5578 truncates the protein upstream of the motif. A transgene expressing only MNM/Mod(mdg4)56.3 was found to fully suppress the meiotic phenotypes of both alleles, indicating that the MNM isoform is solely responsible for their meiotic effects (Thomas, 2005).

Single base mutations resulting in five other Z3 alleles were identified within CG13916. All five mutations are predicted to disrupt the coding sequence by introducing nonsense or missense codons or by disrupting a splice signal. A transgene containing wild-type CG13916 genomic sequences suppressed X-Y NDJ in Z3-0317/Z3-2138 males to a frequency below 0.5%, confirming that the identified mutations in CG13916/snm are responsible for the meiotic phenotypes (Thomas, 2005).

snm is predicted to encode a 973 amino acid protein with 27% identity to D. melanogaster Stromalin (SA), one of four components (along with SMC1, SMC3, and Rad21) of the mitotic cohesin complex responsible for sister chromatid cohesion. To determine the evolutionary relationships among SNM, SA, and other SCC3/STAG family proteins, a phylogenetic analysis of SCC3/SA family protein sequences was conducted, including two previously described meiosis-specific SCC3 proteins: REC11 of S. pombe and STAG3 of mice and humans. The phylogenetic analysis revealed that the three meiotic SCC3/SA family members (SNM, REC11, and STAG3) are more closely related to their respective mitotic paralogs (SA, PSC3, and STAG1/STAG2) than to each other, indicative of independent evolutionary origins for the meiotic SCC3/STAG proteins (Thomas, 2005).

Two additional observations support the hypothesis of an independent origin for snm. (1) Only one SCC3 family member is present in the completed Anopheles genome. Since Anopheles, like Drosophila, belongs to the order Diptera, this observation suggests that snm arose via a duplication event within the Dipteran lineage after the divergence of Anopheles. (2) Three of the five snm introns are located at matching sites in the Drosophila snm and SA genes, consistent with the notion that the two genes arose by a relatively recent duplication event (Thomas, 2005).

The mechanisms underlying pairing and segregation of achiasmate homologs in male Drosophila have eluded geneticists for nearly a century largely because, until recently, no genes required for this pathway had been identified. Numerous candidate genes involved in homology-dependent pathways such as DNA repair, meiotic recombination, synapsis, and even distributive disjunction (a female meiosis-specific pathway for segregating achiasmate bivalents) have proven dispensable for homolog segregation in male meiosis, highlighting the uniqueness of this pathway (Thomas, 2005).

However, a screen of the Zuker collection of EMS-mutagenized 2nd and 3rd chromosomes for mutations that cause elevated frequencies of paternal chromosome 4 loss has led to the identification of three genes specifically required for homolog segregation in male meiosis. teflon is required for the segregation of autosomes but dispensable for X-Y segregation, whereas the two genes described in this report, snm and mnm, have similar but more global roles, being required for proper segregation of all four pairs of homologs. The primary phenotype of mutations in both genes is a high frequency of univalents at PM I and Meta I, leading to virtually random assortment of homologs (Thomas, 2005).

The meiotic phenotypes of snm and mnm mutants indicate a critical role for SNM and MNM in establishing or maintaining interhomolog bonds but do not discriminate between direct versus indirect, i.e., regulatory, roles. A role in regulating expression of other meiotic genes seems plausible a priori for MNM since other mod(mdg4) mutations display a broad range of regulatory phenotypes including modifying gypsy-induced mutations, enhancing PEV, misregulating homeotic genes, and disrupting morphology of neuromuscular synapses. However, the intracellular localization patterns of SNM and MNM do not support an indirect role in homolog conjunction. Both SNM and MNM localize most prominently to the nucleolus during G2, where a role in promoting expression of other meiotic genes seems unlikely. They remain associated with condensed meiotic chromosomes, although such chromosomes have been shown to be transcriptionally inactive. Even more telling, both the X-Y spot and the autosomal foci disappear at the Meta I-Ana I transition, arguing strongly for direct roles of SNM and MNM in conjoining achiasmate bivalents (Thomas, 2005).

Previous results have established an important role for cis-acting 'pairing sites' in segregation of achiasmate homologs, especially for the X-Y pair. Proteins that function directly in conjoining homologs might be expected to localize to those sites. Indeed, it was found that the prominent SNM and MNM foci on the X-Y bivalent at PM I completely overlap the FISH signal from the 240 bp IGS repeats, which are the main X-Y pairing sites. Moreover, G2 nucleoli exhibited strong, albeit imperfect colocalization of SNM and MNM with the 240 bp repeat signal. This observation strongly indicates that SNM and MNM localize to the nucleolus to bind to the X-Y pairing sites (Thomas, 2005).

The data suggest that MNM and SNM may localize to the autosomes by a different mechanism than to the X-Y pair. In tef males, MNM-GFP accumulates to reasonably normal levels in the nucleolus during G2 and on the X-Y bivalent during PM I, but no hint was seen of an autosomal MNM-GFP focus at any stage. Thus TEF is required for autosomal recruitment or stabilization of MNM (and perhaps SNM) but not for their recruitment to the X-Y pair. The basis for this difference is unknown. One possibility is that SNM or MNM recognizes and binds to a sequence within the 240 bp repeat DNA or RNA that is not present on autosomes. An alternative is that a nucleolar protein different from TEF is responsible for recruiting SNM and MNM to the sex chromosomes (Thomas, 2005).

The molecular basis for chromosomal localization of SNM and MNM is unknown. The fact that SNM is required for stable nucleolar and chromosome localization of MNM during G2, but not vice versa, might suggest a primary role of SNM in localizing to pairing sites. However, it is unclear whether SNM can actually associate with pairing sites on its own, as opposed to localizing in their vicinity. MNM provides some function essential for stable localization following condensation, a function dependent upon integrity of the zinc-fingerlike C2H2 motif at its C terminus. It will be important to ascertain what this motif interacts with (Thomas, 2005).

The identification of SNM and MNM sets the stage for the molecular characterization of the interhomolog bonds they mediate. One model is considered in this study: that homologs are connected by a meiosis-specific cohesin complex involving the SCC3/SA homolog SNM and its partner, MNM. A new antibody against the cohesin protein SMC1 gave robust staining of the heterochromatic domains of meiosis I chromosomes. However, SMC1 staining did not colocalize to a significant degree with MNM within the nucleolus or on the X-Y bivalent, whereas SNM and MNM colocalize at those sites almost perfectly. While the presence of subthreshold amounts of SMC1 within the SNM-MNM domain on the X-Y bivalent cannot be ruled out, the data argue against the idea that most of the SNM and MNM proteins on the X-Y bivalent are components of a cohesin complex involving equimolar ratios of cohesin proteins (Thomas, 2005).

An alternative is that SNM and MNM connect homologs directly, perhaps utilizing the BTB domain of MNM. BTB domains are potent dimerization/multimerization domains found in many transcriptional regulatory proteins. Interactions among Mod(mdg4) proteins through these domains is thought to underlie the formation of coalesced 'insulator bodies' in somatic nuclei. It is speculated that interactions of this type among multiple SNM-MNM complexes attached to allelic chromosomal sites might provide a cohesive force sufficient to prevent dissociation of achiasmate homologs. This model is broadly consistent with cytological observations. MNM localizes during early G2 to multiple dispersed nucleolar and chromosomal foci, but to only one or two larger foci per PM I bivalent. It is speculated that this change reflects coalescence of the foci during the G2-M transition, driven by the self-associative potential of the BTB domains of MNM (Thomas, 2005).

The presence of SNM and MNM on chromosomes until the programmed breakdown of bivalents at the Meta I-Ana I transition implies that they are required at least for the maintenance of homolog conjunction. SNM and MNM might also participate directly in initiating conjunction as they are present on meiotic chromosomes by the earliest stages of meiotic prophase. However, it is not known when conjunction is initiated or, except for the X-Y pair, where the conjunction sites on chromosomes are located. The fact that individual euchromatic loci are unpaired while still constrained to a chromosome territory in wild-type has stimulated the suggestion that conjunction occurs only at discrete heterochromatic domains. The data neither support nor refute this model but add a concrete prediction, i.e., that heterochromatic conjunction during late G2 (which might be detectable by FISH or by the GFP-LacI method) should be disrupted by snm and mnm mutations (Thomas, 2005).

The results also suggest an alternative model: that true conjunction does not occur at all until the onset of chromosome condensation and that instead the homologs are maintained in proximity of one another during late prophase by transient homologous interactions at MNM-SNM foci distributed throughout the euchromatin (and perhaps the heterochromatin as well). This would be consistent with the findings that homologous loci wander farther from each other and that DAPI-stained territories are broader and more diffuse in snm and mnm mutants than in wild-type. Coalescence of the foci within each homologous territory at the G2-M transition would then underlie the conjunction (Thomas, 2005).

The data indicate that SNM originated from an SA-like homolog but lost its ancestral function in cohesion and evolved a novel function in stabilizing achiasmate bivalents not involving other cohesins. The absence of an SNM ortholog in the genome of the Dipteran Anopheles giambiae suggests that SNM originated within the Dipteran lineage, although additional insect sequences will be needed to verify this hypothesis. It will be of considerable interest to date the SA-SNM split as accurately as possible. Anopheles belongs to the Nematocera, the 'lower' Dipteran suborder; some members of this suborder, including mosquitoes such as Aedes aegypti, exhibit unmistakable evidence for chiasmate meiosis in both sexes, including well-developed SC in primary spermatocytes. However, males of Drosophila species, which belong to the 'higher' Dipteran suborder Brachycera, are all achiasmate and lack SC. The duplication that gave rise to snm could have played a major role in the evolution of achiasmy within the Diptera by permitting development of a mechanism for creating stable bonds between achiasmate homologs (Thomas, 2005).

The role of the Suppressor of Hairy-wing insulator protein in Drosophila oogenesis

The Drosophila Suppressor of Hairy wing [Su(Hw)] insulator protein has an essential role in the development of the female germline. This study investigated the function of Su(Hw) in the ovary. Su(Hw) is universally expressed in somatic cells, while germ cell expression is dynamic. Robust levels accumulate in post-mitotic germ cells, where Su(Hw) localization is limited to chromosomes within nurse cells, the specialized cells that support oocyte growth. Although loss of Su(Hw) causes global defects in nurse cell chromosome structure, it was demonstrated that these architectural changes are not responsible for the block in oogenesis. Connections between the fertility and insulator functions of Su(Hw) were investigated through studies of the two gypsy insulator proteins, Modifier of (mdg4)67.2 (Mod67.2) and Centrosomal Protein of 190kDa (CP190). Accumulation of these proteins is distinct from Su(Hw), with Mod67.2 and CP190 showing uniform expression in all cells during early stages of oogenesis that diminishes in later stages. Although Mod67.2 and CP190 extensively co-localize with Su(Hw) on nurse cell chromosomes, neither protein is required for nurse cell chromosome development or oocyte production. These data indicate that while the gypsy insulator function requires both Mod67.2 and CP190, these proteins are not essential for oogenesis. These studies represent the first molecular investigations of Su(Hw) function in the germline, which uncover distinct requirements for Su(Hw) insulator and ovary functions (Baxley, 2011).

Thus, during oogenesis somatic cells uniformly express Su(Hw). In contrast, germ cell accumulation is temporally and spatially regulated. Robust accumulation of Su(Hw) begins upon formation of egg chambers, where Su(Hw) localization is restricted to nurse cell (NC) nuclei and is absent from the oocyte nucleus, a distribution that is maintained throughout oogenesis. Other nuclear factors do not show dynamic expression in the germarium, suggesting that regulated accumulation of Su(Hw) may be important for its role in oogenesis (Baxley, 2011).

Phenotypic defects caused by loss of Su(Hw) were investigated through studies of ovaries obtained from females carrying different heteroallelic combinations of su(Hw) mutant alleles. These investigations demonstrate that NC chromosome development is delayed, but not blocked as previously reported. Structural defects in NC chromosomes appear to be independent of known genes involved in NC chromosome development, because transcription of these genes was largely maintained in su(Hw) null backgrounds. Finally, these studies addressed the long-standing hypothesis that the sterility in su(Hw) mutant females is caused by retention of the five-blob chromosome state that affects ribosome biogenesis, oocyte growth, and activates apoptosis. This study demonstrated that rRNA processing occurs normally in su(Hw) mutants, suggesting that ribosome biogenesis is not impaired. Further, it was discovered that NC chromosomes never disperse in ovaries obtained from fertile su(Hw)f/v females, even though oocyte growth, rRNA processing and fecundity are wild type. These observations establish that decondensation and dispersal of NC chromosomes is not the cause of sterility in su(Hw) mutants (Baxley, 2011).

The uncoupling of the NC structural defects and Su(Hw) dependent fertility raises the question of which cells require Su(Hw) function for completion of oogenesis. At present, it is not known whether the essential function resides in NCs or the surrounding follicle cells, as these latter cells provide signals to the germline needed for egg chamber development. Additional investigations are required to resolve this issue (Baxley, 2011).

Mod67.2 and CP190 are BTB-domain proteins that are required for enhancer blocking by the gypsy insulator. The role of these proteins in oogenesis was investigated, to gain insights into the connections between the insulator and fertility functions of Su(Hw). It was found that during oogenesis Mod67.2 and CP190 show parallel accumulation, with these BTB-domain proteins found in all somatic and germ cells, including cells that lack Su(Hw) such as the oocyte nucleus. In early egg chambers, Mod67.2 and CP190 extensively co-localize with Su(Hw), while older egg chambers display diminished levels. NC chromosome association of Mod67.2 and CP190 is largely dependent on Su(Hw), although both proteins retain NC chromosome binding in su(Hw) mutants. These findings are consistent with genome-wide studies of protein binding in somatic cells that show that chromosome association of Mod67.2 and CP190 does not always overlap with Su(Hw) binding sites (SBSs) (Baxley, 2011).

Extensive co-localization of Su(Hw), Mod67.2 and CP190 is present in stages of oogenesis where the su(Hw) mutant phenotype becomes evident. Even so, null or nearly null mod(mdg4) and Cp190 single and double mutant females lay eggs of normal size. No evidence was observed for defects in NC chromosome development or increased apoptosis, implying that Mod67.2 and CP190 are not required for oogenesis. These observations imply that the fertility and insulator functions of Su(Hw) are different. Such findings may be explained if lower levels of BTB domain proteins are needed for oogenesis than are needed to establish an insulator. It is noted that while mod(mdg4) allele was used that fails to produce any of the Mod67.2 isoform, the Cp190 heteroallelic combinations studied were hypomorphic, because null alleles are pharate lethal. As such, Cp190 mutant ovaries may have enough CP190 activity to support Su(Hw) functions. However, CP190 null embryos have been generated from germline clones, implying that oogenesis is not blocked when CP190 is absent from germ cells. These data indicate that Mod67.2 and CP190 are not essential for oogenesis. It is predicted that Su(Hw) has Mod67.2 and CP190 independent functions. Support for this postulate comes from genome-wide studies that demonstrate that ~ 50% of SBSs do not bind Mod67.2 or CP190 (Baxley, 2011).

The different requirements for Su(Hw) in insulation and fertility raise the question of whether the essential role of Su(Hw) in oogenesis involves formation of chromatin insulators. It is predicted that if insulator function is involved, then novel interaction partners may be required for Su(Hw) to demarcate chromatin domains. Alternatively, Su(Hw) function may extend beyond that of an insulator protein, a possibility that is supported by recent genome wide studies of SBSs. The vast majority of non-gypsy SBSs contain a single motif, in contrast to the twelve Su(Hw) binding motifs found in the gypsy insulator. This observation is striking considering that enhancer blocking by the gypsy insulator requires at least four tightly spaced SBSs. Direct tests of the insulator activity of individual SBSs in transgene assays have shown that ~ 40% block enhancer action, suggesting that not all SBSs are insulators. If the formation of chromatin insulators by Su(Hw) is not required for fertility, then how does this protein contribute to nuclear functions during oogenesis? It is possible that Su(Hw) has the capacity to directly to modulate transcription of target genes. For example, studies of the function of one SBS revealed that this SBS was required for activation of transcription of the adjacent gene. Further, a repressor activity is suggested by genome-wide studies that correlate Su(Hw) localization with repressive chromatin and gene silencing. Interestingly, diverse regulatory functions have been documented for the major vertebrate insulator protein, CCCTC binding factor (CTCF). While CTCF is best known as an insulator protein, early studies of CTCF documented direct involvement in transcriptional activation and repression. More recent genetic studies in transgenic mice provide additional support for direct regulation of gene expression. These observations suggest that Su(Hw) may be similar to CTCF, functioning as a multi-faceted transcriptional regulator (Baxley, 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).

RNA helicase Belle/DDX3 regulates transgene expression in Drosophila

Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. This study reports a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dicer-1 (Dcr-1) also alleviates the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally genetic inhibition of Bel function was shown to lead to de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited (Lo, 2016).

Transgene silencing refers to the activity of various host defense responses that ordinarily act on natural foreign or parasitic sequences such as transposable elements (TEs), viroids, RNA and DNA viruses, and bacterial DNA. Since transgenes or their transcripts can resemble these cellular invaders in a number of ways, they naturally become the targets of host protective reactions. There are at least two distinct host defense systems responsible for silencing transgenes. One performs its effect via de novo DNA methylation at the genome level. The second defense system operates post-transcriptionally to silence transgenes, which involves sequence-specific RNA degradation in the cytoplasm. Therefore, transgene silencing involves complex cell immune systems including epigenetic and RNA silencing mechanisms. Although many factors involved in transgene silencing have been identified, and several mechanisms have been proposed, there remains much to understand regarding this vital aspect of the cell immune system (Lo, 2016).

Drosophila oogenesis, which involves the generation of the female gamete (oocyte), nurse cells, and follicle cells, is an excellent system for the study of TE and transgene silencing. The egg chamber, the developmental unit of oogenesis, contains the germline cells (one oocyte and 15 nurse cells) and a layer of surrounding somatically derived epithelial follicle cells. Both the germline cells and follicle cells can produce small RNAs to silence TE expression. The nuage, a perinuclear structure within Drosophila nurse cells, is an RNA-rich organelle unique to the germline. The nuage is required for the processing and localization of germline mRNAs and for the biogenesis of PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs that function as the cell immune system for silencing TEs. In D. melanogaster, most primary piRNAs are produced from discrete pericentromeric and telomeric heterochromatic loci (called piRNA clusters) containing damaged repeated TE sequences. In fly germline cells, an additional step of piRNA biogenesis, the 'ping-pong cycle' mechanism, is employed to generate the secondary piRNAs. Multiple factors localized in the nuage of germline cells have been discovered to be essential for secondary piRNA biogenesis, including Aub, AGO3, Spindle-E (Spn-E), and Vasa. In follicle cells, piRNAs are only produced from piRNA clusters (e.g., flamenco ) via PIWI and other related nuclear factors, and there is no secondary piRNA biogenesis involved. Intriguingly, besides piRNA clusters, euchromatic transposon insertion sites have been identified as another origin to produce piRNAs and endo-siRNAs. This mechanism provides another layer of defense to suppress TE activity and can also serve as a way to affect expression of coding genes and microRNA (miRNA) genes adjacent to inserted TEs (Lo, 2016).

Vasa and Spn-E belong to a family of DEAD-box proteins defined by multiple distinct conserved motifs including the D-E-A-D (Asp-Glu-Ala-Asp) motif. Among the identified DEAD-box proteins, one subfamily is highly conserved from yeast to human, which includes orthologs in yeast (DED1), Drosophila (Belle (Bel)), Xenopus (An3), mice (PL10), and humans (DDX3). These DEAD-box subfamily proteins possess the ATP-dependent RNA helicase activity to unwind double-stranded RNA and remodel RNA-protein interactions. Yeast DED1 is a multifunctional protein that functions to regulate multiple stages of RNA processing and translation. DED1 has also been shown to play a specific role in cell-cycle control. DDX3, the human homolog of DED1, is known to be involved in modulating multiple biological processes, including antiviral innate immunity, mitotic chromosome segregation in somatic cells, the suppression of spermatogenesis, G1-S transition of the cell cycle, epithelial-mesenchymal transition (EMT), a bona fide component of the RNAi pathway, TNF-related apoptosis, and WNT signaling (Lo, 2016 and references therein).

Vasa, a paralog of Bel, is required for the formation and function of nuage to suppress TE expression by being involved in the production of piRNAs. Most recently, it has been reported that Vasa is a key component in the piRNA amplifier complex in the nuage and serves as a protein platform to recruit PIWI proteins, the Tudor protein Qin/Kumo and antisense piRNA guides in an ATP-dependent manner for the ping-pong-loop amplification of secondary piRNAs. Bel colocalizes with Vasa in the nuage and at the oocyte posterior during oogenesis, and is required for female fertility. Recent findings have shown that loss of bel delays activation of Notch signaling in follicle cells, which in turn leads to delayed cell differentiation and defects in the switch from the mitotic cycle to the endocycle. However, unlike Vasa, the specific roles of Bel in the nuage of germline cells, and whether it is involved in piRNA biogenesis, remain unknown (Lo, 2016).

From previous studies, it was unexpectedly found that the Gal4-driven expression of a UASp-Bel:GFP transgene was silenced in bel mutant germline cells. This silencing effect was not specific to bel-based transgenes because 13 out of 22 different transgenic lines tested could be silenced in either germline or somatic bel mutant cells, or both. Subsequently the RNA helicase Spn-E, the epigenetic regulator Modifier of mdg4 [Mod(mdg4)] and the miRNA biogenesis enzyme Dcr-1 was identified as crucial factors for this bel-related transgene silencing. Their abrogation could either partially or completely rescue the transgene-silencing phenotype induced by loss of bel. Importantly, small RNA deep sequencing analysis suggests that a piRNA-mediated mechanism is potentially involved in Bel-inactivation-induced transgene silencing. Together, these studies genetically link the function of Bel to Spn-E, Mod(mdg4), and Dcr-1, and suggest that transgene silencing induced by Bel inactivation may involve RNA processing, piRNA, miRNA, and epigenetic mechanisms (Lo, 2016).

This article reports that loss-of-bel function triggers transgene silencing, which occurs through reduction in transgene RNA levels. Furthermore, genetic studies indicate that this transgene silencing effect induced by bel abrogation requires the RNA helicase Spn-E, the insulator modulator Mod(mdg4) and/or the miRNA biosynthesis enzyme Dcr-1. Based on the functional roles of these three molecules, the data suggest that this transgene silencing effect may involve RNA processing, chromatin remodeling and/or miRNA biogenesis. This transgene silencing event occurring under various bel mutant backgrounds implies that Bel may regulate these three molecular mechanisms to sustain transgene expression in the normal physiological condition. Intriguingly, these studies also identified additional complexity in the relationship between Bel and Spn-E because the mislocalization of nuage components in spn-E mutants requires Bel. Therefore, these findings, taken together, provide new insight into Bel function and expand the molecular interaction network radiating from Bel (Lo, 2016).

These studies show that loss of bel gave rise to transgene silencing via decreased transgene RNA levels. This phenomenon could be attributable to either transcriptional suppression or increased RNA degradation. Some support for an RNA degradation/targeting mechanism comes from the finding that Spn-E is required for transgene silencing induced by loss of bel. In germline nurse cells, Spn-E, which is located in the cytoplasmic nuage, is crucial for properly maintaining the subcellular localization of piRNA-related protein factors in the nuage, the ping-pong reaction of piRNA biogenesis, and silencing of TEs. Spn-E is also required for the proper localization of RNA transcripts (e.g., Bicoid and Oskar) during oogenesis, which might be related to its role in organizing a cytoskeletal framework. Therefore, it is plausible that the Spn-E-dependent RNA processing activity and/or Spn-E-generated piRNAs mediate transgene RNA degradation. In addition, it is possible that piRNAs can also elicit transcriptional silencing of transgenes based on their nuclear epigenetic role in TE silencing. Another striking finding from these studies shows that Bel is involved in disrupting the subcellular localization of nuage components when Spn-E is abrogated. In contrast, loss of Bel alone had no impact on the localization of piRNA-related nuage protein components. These findings suggest that there could be a competitive relationship between Bel and Spn-E, where these two molecules negatively regulate each other. This hypothesis is also supported by observations that Spn-E is required for transgene silencing when Bel is abrogated. According to these findings, a model is envisioned that Bel may function as a negative regulator for ping-pong-cycle-mediated piRNA biogenesis via disrupting the nuage localization of piRNA-related proteins as Spn-E function is abrogated, whereas Spn-E may be aberrantly activated by loss of Bel, in turn leading to transgene silencing. Whether piRNAs generated from the Spn-E-mediated ping-pong cycle are involved in transgene silencing is currently unknown. However, a recent study has shown that piRNAs can be generated directly from the transposon-derived transgene insertion area located in the euchromatic genome, which have been proposed to be involved in transgene silencing. This finding links piRNAs to transgene silencing. Preliminary small RNA deep sequencing analysis showed that the viable trans-heterozygous bel74407/neo30 mutant ovaries, which manifest a partial transgene silencing phenotype, displayed no significant defect in overall piRNA biogenesis from piRNA clusters and the ping-pong cycle, indicating that unlike Vasa, Bel is not involved in regulating piRNA generation. This finding is consistent with the result that homozygous bel mutants had no defect in nuage protein localization, which is different from other piRNA-related nuage proteins whose defects can significantly disturb the localization of other nuage protein components. Nevertheless, deep sequencing analysis also identified de novo piRNAs generated in bel74407/neo30 mutant ovaries (but not in wild-type ovaries) that could be mapped to the integrated P-element-derived transgene sequence area (P[LacW]). This result is in line with previous findings and indicates that the de novo generation of piRNAs from the inserted transgene region in the genome occurs under the bel mutant background. Given that Bel is a paralog of Vasa and the current genetic findings also suggest a competitive relationship between Bel and Spn-E, loss of Bel may disrupt its normal regulation of some small RNA-related helicases and co-factors, which in turn aberrantly activates the small RNA pathway(s). Therefore, it is possible that loss of Bel may promote de novo piRNA biogenesis from the transgene insertion sites by freeing these small RNA regulators and provoking the activation of their related small RNA pathway(s), which is one possible mechanism leading to transgene silencing. Since it was not possible create viable mutant progeny bearing mutations at both bel and spn-E gene loci, it is still uncertain whether Spn-E is involved in regulating de novo piRNA biogenesis from transgene insertion sites. Although it was not possible to elucidate the detailed mechanism due to technical hurdles, exploring the regulatory roles of Bel and Spn-E in this new type of piRNA biogenesis will be key, interesting research for understanding molecular mechanisms underlying transgene silencing (Lo, 2016).

Another unexpected finding is that the spn-E mutant rescue of transgene silencing associated with loss of bel also occurs in somatic follicle cells. This indicates that in addition to its well-known function in germline cells, Spn-E may have a somatic function. As Spn-E is not implicated in somatic piRNA biogenesis (e.g., flamenco), it is uncertain how Spn-E mediates somatic transgene silencing and whether piRNAs participate in this event. Therefore, it will be important in the future to unravel whether the mechanism by which Spn-E facilitates transgene silencing in bel mutant somatic cells is the same as that in germline cells (Lo, 2016).

The common feature for transgenes silenced by Bel abrogation is their P-element-based integration into genomic DNA. The observation that some P-element-based transgenes were not silenced in bel mutant cells raises an interesting question about what factors can determine whether a transgene can be silenced or not. From genetic analysis of a series of transgenes, it was observed that six examined transgenes (ci-LacZ, dMyc-LacZ, dom-LacZ, C306-Gal4, ptc-Gal4, Tj-Gal4), which were generated by the insertion of two different P-element-derived vector sequences (P[LacW] and P[GawB]), were silenced under the bel mutant background. In contrast, two transgenes (histone-GFP, histone-RFP) generated by the insertion of P[His2Av]-derived vector sequences were not silenced by Bel inactivation. Although these data are not a conclusive result, they imply that the inserted transgene sequence itself, not the insertion location in the genome, may be a critical determinant for Bel-dependent transgene silencing since this silencing phenotype seems to be transgene-specific and a change in the transgene insertion location in the genome has no influence on whether this transgene can be silenced or not when Bel function is inhibited. It is possible that the transgene sequence determines a local chromosomal conformation and whether the transgene can be silenced under the bel mutant background is determined by whether its chromosomal structure can be recognized by epigenetic regulators involved in transgene silencing. Although further investigations are still needed to verify this hypothesis due to limited cases in this study, it raises the possibility that epigenetic regulation at the chromatin level may be involved in Bel-dependent transgene silencing. Indeed, besides Spn-E, the genetic study identified Mod(mdg4) as another crucial factor required for transgene silencing in both germline and somatic cells when Bel is abrogated. The mod(mdg4) gene encodes multiple nuclear factors through trans-splicing and this protein family is functionally involved in the modification of the properties of insulators, which are genomic elements that regulate gene expression. Mod(mdg4) proteins can function as chromatin modulators engaged in the organization of highly ordered chromatin domains. The involvement of Mod(mdg4) in transgene silencing suggests that nuclear epigenetic events are also crucial for induction of transgene silencing when Bel is inactivated. However, the role of Mod(mdg4) in transgene silencing might also be indirect, such as through its regulation of other genes that could contribute to silencing. Nevertheless, the findings suggest that the co-ordination between nuclear and cytoplasmic events mediated by Mod(mdg4) and Spn-E, respectively, is mandatory for induction of transgene silencing when Bel is functionally inhibited (Lo, 2016).

The miRNA biogenesis enzyme Dcr-1 is the third factor identified from these studies that is crucial for transgene silencing in the absence of Bel. Interestingly, the block in transgene silencing in this case (double mutant for bel and Dcr-1) only occurred in germline cells, but not in somatic cells. This discovery raises the possibility that there are additional miRNA-targeted proteins present in germline cells, but not in somatic cells, and they can interact with factors essential for germline transgene silencing. If this is a case, aberrantly elevated levels of these miRNA-targeted proteins in Dcr-1 mutant germline cells might interfere with transgene silencing. Besides this possible indirect role, another possibility is that the Dcr-1-dependent miRNA pathway may play a direct role in transgene silencing in germline cells as Bel is abrogated. The miRNA pathway has been shown to be implicated in transgene silencing in Drosophila S2 cells. Although the silencing mechanism is unclear, this finding raises a possible direct role of the Dcr-1-dependent miRNA pathway in bel-mutant transgene silencing. A study from Zhou has shown that Bel proteins were cofractionated with the miRNA-dependent RNA-induced silencing complexes (miRISCs) and co-immunoprecipitated with Ago1, the protein component of miRISCs. This finding suggests a compelling possibility that Bel may be directly involved in the miRNA pathway to regulate miRISC-dependent RNA silencing and Bel inactivation may result in the aberrant functionality of miRISC and its related RNA silencing. Since miRNAs can target mRNAs via their short seed sequences, a possibility which cannot be ruled out is that some miRNAs may directly target transgene RNAs to regulate their levels. Future investigation is needed to reveal which possibility is more relevant (Lo, 2016).

In conclusion, these findings provide novel insights into the regulatory role of Bel in the expression of transgenes in Drosophila and its functional linkage to crucial factors implicated in RNA processing, chromatin remodeling and miRNA biogenesis. These findings further advance understanding of the complex cellular functions of Bel. These studies of the role for Bel in transgene expression may have important, future implications for understanding the regulation of expression of newly invaded or transposed TEs and virus-retrotransposon DNA chimeras generated from viral infection as they may share the similar scenario as transgene integration (Lo, 2016).


Bartkuhn, M., et al. (2009). Active promoters and insulators are marked by the centrosomal protein 190. EMBO J. 28: 877-888. PubMed Citation: 19229299

Baxley, R. M., et al. (2011). The role of the Suppressor of Hairy-wing insulator protein in Drosophila oogenesis. Dev. Biol. 356(2): 398-410. PubMed Citation: 21651900

Bezborodova, E., Kulikov, A. and Georgiev, P. (1997). A new family of genes which, when mutated, suppress the inhibitory effect of the mod(mdg4)1u1 mutation on y2 expression in Drosophila melanogaster. Mol. Gen. Genet. 257(1): 83-90. PubMed Citation: 9439572

Buchner, K., et al. (2000). Genetic and molecular complexity of the position effect variegation modifier mod(mdg4) in Drosophila. Genetics 155: 141-157. PubMed Citation: 10790390

Bushey, A. M., Ramos, E., and Corces, V. G. (2009). Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions. Genes Dev. 23: 1338-1350. PubMed Citation: 19443682

Byrd, K. and Corces, V. G. (2003). Visualization of chromatin domains created by the gypsy insulator of Drosophila. J. Cell. Biol. 162(4): 565-74. 12925706

Cai, H. N. and Levine, M. (1997). The gypsy insulator can function as a promoter-specific silencer in the Drosophila embryo. EMBO J. 16: 1732-41. PubMed Citation: 9130717

Capelson, M. and Corces, V. G. (2005). The ubiquitin ligase dTopors directs the nuclear organization of a chromatin insulator. Mol. Cell 20(1): 105-16. 16209949

Capelson, M. and Corces V. G. (2006). SUMO conjugation attenuates the activity of the gypsy chromatin insulator. EMBO J. 25(9): 1906-14. Medline abstract: 16628226

Chen, S. and Corces, V. G. (2001). The gypsy insulator of Drosophila affects chromatin structure in a directional manner. Genetics 159: 1649-1658. 11779804

Chopra, V. S., Cande, J., Hong, J. W. and Levine, M. (2009). Stalled Hox promoters as chromosomal boundaries. Genes Dev. 23: 1505-1509. PubMed Citation: 19515973

Dorn, R., et al. (1993). The enhancer of position-effect variegation of Drosophila, E(var)3-93D, codes for a chromatin protein containing a conserved domain common to several transcriptional regulators. Proc. Natl. Acad. Sci. 90(23): 11376-11380. PubMed Citation: 8248257

Dorn, R., Reuter, G. and Loewendorf, A. (2001). Transgene analysis proves mRNA trans-splicing at the complex mod(mdg4) locus in Drosophila. Proc. Natl. Acad. Sci. 98: 9724-9729. 11493677

Erokhin M., Parshikov A., Georgiev P. and Chetverina D. (2010). E(y)2/Sus1 is required for blocking PRE silencing by the Wari insulator in Drosophila melanogaster. Chromosoma 119: 243-253. PubMed Citation: 20082086

Erokhin, M., et al. (2011). Insulators form gene loops by interacting with promoters in Drosophila. Development 138(18): 4097-106. PubMed Citation: 21862564

Gabler, M., et al. (2005). Trans-splicing of the mod(mdg4) complex locus is conserved between the distantly related species Drosophila melanogaster and D. virilis. Genetics 169(2): 723-36. Medline abstract: 15520256

Gause, M., et al. (1998). hobo induced rearrangements in the yellow locus influence the insulation effect of the gypsy su(Hw)-binding region in Drosophila melanogaster. Genetics 149(3): 1393-1405. PubMed Citation: 9649529

Gause, M., Morcillo, P. and Dorsett, D. (2001). Insulation of enhancer-promoter communication by a gypsy transposon insert in the Drosophila cut gene: Cooperation between Suppressor of Hairy-wing and Modifier of mdg4 proteins. Mol. Cell. Bio. 21: 4807-4817. 11416154

Gdula, D. A., and Corces, V. G. (1997). Characterization of functional domains of the su(Hw) protein that mediate the silencing effect of mod(mdg4) mutations. Genetics 145 (1): 153-161

Georgiev, P. G. and Corces, V. G. (1995). The su(Hw) protein bound to gypsy sequences in one chromosome can repress enhancer-promoter interactions in the paired gene located in the other homolog. Proc. Natl. Acad. Sci. 9295: 5184-5188

Gerasimova, T. I., et al. (1995). A Drosophila protein that imparts directionality on a chromatin insulator is an enhancer of position-effect variegation. Cell 82: 587-597

Gerasimova, T. I. and Corces, V. G. (1998). Polycomb and trithorax group proteins mediate the function of a chromatin insulator. Cell 92(4): 511-521

Gerasimova, T. I., Byrd, K. and Corces, V. G. (2000). A chromatin insulator determines the nuclear localization of DNA. Mol. Cell 6: 1025-1035. PubMed ID: 11106742

Ghosh, D., Gerasimova, T. I. and Corces, V. G. (2001). Interactions between the Su(Hw) and Mod(mdg4) proteins required for gypsy insulator function. EMBO J. 20: 2518-2527. 11350941

Golovnin, A., et al. (2003). An endogenous Su(Hw) insulator separates the yellow gene from the Achaete-scute gene complex in Drosophila. Development 130: 3249-3258. 12783795

Golovnin, A., Volkov, I. and Georgiev, P. (2012). SUMO conjugation is required for the assembly of Drosophila Su(Hw) and Mod(mdg4) into insulator bodies that facilitate insulator complex formation. J. Cell Sci. 125(Pt 8): 2064-74. PubMed Citation: 22375064

Golovnin, A., Melnikova, L., Shapovalov, I., Kostyuchenko, M. and Georgiev, P. (2015). EAST organizes Drosophila insulator proteins in the interchromosomal nuclear compartment and modulates CP190 binding to chromatin. PLoS One 10: e0140991. PubMed ID: 26489095

Harvey, A. J., Bidwai, A. P. and Miller, L. K. (1997). Doom, a product of Drosophila mod(mdg4) gene induces apoptosis and binds to baculovirus inhibitor-of-apoptosis proteins. Mol. Cell. Biol. 17: 2835-43

Hay, B. A., Wassarman, D. A. and Rubin, G. M. (1995). Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83: 1253-1262

Karakozova, M., Savitskaya, E., Melnikova, L., Parshikov, A. and Georgiev, P. (2004). The Mod(mdg4) component of the Su(Hw) insulator inserted in the P transposon can repress its mobility in Drosophila melanogaster. Genetics 167(3): 1275-80. 15280241

Kravchenko, E., et al. (2005). Pairing between gypsy insulators facilitates the enhancer action in trans throughout the Drosophila genome. Mol. Cell, Biol. 25(21): 9283-91. 16227580

Labrador, M., Mongelard, F., Plata-Rengifo, P., Baxter, E. M., Corces, V. G., and Gerasimova, T. I. (2001). Protein encoding by both DNA strands. Nature 409: 1000. 11234000

Lo, P. K., Huang, Y. C., Poulton, J. S., Leake, N., Palmer, W. H., Vera, D., Xie, G., Klusza, S. and Deng, W. M. (2016). RNA helicase Belle/DDX3 regulates transgene expression in Drosophila. Dev Biol 412: 57-70. PubMed ID: 26900887

Matzat, L. H., Dale, R. K., Moshkovich, N. and Lei, E. P. (2012). Tissue-specific regulation of chromatin insulator function. PLoS Genet 8: e1003069. PubMed ID: 23209434

Mongelard, F., et al. (2002). Trans-splicing as a novel mechanism to explain interallelic complementation in Drosophila. Genetics 160: 1481-1487. 11973303

Nabirochkin, S., Ossokina, M. and Heidmann, T. (1998). A nuclear matrix/scaffold attachment region co-localizes with the gypsy retrotransposon insulator sequence. J. Biol. Chem. 273(4): 2473-2479

Namciu, S. J., Blochlinger, K. B. and Fournier, R. E. (1998). Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster. Mol. Cell. Biol. 18(4): 2382-2391. Medline reference:

Nègre, N., et al. (2010). A comprehensive map of insulator elements for the Drosophila genome. PLoS Genet. 6: e1000814. PubMed Citation: 20084099

Pai, C. Y., Lei, E. P., Ghosh, D. and Corces, V. G. (2004). The centrosomal protein CP190 is a component of the gypsy chromatin insulator. Mol. Cell 16(5): 737-48. 15574329

Richter, C., Oktaba, K., Steinmann, J., Muller, J. and Knoblich, J. A. (2011). The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements. Nat Cell Biol 13: 1029-1039. Pubmed: 21857667

Smith, S. T., et al. (2009). Genome wide ChIP-chip analyses reveal important roles for CTCF in Drosophila genome organization. Dev. Biol. 328: 518-528. PubMed Citation: 19210964

Soshnev, A. A., He, B., Baxley, R. M., Jiang, N., Hart, C. M., Tan, K. and Geyer, P. K. (2012). Genome-wide studies of the multi-zinc finger Drosophila Suppressor of Hairy-wing protein in the ovary. Nucleic Acids Res 40: 5415-5431. PubMed ID: 22406832

Soshnev, A. A., Li, X., Wehling, M. D. and Geyer, P. K. (2008). Context differences reveal insulator and activator functions of a Su(Hw) binding region. PLoS Genet 4: e1000159. PubMed ID: 18704163

Thomas, S. E., Soltani-Bejnood, M., Roth, P., Dorn, R., Logsdon, J. M. and McKee, B. D. (2005). Identification of two proteins required for conjunction and regular segregation of achiasmate homologs in Drosophila male meiosis. Cell 123(4): 555-68. 1628600

Zorin, I. D., Gerasimovaa, T. I. and Corcesa, V. G. (1999). The lawc gene is a new member of the trithorax-Group that affects the function of the gypsy insulator of Drosophila. Genetics 152: 1045-1055. PubMed Citation: 10388823

modifier of mdg4: Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 April 2017

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