suppressor of Hairy wing
The gypsy insulator is thought to play a role in nuclear organization and the establishment of higher order chromatin
domains by bringing together several individual insulator sites to form rosette-like structures in the interphase nucleus. The
Su(Hw) and Mod(mdg4) proteins are components of the gypsy insulator required for its effect on enhancer-promoter
interactions. Using the yeast two-hybrid system, it has been shown that the Mod(mdg4) protein can form homodimers, which can
then interact with Su(Hw). The BTB domain of Mod(mdg4) is involved in homodimerization, whereas the C-terminal
region of the protein is involved in interactions with the leucine zipper and adjacent regions of the Su(Hw) protein. Analyses
using immunolocalization on polytene chromosomes confirm the involvement of these domains in mediating the interactions between these proteins.
Studies using diploid interphase cells further suggest the contribution of these domains to the formation of rosette-like structures in the nucleus. The
results provide a biochemical basis for the aggregation of multiple insulator sites and support the role of the gypsy insulator in nuclear organization (Ghosh, 2001).
The formation of loops or higher order domains of chromatin structure requires the individual insulator sites from different chromosomal locations to
come together in the nucleus. This organization must be mediated by interactions among protein components of the insulator. These
interactions are indeed possible and take place in vivo in the case of the gypsy insulator of Drosophila. Mapping the domains of the Su(Hw) and
Mod(mdg4) proteins involved in this interaction might shed light on how insulators could be involved in the establishment of higher order chromatin
organization. Disruption of the leucine zipper and regions B and C of Su(Hw) renders the gypsy insulator unable to interfere with enhancer-promoter
interactions. Results presented here indicate that disruption of this region of Su(Hw) also abolishes its interaction with Mod(mdg4) and eliminates the punctate nuclear staining pattern, suggesting that interaction between the two proteins is
required for establishing domains in the nucleus, and that the establishment of these domains correlates with the functionality of the insulator (Ghosh, 2001).
Mod(mdg4) has at least 21 different isoforms generated by alternative splicing. All the proteins contain a common N-terminus of 402 amino acids that includes a BTB/POZ domain, whereas the C-terminus of the protein is variable. Most of these Mod(mdg4) proteins are present in a few sites on polytene chromosomes and only the Mod(mdg4) 2.2 protein (the product of a splice variants, the 2.2 kb transcript that is the major form in the wild-type Canton S strain) appears to be a general component of the gypsy insulator. The Su(Hw)
protein interacts with Mod(mdg4) 2.2 through the C-terminal domain of the Mod(mdg4) 2.2 protein. Since this domain is specific to this form of the
protein and it is not present in any of the other variants, this result supports the idea that Mod(mdg4) 2.2 is the component of the gypsy insulator,
whereas other mod(mdg4)-encoded proteins might have more specific roles in the cell. Deletion of the BTB domain eliminates homodimeric
interactions between Mod(mdg4) 2.2 and results in weakened interactions between Su(Hw) and Mod(mdg4) 2.2. This result could be interpreted as
suggesting that Su(Hw) and Mod(mdg4) 2.2 interact through the BTB domain. However, this domain by itself is not able to interact with the full-length
Su(Hw) protein or with the LZ-B-C region; this is not due to incorrect folding of the protein, since the BTB domain by itself is able to fold properly
and mediate interaction with full-length Mod(mdg4) or another BTB domain. These results are interpreted to suggest that the BTB domain mediates the
formation of Mod(mdg4) 2.2 dimers, which in turn are required to mediate the interaction with Su(Hw) (Ghosh, 2001).
BTB domain-containing proteins frequently have zinc fingers involved in DNA binding. The Mod(mdg4) 2.2 protein is unusual in the sense that it does
not possess any such DNA-binding domain at the C-terminus. However, the presence of a domain that mediates interactions with Su(Hw), which
binds DNA through its zinc fingers, might serve the purpose of recruiting this protein to chromatin. The BTB domain is responsible for
self-oligomerization of proteins such as GAGA, promyelocytic leukemia zinc finger protein (PLZF) and ZID in vitro. Interestingly, although the BTB domain-containing promyelocytic leukemia zinc
finger protein appears to form only dimers in solution, a short four-stranded antiparallel ß-sheet between two symmetry-related dimers can be
observed in the crystal. This interaction involves four different peptide chains and, therefore, can give rise to the formation of
tetramers and oligomers of higher stoichiometry, suggesting that BTB-containing proteins can form large multimers. This observation is especially
significant in the context of proposed models for insulator function, which require multiple insulator sites to come together in one large aggregate. It
might be possible for Mod(mdg4) 2.2 to interact with several Mod(mdg4) 2.2 molecules, thus helping to bring together several Mod(mdg4) binding
sites to form insulator aggregates as observed in interphase diploid cells. Alternatively, Mod(mdg4) 2.2 might interact with other BTB
domain-containing proteins, which might be an integral part of the gypsy insulator complex. The BTB domain forms an extensive dimer interface that is
a possible binding site for other proteins. Since the presence of the BTB domain is only partially required for binding of Su(Hw), there might possibly
be other as yet unidentified partners of Mod(mdg4) that interact with the BTB domain. Alternatively, the BTB dimer interface might stabilize the
interaction of Su(Hw) with the C-terminal region of Mod(mdg4) (Ghosh, 2001).
The finding of specific domains of the Su(Hw) and Mod(mdg4) proteins that mediate intermolecular interactions provides a strong biochemical
foundation for the involvement of these proteins in the establishment of chromosomal loops. These loops are the basis for the proposed role of
insulators in the formation of higher order chromatin domains and nuclear organization of the chromosomes during interphase. These studies also
provide support for the involvement of other proteins in insulator function. The identification of these proteins will provide additional evidence to
understand the mechanisms by which these important sequences control eukaryotic gene expression (Ghosh, 2001).
The Drosophila mod(mdg4) gene products counteract heterochromatin-mediated silencing of the white gene and help activate genes of the bithorax complex. They also regulate the insulator activity of the gypsy transposon when gypsy inserts between an enhancer and promoter. The Su(Hw) protein is required for gypsy-mediated insulation, and the Mod(mdg4)-67.2 protein binds to Su(Hw). The aim of this study was to determine whether Mod(mdg4)-67.2 is a coinsulator that helps Su(Hw) block enhancers or a facilitator of activation that is inhibited by Su(Hw). Evidence is provided that Mod(mdg4)-67.2 acts as a coinsulator by showing that some loss-of-function mod(mdg4) mutations decrease enhancer blocking by a gypsy insert in the cut gene. The C terminus of Mod(mdg4)-67.2 binds in vitro to a region of Su(Hw) that is required for insulation, while the N terminus mediates self-association. The N terminus of Mod(mdg4)-67.2 also interacts with the Chip protein, which facilitates activation of cut. Mod(mdg4)-67.2 truncated in the C terminus interferes in a dominant-negative fashion with insulation in cut but does not significantly affect heterochromatin-mediated silencing of white. It is inferred that multiple contacts between Su(Hw) and a Mod(mdg4)-67.2 multimer are required for insulation. It is theorized that Mod(mdg4)-67.2 usually aids gene activation but can also act as a coinsulator by helping Su(Hw) trap facilitators of activation, such as the Chip protein (Gause, 2001).
This study found that certain loss-of-function alleles of mod(mdg4) reduce insulation by the Su(Hw) protein in the cut gene. This is evidence that mod(mdg4) products are not simply targets of Su(Hw) insulator activity but contribute to the insulator activity of Su(Hw). Wild-type Mod(mdg4)-67.2, the major protein product of mod(mdg4), interacts with a region of Su(Hw) that has been shown to be required for insulation in vivo, but the truncated versions of the Mod(mdg4)-67.2 proteins produced by the viable mod(mdg4)u1 and mod(mdg4)T6 alleles did not. This is consistent with the observation that binding of Mod(mdg4) proteins to Su(Hw) binding sites on salivary gland polytene chromosomes is greatly reduced in mod(mdg4)u1 mutants. mod(mdg4)u1 and mod(mdg4)T6 more strongly reduce insulator activity than do null alleles of mod(mdg4) and that this antimorphic nature of mod(mdg4)u1 may stem from the ability of the mutant protein to interact with wild-type Mod(mdg4)-67.2 protein. To explain these observations, a model is proposed in which a multimer of Mod(mdg4)-67.2 interacts with more than one Su(Hw) molecule to form the active insulator complex, and the truncated Mod(mdg4)-67.2 proteins produced by mod(mdg4)u1 and mod(mdg4)T6 destabilize this complex (Gause, 2001).
The evidence that Mod(mdg4)-67.2 is an active component of the gypsy insulator that blocks gene activation appears at first glance to be contradictory to the evidence indicating that the mod(mdg4) gene is a member of the trxG of genes that activate genes in the bithorax complex. Another trxG protein, however, also appears to have insulator activity. The GAGA factor encoded by the Trithorax-like (Trl) gene is similar to Mod(mdg4)-67.2 in that it contains a BTB/POZ motif at the N terminus, self-interacts, and supports activation of the bithorax complex. GAGA factor is also required for enhancer blocking by the insulator associated with the even-skipped promoter. This insulator activity requires GAGA binding sites just proximal to the transcription start site and is diminished by Trl mutations. Potential GAGA binding sites are found just proximal to many promoters in Drosophila, including sequences associated with insulator activity in the alpha1 tubulin gene promoter. The GAGA-dependent insulator just proximal to the eve promoter does not prevent activation of the eve promoter by upstream enhancers even though it is positioned between them. Indeed, GAGA binding sites just proximal to the engrailed gene promoter potentiate activation by an upstream enhancer. To resolve the paradoxical insulator and activator activities of the GAGA and Mod(mdg4)-67.2 BTB/POZ proteins, therefore, it must be theorized that the function of promoter-proximal insulators is to aid activation of the promoters that contain them by helping to capture and anchor distal activator or facilitator proteins near the promoter. If so, it is feasible that the Mod(mdg4)-67.2 protein has a promoter-anchoring function in the bithorax complex, but when bound to Su(Hw), it anchors activator or facilitator proteins far from the promoter, thereby preventing activation (Gause, 2001).
Chromatin insulators, or boundary elements, affect promoter-enhancer interactions and buffer transgenes from position effects. The gypsy insulator of Drosophila is bound by a protein complex with two characterized components, the zinc finger protein Suppressor of Hairy-wing [Su(Hw)] and Mod(mdg4)2.2, which is one of the multiple spliced variants encoded by the modifier of mdg4 [mod(mdg4)] gene. A genetic screen for dominant enhancers of the mod(mdg4) phenotype identified the Centrosomal Protein 190 (CP190) as an essential constituent of the gypsy insulator. The function of the centrosome is not affected in CP190 mutants whereas gypsy insulator activity is impaired. CP190 associates physically with both Su(Hw) and Mod(mdg4)2.2 and colocalizes with both proteins on polytene chromosomes. CP190 does not interact directly with insulator sequences present in the gypsy retrotransposon but binds to a previously characterized endogenous insulator, and it is necessary for the formation of insulator bodies. The results suggest that endogenous gypsy insulators contain binding sites for CP190, which is essential for insulator function, and may or may not contain binding sites for Su(Hw) and Mod(mdg4)2.2 (Pai, 2004).
A genetic screen for dominant enhancers of mod(mdg4) has resulted in the identification of CP190 as a third component of the gypsy insulator. CP190 is present at gypsy retrotransposon insulator sites and overlaps extensively with Su(Hw) and Mod(mdg4)2.2 at presumed endogenous insulators. CP190 displays a specific distribution pattern on polytene chromosomes, showing significant overlap with Su(Hw) and Mod(mdg4)2.2 at the junctions between transcriptionally inert bands and transcriptionally active interbands. Similar localization patterns have been reported for other insulators. For example, the faswb insulator at the notch locus and the BEAF-32 protein of the scs' insulator are also present at the boundaries between bands and interbands. Results suggest that CP190 can bind DNA on its own or can be tethered to the chromosome through interactions with Su(Hw). Mutations in the CP190 gene impair the function of the insulator present in the gypsy retrotransposon without affecting the presence of Su(Hw) and Mod(mdg4)2.2, suggesting an essential task for CP190 in the activity of this insulator. In addition, the lethality of CP190 mutants suggests a critical role for the CP190 protein in the function of gypsy endogenous insulators. This essential role may be a consequence of the requirement of CP190 for the formation of insulator bodies in the nuclei of diploid cells (Pai, 2004).
The insulator present in the gypsy retrotransposon contains only Su(Hw) binding sites, and CP190 is present in this insulator through direct interactions with Su(Hw). The gypsy insulator contains 12 Su(Hw) binding sites, and at least four are needed for insulator activity. However, clusters of three or more Su(Hw) binding sites are rare in the genome. Therefore, a critical question is whether the sites of Su(Hw) and Mod(mdg4)2.2 localization present throughout the genome truly function as insulators. The presence of CP190 at these sites and its ability to bind DNA might explain this apparent paradox. For example, the endogenous insulator present in the yellow-achaete region has only two binding sites for Su(Hw). Nevertheless, the y454 fragment containing this insulator is able to bind CP190, suggesting that this protein might act in concert with Su(Hw) to confer insulator activity. It is therefore possible that endogenous gypsy insulators are composed of binding sites for Su(Hw) and/or for CP190 and, together with Mod(mdg4)2.2, form a complex. Endogenous gypsy insulators may have few or no Su(Hw) binding sites, and they may rely on CP190 to bind DNA and tether other insulator components such as Mod(mdg4)2.2 via protein-protein interactions (Pai, 2004).
Previous studies have suggested that gypsy insulators separated at a distance in the genome may come together and form large insulator bodies in the nucleus during interphase. These aggregates represent higher order structures of chromatin and are implicated in the regulation of gene expression by compartmentalizing the genome into transcriptionally independent domains. The formation of these aggregates appears to require Mod(mdg4) function because the large aggregates are missing in mod(mdg4) mutants. The formation of gypsy insulator bodies is severely impaired also in CP190 mutants, suggesting that CP190 plays an essential role in the formation of these bodies and in the establishment of the chromatin domain organization mediated by gypsy endogenous insulators. It is possible that the BTB/POZ protein-protein interaction domains of both CP190 and Mod(mdg4)2.2 are required for and contribute to the stability of the interactions among insulator sites. In vitro-expressed CP190 lacking the BTB/POZ domain is soluble, whereas the wt protein is not, further suggesting that CP190 might exist as a complex with itself or other proteins in vivo, and the formation of this complex is likely mediated by the BTB/POZ domain. However, because CP190 is present at the gypsy insulator in the absence of Mod(mdg4)2.2 protein, the interaction between these two proteins may not be crucial for CP190 recruitment to the insulator (Pai, 2004).
Previous studies have identified CP190 as a centrosome-specific protein during mitosis that also associates with chromatin during interphase. Although many of these studies have focused on the possible role of CP190 during cell division, the current results suggest that centrosomal function and cell division are not affected in CP190 mutants. This conclusion is supported by independent studies of CP190 function during the cell cycle. The main function of CP190 might then be to regulate chromosome-related processes during interphase. Several lines of evidence suggest that this role is related to the function of the gypsy insulator: mutations in CP190 alter gypsy-induced phenotypes; CP190 colocalizes with Su(Hw) and Mod(mdg4)2.2 on polytene chromosomes and in diploid cell nuclei, and CP190 associates physically with gypsy insulator components in vitro and in vivo. However, the centrosomal localization of CP190 might also be important for its role in the gypsy insulator despite being unnecessary for cell cycle progression. The centrosome could either be a temporary storage site for CP190 during mitosis, or a site for a mitosis-specific modification that could be important for CP190 reassociation with chromosomes later in the cell cycle. The presence of CP190 in the centrosome could also be related to the regulation of the level of this protein in the cell. In fact, it has been shown that some chromatin-binding proteins are targeted to the centrosome for degradation. Alternatively, the presence of CP190 at the centrosome might be related to a possible role in the ubiquitin modification pathway. Recent findings have linked BTB/POZ domain proteins to ubiquitin E3 ligase function, some of which are known to be present at the centrosome. CP190 may be involved in similar types of interactions as an adaptor for ubiquitin E3 ligases and might target associated insulator proteins to the centrosome during mitosis for ubiquitination and/or degradation, which in turn may be required for properly reestablishing chromosome domain boundaries after mitosis (Pai, 2004).
Chromatin insulators are gene regulatory elements implicated in the establishment of independent chromatin domains. The gypsy insulator of D. melanogaster confers its activity through a protein complex that consists of three known components, Su(Hw), Mod(mdg4)2.2 (a spliced variant encoded by the modifier of mdg4), and CP190. Drosophila Topoisomerase I-interacting RS protein (dTopors) interacts with the insulator protein complex and is required for gypsy insulator function. In the absence of Mod(mdg4)2.2, nuclear clustering of insulator complexes is disrupted and insulator activity is compromised. Overexpression of dTopors in the mod(mdg4)2.2 null mutant rescues insulator activity and restores the formation of nuclear insulator bodies. dTopors associates with the nuclear lamina, and mutations in lamin disrupt dTopors localization as well as nuclear organization and activity of the gypsy insulator. Thus, dTopors appears to be involved in the establishment of chromatin organization through its ability to mediate the association of insulator complexes with a fixed nuclear substrate (Capelson, 2005).
A yeast two-hybrid screen for proteins that interact with Mod(mdg4)2.2 resulted in identification of dTopors as a factor involved in the activity of the gypsy insulator. dTopors was found to interact with the three known insulator components, Su(Hw), Mod(mdg4)2.2, and CP190, and to associate with the gypsy insulator complex on chromosomes and in diploid nuclei. Additionally, dTopors appears to physically associate with the nuclear lamina. Genetically, dTopors was shown to behave as a positive factor involved in gypsy insulator activity. Consistently, reduction in levels of dTopors, observed in the background of a dTopors-spanning deletion or of an inducible dTopors RNAi construct, results in the disruption of insulator activity. The effects of elevated levels of dTopors are particularly dramatic as they restore the activity of a compromised gypsy insulator on multiple levels. The enhancer blocking function of the insulator, the binding of Su(Hw) to chromatin, and the formation of insulator bodies in cell nuclei -- all compromised in mod(mdg4)u1 mutants -- are rescued by overexpression of dTopors (Capelson, 2005).
These effects can be explained by a model in which dTopors acts as a nuclear lamina-associated factor that serves to tether the gypsy insulator complexes to a fixed substrate. In the wild-type situation, Mod(mdg4)2.2 mediates the coalescence of distant insulator sites and the subsequent establishment of chromatin compartments, whereas dTopors may be involved in further organization of insulator bodies at specific nuclear attachment points through its direct interaction with both Mod(mdg4)2.2 and Su(Hw). The absence of Mod(mdg4)2.2 leads to the breakdown of nuclear organization and the destabilization of Su(Hw)-chromatin association. Through tethering distant insulator sites to a nuclear substrate, dTopors, when present at elevated levels, may be able to compensate for the loss of a component such as Mod(mdg4)2.2. By stabilizing the nuclear organization of insulator complexes, dTopors may also promote the binding of Su(Hw) to chromatin. This explanation is further reinforced by the observed disruptive effects of a lamin mutation on the nuclear organization and the enhancer blocking activity of the gypsy insulator (Capelson, 2005).
The connection between gypsy insulator activity and nuclear insulator bodies has relied predominantly on the effects of the mutations in Mod(mdg4)2.2 and CP190 on both enhancer blocking function and insulator body integrity. The activity of dTopors provides further evidence for a functional relationship between insulators and their nuclear localization, since rescue of insulator phenotypes by dTopors is accompanied by the recovery of insulator bodies. Establishment of independent chromatin domains, which has been proposed as the main function of insulators, is thought to rely on structural partitioning of chromatin through physical interactions between distant loci or through interactions with a fixed nuclear substrate. It has been previously intimated that gypsy insulators may employ both types of structural organization to ensure the establishment of domain autonomy. This work suggests that the gypsy insulator may undergo physical clustering through the BTB domains of Mod(mdg4)2.2 and of CP190 and may utilize the attachment to the nuclear lamina via dTopors. The interaction of the insulator with a nuclear substrate is further supported by a recent report that gypsy insulator proteins associate with the nuclear matrix, of which lamin is a principal component. Tethering to a subnuclear surface has also been implicated in the activity of the chicken β-globin insulator, where β-globin insulator loci were observed to interact with the nucleolar surface, perhaps via a direct association between the insulator protein CTCF and the nucleolar component nucleophosmin (Capelson, 2005).
The E3 ubiquitin ligase activity of dTopors was not found to act directly on the known insulator proteins, yet the RING domain of dTopors appears to be essential for its positive effect on the gypsy insulator. It thus remains possible that an unknown factor involved in insulator activity may be a substrate for dTopors-mediated ubiquitination. A connection between the gypsy insulator complex and the ubiquitin conjugation pathway is also suggested by the presence of BTB domains in Mod(mdg4)2.2 and CP190, since BTB domain proteins have been proposed to act as substrate adaptors for the ubiquitin RING E3 ligases. It is feasible that BTB-containing insulator proteins and RING-containing dTopors are involved in ubiquitin conjugation with functional consequences for the insulator (Capelson, 2005).
The association of dTopors with a subset of insulator binding sites on polytene chromosomes implies that its presence is not required by all insulator complexes. This may be a consequence of the proposed function of dTopors as a tethering factor, such that the interaction between distant insulator loci may alleviate the need for dTopors at every binding site of the insulator complex. Alternatively, it may suggest that endogenous insulator complexes are not all functionally equivalent, and that the enzymatic properties of dTopors may be important for specific insulator complexes. The ubiquitin ligase activity of dTopors may be involved in regulation of insulator complexes, such that modification of a yet uncharacterized component by ubiquitin can lead to variation in function of endogenous insulators (Capelson, 2005).
Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type-specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. This study identified the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. A novel, quantitative tissue-specific barrier assay was developed to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP-seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator (Matzat, 2012).
Chromatin insulators are DNA-protein complexes that influence eukaryotic gene expression by organizing the genome into distinct transcriptional domains. Functionally conserved from Drosophila to humans, insulators regulate interactions between regulatory elements such as enhancers and promoters and demarcate silent and active chromatin regions. Chromatin insulators are thought to exert effects on gene expression by constraining the topology of chromatin and facilitating the formation of intra- and inter-chromosomal looping. These higher order interactions can vary between cell types, thereby facilitating tissue-specific transcriptional output (Matzat, 2012).
Drosophila harbor several distinct classes of chromatin insulators, including the well studied gypsy insulator, also known as the Suppressor of Hairy wing (Su(Hw)) insulator. The zinc-finger DNA-binding protein, Su(Hw), recognizes a particular motif, imparting specificity to the gypsy insulator. In addition to Su(Hw), the core gypsy insulator complex contains Centrosomal protein 190 (CP190), which also harbors a zinc finger domain, and the non-DNA-binding protein, Modifier of mdg4<;S> 2.2 (Mod(mdg4)2.2). These core proteins are required for gypsy insulator activity. Both CP190 and Mod(mdg4)2.2 contain broad complex, tramtrack, bric-a-brac (BTB) dimerization domains that have been suggested to mediate insulator-insulator interactions and facilitate the formation of long range insulator-mediated loops along the chromatin fiber (Matzat, 2012).
Specialized nuclear arrangement of gypsy insulator complexes correlates tightly with insulator function. The gypsy insulator proteins bind to thousands of sites throughout the genome with more than half of Su(Hw) binding sites occurring in intergenic regions and a large number of sites located within introns. Consistent with a role in boundary formation, Su(Hw) sites are positively correlated with both Lamin-associated domains and boundaries between transcriptionally active and silent chromatin. It has been shown that gypsy insulator proteins coalesce at a small number of foci in diploid nuclei, termed insulator bodies, which have been proposed to act either as hubs of higher order chromatin domains or storage sites for insulator proteins. Importantly, mutation of certain insulator components results in impaired insulator activity coincident with diffuse or smaller, more numerous insulator bodies. However, formation of insulator bodies is not sufficient for gypsy insulator activity, and a detailed mechanistic understanding of insulator bodies is still lacking. Nevertheless, the tight correlation between gypsy insulator function and insulator body localization suggests an important role for these structures. Finally, in addition to a variety of accessory proteins, a role for RNA in insulator function and insulator body organization was suggested based on RNA-dependent protein interaction with insulator complexes (Matzat, 2012).
Genome-wide studies indicate that the locations of insulator protein binding sites are mainly consistent across different cell types but that insulator-dependent looping configurations may dictate differences in gene expression. In Drosophila, it has been shown that external stimuli can alter chromatin association of CP190, possibly leading to a change in chromatin looping. Recent large-scale chromatin conformation capture (3C)-based studies have implicated insulator protein binding sites as key contact points mediating looping throughout the genome. In several studies across species, specific chromatin conformations are observed in loci that produce tissue- or cell-type specific transcripts. Whether insulators either establish tissue-specific chromatin organization or maintain configurations established via transcription is unclear. Furthermore, factors that control tissue-specific insulator-dependent chromatin organization remain unknown (Matzat, 2012).
This study identifies a CNS enriched, RNA recognition motif (RRM) containing protein, Alan Shepard (Shep), as the first tissue-specific regulator of gypsy insulator activity and insulator body localization. Shep interacts directly with Mod(mdg4)2.2 and Su(Hw) and also associates with gypsy insulator proteins in vivo. Using a novel quantitative, tissue-specific insulator assay, it was found that Shep negatively regulates gypsy insulator activity in the CNS. In addition, mutation of Shep improves compromised insulator function and insulator body formation. Finally, genome-wide localization in the CNS-derived BG3 cell line reveals enrichment of overlap between Shep and Mod(mdg4)2.2 but less frequent than expected overlap among Shep, Su(Hw) and Mod(mdg4)2.2 together. These data suggest that gypsy chromatin insulator function can be regulated in a tissue-specific manner (Matzat, 2012).
Two lines of evidence indicate that Shep affects insulator activity in a tissue-specific manner. First, insulator body localization is altered in CNS but not other tissues of shep mutants. Second, barrier activity is improved in CNS but not muscle tissue when Shep levels are reduced. Finally, genome-wide mapping of Shep and gypsy insulator proteins in BG3 cells reveals substantial overlap with individual insulator proteins but lack of three-way overlap, further supporting a role for Shep in negative regulation of insulator activity in certain tissues (Matzat, 2012).
Shep acts as a tissue-specific negative regulator of gypsy insulator function and insulator body localization. Shep localization is most enriched in the CNS at both embryonic and larval stages; however, it is also expressed at lower levels in additional tissues. Although this study has demonstrated that Shep functions in the CNS, Shep can also repress enhancer blocking activity in the wing and could possibly affect insulator activity in other tissues. For example, ubiquitous reduction of Shep levels strongly improves overall barrier activity, suggesting that tissues outside of the CNS may also harbor Shep activity. Nonetheless, Shep does not appear to function in all tissues; knockdown of Shep does not affect barrier activity in muscle tissue, no changes in insulator body localization are observed in eye or leg tissue of shep mutants, and no effect is observed for y2 enhancer blocking in pigment cells of shep mutants. Interestingly, when Shep is overexpressed in muscle tissue, reduction of barrier activity is observed, suggesting that a certain threshold of Shep protein is needed to repress insulator activity. Since Shep protein can be detected at least at low levels in all tissues tested thus far, it is unlikely that the mere presence of Shep protein is sufficient to disrupt gypsy insulator activity. It remains to be determined what other cofactors, such as proteins or RNAs, may contribute to Shep activity (Matzat, 2012).
Shep may negatively regulate insulator activity by interfering with insulator protein interactions required for their activity. ChIP-seq analyses shows that the genome-wide binding profile of Shep in CNS-derived BG3 cells overlaps substantially with that of Mod(mdg4)2.2 but not extensively with both Su(Hw) and Mod(mdg4)2.2 combined. Lack of three-way overlap is not entirely unexpected given that Shep is a negative regulator of gypsy insulator activities. Shep coimmunoprecipitation experiments copurify only a small fraction of total insulator proteins present in nuclear extracts, suggesting that Shep-insulator complexes are not abundant or not stable in vivo. Since Shep can bind either Mod(mdg4)2.2 or Su(Hw) in vitro at a 1:1 ratio, Shep binding could compete with direct interaction between Mod(mdg4)2.2 and Su(Hw) or their interactions with other factors such as CP190. Moreover, the finding that mod(mdg4) mutants are highly sensitive to Shep dosage suggests an antagonistic functional relationship between Mod(mdg4)2.2 and Shep. Specifically, Shep may negatively regulate higher order insulator-insulator complex interactions, which appear to be mediated by direct interaction between Mod(mdg4)2.2 and CP190. Insulator body localization in larval brains of shep, mod(mdg4)u1 mutants reverts back to a wildtype pattern compared to compromised mod(mdg4)u1 mutants, perhaps indicating that the normal function of Shep may be to prevent larger insulator complexes from forming in these cell types (Matzat, 2012).
The results are consistent with the possibility that Shep promotes tissue-specific chromatin configurations by modulating insulator complexes. While differential occupancy of insulator proteins at their respective binding sites may play a role in regulating certain loci, occupancy throughout the genome does not differ extensively between cell types. Therefore, alternate mechanisms to control insulator activity likely exist. Shep activity could prevent insulator-insulator contacts otherwise present in tissues that do not express shep, resulting in relief of enhancer blocking or repression by silencers. Interestingly, shep was identified as a regulator of complex behavioral traits in screens for altered sensory-motor responsiveness to gravity and aggressive behavior), suggesting the possibility that regulation of an insulator-based mechanism could exist to effect changes in neurological function (Matzat, 2012 and references therein).
Given that Shep is an RRM-containing protein, RNA-binding may contribute to the ability of Shep to associate with insulator complexes in vivo. Shep RRMs are highly conserved, and lethality caused by Shep overexpression in the mod(mdg4) mutant background is not observed when the RRMs are mutated. This result suggests that Shep RRMs may be functional with respect to insulator activity. One possibility is that the specific RNA bound by Shep could affect targeting of Shep to insulator sites. Another not mutually exclusive prospect is that Shep is recruited to chromatin cotranscriptionally by binding nascent transcripts. It will be important to determine in future studies if Shep binds RNA while in complex with gypsy insulator proteins as well as the identities of Shep and insulator-associated RNA. These results point to a novel role for Shep and possibly RNA to regulate insulator activity in a tissue-specific manner (Matzat, 2012).
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