suppressor of Hairy wing

REGULATION (part 1/2)

Targets of Activity

Gypsy encodes a full-length 7.0-kb RNA expressed in the salivary gland precursors and fat body of the fly embryo, imaginal discs and fat body of larvae, and fat body and ovaries of adult females. The SU(HW)-binding region inserted upstream of the promoter of a lacZ reporter gene can induce beta-galactosidase expression in a subset of the embryonic and larval tissues where gypsy is normally transcribed. This expression is dependent on the presence of SU(HW) protein, suggesting that SU(HW) is a positive activator of gypsy transcription. Flies transformed with a construct in which the 5' LTR and leader sequences of gypsy are fused to lacZ show beta-galactosidase expression in all tissues where gypsy is normally expressed, indicating that sequences other than the SU(HW)-binding site are required for proper spatial and temporal expression of gypsy. Mutations in the zinc fingers of SU(HW) affect its ability to bind DNA and to induce transcription of the lacZ reporter gene. Two other structural domains of SU(HW) also play an important role in transcriptional regulation of gypsy. Deletion of the amino-terminal acidic domain results in the loss of lacZ expression in larval fat body and adult ovaries, whereas mutations in the leucine zipper region result in an increase of lacZ expression in larval fat body and a decrease in adult ovaries. These effects might be the result of interactions of SU(HW) with activator and repressor proteins through the acidic and leucine zipper domains to produce the final pattern of tissue-specific expression of gypsy (Smith, 1995).

The 5'-untranslated region of the Drosophila gypsy retrotransposon contains an "insulator," which disrupts the interactions between distally located enhancers and proximal promoter elements. The insulator effect is dependent on the suppressor of Hairy-wing (su[Hw]) protein, which binds to reiterated sites within the 350 base pairs of the gypsy insulator, and additionally acts as a transcriptional activator of gypsy. This study shows that the 350-base pair su(Hw) binding site-containing gypsy insulator behaves as a matrix/scaffold attachment region (MAR/SAR), involved in interactions with the nuclear matrix. In vitro experiments using nuclear matrices from Drosophila, murine, and human cells demonstrate specific binding of the gypsy insulator, not observed with any other sequence within the retrotransposon. Moreover, it is shown that the gypsy insulator, like previously characterized MAR/SARs, specifically interacts with topoisomerase II and histone H1, i.e. with two essential components of the nuclear matrix. Experiments within cells in culture demonstrate differential effects of the gypsy MAR sequence on reporter genes, namely no effect under conditions of transient transfection and a repressing effect in stable transformants, as expected for a sequence involved in chromatin structure and organization (Nabirochkin, 1998).

The presence of a MAR/SAR within gypsy is not totally unexpected, since 'boundary' elements are in general regions that contain not only enhancer and insulating elements, but also matrix attachment domains. The rather original feature of the gypsy sequence is that all three domains, which in general are sufficiently "dispersed" so as to allow isolation of "pure" enhancers, MAR/SAR, or insulators, are in the present case "gathered" within a single and relatively short (350 bp) sequence. This rather uncommon situation might in fact be relevant to the pressure for compactness within retroviral sequences, as it is known that retroviruses can only package a limited amount of genetic information. A consequence of compaction is that the gypsy insulator and its associated components are most probably interacting, in vivo, with elements of the nuclear matrix. Accordingly, proteins of the nuclear matrix might play a role in the insulation process, and conversely the su(Hw) protein (which is essential for insulation) might interact with proteins of the matrix. Such interactions could actually account for the data on gypsy insulation and fit with previously proposed models for the gypsy effects (Nabirochkin, 1998).

A first series of data strongly suggested that the gypsy insulator, like all previously characterized insulators, essentially prevents interactions between distal enhancer and promoter, without any direct repressing effect on the enhancer itself. This directional effect can most easily be accounted for by the "looping model" involving generation of structural domains isolated one from the other by attachment of boundary sequences (MAR/SAR) to the nuclear matrix. Alternatively, a series of data on gypsy insulation (essentially in mod(mdg4) mutants) discloses bidirectional repressing effects, which can be accounted for by a model involving heterochromatinization. The present data (showing that the gypsy insulator behaves as a MAR/SAR) are clearly in agreement with the structural looping model, but also support the heterochromatinization model. Indeed, the gypsy MAR/SAR DNA per se, in the absence of su(Hw) protein, is involved in histone H1 nucleation (as shown in this paper), and it has been demonstrated that histone H1 nucleation is associated with both DNA compaction and transcriptional silencing. Additionally, Laemmli and co-workers have found that histone H1 can be removed from MAR/SAR domains by distamycin and distamycin-like proteins (D-like proteins, such as the high mobility group proteins); this has led to the proposal that MAR/SARs can activate or repress transcription of adjacent genes depending on the nucleation/depletion of histone H1. The gypsy MAR/SAR could then be responsible for the repressing effect observed in the mod(mdg4) mutants, as well as in the present assay within heterologous cells (assuming further that appropriate D-like proteins are absent in those cells). Taking into account, in addition, that mutations in the mod(mdg4) or the su(Hw) genes modify position-effect variegation, it could be further hypothesized that the su(Hw)/mod(Mdg4) complex acts as the D-like proteins and modifies the nucleation processes to allow the switch from a repressing to an active state. Accordingly, a model in which the su(Hw) binding sites and the associated su(Hw)/mod(Mdg4) complex modulate the effects of the MAR/SAR DNA sequence could rather simply account for the biological effects of the gypsy insulator in both the wild type and su(Hw)/mod(mdg4) mutants. The proposed model would then reconcile the two previous models for gypsy insulation, i.e. the heterochromatinization and the looping models (Nabirochkin, 1998 and references).

Eukaryotic genomes are divided into independent transcriptional domains by DNA elements known as insulators. The gypsy insulator, a 350-bp element isolated from the Drosophila gypsy retrovirus, contains twelve degenerate binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Su(Hw) associates with over 500 non-gypsy genomic sites, the functions of which are largely unknown. Using a bioinformatics approach, 37 putative Su(Hw) insulators (pSIs) were identified that represent regions containing clustered matches to the gypsy insulator Su(Hw) consensus binding sequence. The majority of these pSIs contain fewer than four Su(Hw) binding sites, with only seven showing in vivo Su(Hw) association, as demonstrated by chromatin immunoprecipitation. To understand the properties of the pSIs, these elements were tested for enhancer-blocking capabilities using a transgene assay system. In a complementary set of experiments, effects of the pSIs on transcriptional regulation of genes at the natural genomic location were determined. The data suggest that pSIs have complex genomic functions and, in some cases, establish insulators. These studies provide the first direct evidence that the Su(Hw) protein contributes to the regulation of gene expression in the Drosophila genome through the establishment of endogenous insulators (Parnell, 2006; full text of article).

Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions

Insulators are protein-bound DNA elements that are thought to play a role in chromatin organization and the regulation of gene expression by mediating intra- and interchromosomal interactions. Suppressor of Hair-wing [Su(Hw)] and Drosophila CTCF (dCTCF) insulators are found at distinct loci throughout the Drosophila genome and function by recruiting an additional protein, Centrosomal Protein 190 (CP190). Chromatin immunoprecipitation (ChIP) and microarray analysis (ChIP-chip) experiments were performed with whole-genome tiling arrays to compare Su(Hw), dCTCF, boundary element-associated factor (BEAF), and CP190 localization on DNA in two different cell lines; evidence was found that BEAF is a third subclass of CP190-containing insulators. The DNA-binding proteins Su(Hw), dCTCF, and BEAF show unique distribution patterns with respect to the location and expression level of genes, suggesting diverse roles for these three subclasses of insulators in genome organization. Notably, cell line-specific localization sites for all three DNA-binding proteins as well as CP190 indicate multiple levels at which insulators can be regulated to affect gene expression. These findings suggest a model in which insulator subclasses may have distinct functions that together organize the genome in a cell type-specific manner, resulting in differential regulation of gene expression (Bushey, 2009).

Su(Hw), dCTCF, and BEAF have all been implicated in chromatin loop formation, and the interaction of these different DNA-binding proteins with CP190 could have functional implications for the arrangement of the chromatin fiber within the nucleus. The work presented in this study provides critical insight into the genome-wide distribution of these four insulator proteins and is a first, crucial step toward understanding the role that they play in chromatin organization and the regulation of gene expression (Bushey, 2009).

Although insulator elements containing Su(Hw), dCTCF, and BEAF could, in principle, play similar roles, it was found that they have very different distribution patterns with respect to gene location. Only 20% of Su(Hw) sites are located within 1 kb of the 5' or 3' ends of genes. In contrast, 47% of dCTCF sites and 84% of BEAF sites are found within 1 kb of gene ends, and their distributions are highly skewed toward the 5' end of highly expressed genes. dCTCF and BEAF appear to display further functional compartmentalization in their roles, since BEAF tends to be present at the 5' end of genes involved in metabolic processes and dCTCF is enriched near genes involved in developmental processes. This could indicate that BEAF plays a specific role in the regulation of gene units consisting of metabolic genes, whereas Su(Hw) may play a more general role by setting the foundation for chromatin organization. dCTCF, which shows an intermediate distribution compared with Su(Hw) and BEAF, may sometimes function in large-scale organization and sometimes work at the level of individual developmental gene units. Together, the three CP190 insulator subclasses could create a chromatin web that is part of the framework organizing DNA in the nucleus (Bushey, 2009).

Insulators have been typically characterized as sequences capable of regulating interactions between transcriptional regulatory sequences and/or chromatin states. This function can easily be envisioned in the case of Su(Hw) and dCTCF sites located far from genes, where these sites could flank a group of transcription units that would then represent a domain of coregulated genes. If this is the case, what is the function of the remaining dCTCF and BEAF sites located close to the 5' and 3' ends of genes? This distribution is surprising in the context of what is normally think of as insulator function; however, when CTCF-binding sites were mapped in the human genome, a similar distribution pattern was observed. This is suggestive of a wider role for insulator proteins than just the establishment of chromatin domains, and, in fact, alternative insulator protein functions have been suggested. For example, CTCF in humans is present in the Igh locus in many of the VH as well as DH and JH exons, suggesting a role in V(D)J recombination. Additionally, this study provides evidence that insulator proteins near genes play a role in the regulation of expression of specific genes and suggests that the mechanism behind this regulation differs from classic transcription factors, since the same insulator complexes were seen to both activate and repress transcription. These functions could be a consequence of the ability of these proteins to both interact with each other and mediate intra- and interchromosomal loops. Bringing together various insulator protein-binding sites could facilitate localization to either transcriptionally active or transcriptionally repressed regions of the nucleus depending on the genomic context of the sites (Bushey, 2009).

Comparison of the genome-wide distribution of the three insulator subclasses in two different cell lines has led to insights into possible mechanisms employed during cell differentiation to establish different patterns of gene expression. Overall, the analysis suggests that cells may control insulator function at multiple levels and that these forms of regulation occur throughout the genome. Regulation of insulator function seems to begin at the level of DNA binding, since differential binding was observed at 5%-37% of sites for Su(Hw), dCTCF, and BEAF between two different cell lines even with the most conservative statistical analysis. Similar percentages of cell type-specific binding sites were observed for vertebrate CTCF between different cell lines. Previous analysis of Su(Hw) binding in various tissues has not revealed any significant tissue-specific binding sites, perhaps because only a small number of sites was analyzed in these studies. Alternatively, the discrepancy could be due to the use of whole tissues in previous studies that contain multiple cell types, making it difficult to detect cell type-specific sites (Bushey, 2009).

After Su(Hw), dCTCF, and BEAF bind DNA, they are thought to recruit other proteins such as CP190. Regulation at this level was observed throughout the genome, where a subset of the Su(Hw), dCTCF, and BEAF sites recruit CP190 in a cell type-specific manner. The additional Su(Hw), dCTCF, and BEAF sites that do not recruit CP190 in either Kc or Mbn2 cells may do so in other cell types or other growth conditions not tested in this study. This idea is supported by the two dCTCF sites in the bithorax region that were found to contain CP190 in third instar larvae brains but not in the data sets collected in Kc cells or Mbn2 cells. Although further study is needed to determine which sites of insulator protein localization participate in chromatin organization, it is expected that sites lacking CP190 do not, since mutations in CP190 are known to disrupt insulator body formation and only those sites that recruit CP190 seem to affect gene expression. Therefore, these sites may represent insulators that are poised for incorporation into chromatin loops upon recruitment of CP190. On the other hand, these sites could function through the recruitment of an alternative cofactor and in this way represent a functionally distinct subset of Su(Hw)-, dCTCF-, and BEAF-binding sites (Bushey, 2009).

An additional layer of regulation may then occur at the level of protein-protein interactions mediated by CP190. This type of regulation cannot be gleaned from ChIP-chip data, but other experiments have shown that sumoylation of insulator proteins is able to inhibit protein-protein interactions affecting Su(Hw) insulator body formation but not association of insulator proteins with DNA. Similarly, vertebrate CTCF insulator function has been linked to poly(ADP-ribosyl)ation (PARlation), and it has been suggested that PARlation facilitates CTCF self-interaction . Furthermore, the presence of RNA and RNA-binding proteins may also contribute to the formation or maintenance of insulator bodies required to create chromatin loops. Finally, insulator bypass that results in the inactivation of insulator activity through pairing of nearby insulator elements, and specialized sequences such as the promoter targeting sequences (PTS), can allow an enhancer to bypass an insulator. These forms of regulation may alter the ability of insulator proteins to interact with one another to regulate insulator loop formation (Bushey, 2009).

It is expected that these various forms of regulation including DNA binding, CP190 recruitment, and loop formation result in changes in gene expression between different cell lines. However, transcription analysis with insulator proteins is difficult since insulator elements are thought to control regulatory elements such as enhancers and silencers that can be found far away from their target promoters. Therefore, determining which genes are controlled by an insulator site is not a trivial process. In the transcription analysis, genes were considered with a cell type-specific insulator site only within the gene or the 1 kb surrounding region. Despite this limitation, a significant enrichment was still seen for genes that change expression between cell types when they have a cell type-specific insulator site nearby, supporting the idea that insulator proteins are involved in the regulation of gene expression. Genes that did not change expression despite being located near a cell type-specific insulator protein-binding site may not be the actual target genes of the insulator sites; therefore, this analysis probably greatly underestimates the effect of insulator proteins on gene expression. Additionally, it was found that insulator protein-binding sites that localize to genes are enriched at genes with certain expression signals, high expression for dCTCF and BEAF, and low expression for Su(Hw). However, comparison between the two cell lines revealed that expression can be either positively or negatively regulated by sites with each DNA-binding protein. Therefore, although an insulator protein associates with a highly expressed gene, it may lead to either an increase or decrease in transcription of this gene. The observed level of expression may be an additive effect of many different regulatory elements, including multiple insulator sites. Different mechanisms may be used to regulate a highly transcribed gene versus a gene with low levels of transcription, and therefore the different insulator subclasses may target these different mechanisms (Bushey, 2009).

The transcription analysis in this study suggests that insulator proteins play a role in the regulation of gene expression, but has just begun to explore the depth of their effect. Numerous steps at which insulator activation can be subject to regulation allow for a vast amount of variation between different cell types and could play a major role in establishing the diverse patterns of chromatin organization necessary for cell type-specific gene expression. The different CP190 insulator subclasses might have distinct roles in this cell type-specific nuclear organization. In vertebrates, CTCF is the only insulator known thus far, and an important question to address in the future is the apparent disparity between genome complexity and insulator diversification between flies and vertebrates. It is possible that vertebrates have insulator subclasses represented by DNA-binding proteins other than CTCF that have not yet been identified. Alternatively, it is possible that vertebrate CTCF has acquired all the functions of the various Drosophila insulator subclasses. The distribution pattern of dCTCF suggests that it can play a role in both global organization and in the regulation of individual genes, making it the most likely candidate of the three Drosophila subclasses to play this overarching organizational role in vertebrates. Therefore, vertebrates may use methods other than variant DNA-binding proteins to distinguish insulator subclasses, such as recruitment of different CTCF interaction partners at different insulator sites. This would make it difficult to distinguish between the various layers of insulator control in the vertebrate genome. If this is the case, Drosophila could provide a powerful model system to dissect the various functions and levels of regulation of chromatin insulators (Bushey, 2009).

A comprehensive map of insulator elements for the Drosophila Genome

Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. The genome-wide binding sites of 6 insulator associated proteins (dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF) was studied to obtain the first comprehensive map of insulator elements in Drosophila embryos. Over 14,000 putative insulators, including all classically defined insulators, were identified. Two major classes of insulators were defined by dCTCF/CP190/BEAF-32 and Su(Hw), respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters; divide differentially expressed, alternative, and divergent promoters; act as chromatin boundaries; are associated with chromosomal breakpoints among species; and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units and a framework for understanding insulator function during the development and evolution of Drosophila (Négre, 2010).

This study the embryonic binding profile of six factors previously known to be associated with insulator function in Drosophila. The analysis of insulator binding site distributions and protein composition suggest there exist 2 principal categories of insulator elements (Class I and Class II). In particular, it was shown that Class I insulators, identified by the binding of CTCF, CP190 or BEAF-32, segregate differentially expressed genes and delimit the boundaries of chromatin silencing, while they are depleted between known CRMs and their target genes. No evidence was found supporting a significant distinction between CP190/BEAF and CP190/CTCF or CTCF/BEAF. In contrast, the analyses suggest that BEAF-32, CP190, and CTCF are distributed and function quite similarly, while Su(Hw) appears distinct. The Class II insulators, bound by Su(Hw), are often exceptional in this analyses. It is noted that the analysis of genome-wide mapping data, expression data, and genome annotation provides an endogenous boundary assay that demonstrates that, while Su(Hw) has been described as an insulator before, it is not systematically associated with the boundaries of the gene units (Négre, 2010).

By helping to delimit the regulatory boundaries of genes, the Class I insulator map presented in this study will aid in the identification of transcription factor target genes and the construction of transcriptional regulatory networks. As an example of this concept, the distribution of known regulatory elements and insulators across the Antennapedia Complex (ANT-C) of homeotic genes is presented. This region quite strikingly demonstrates the potential utility of insulator binding data for cis-regulatory annotation. Across approximately 500 kb, cis-regulatory elements and their target promoters are found between insulator pairs. For example, a single insulator separates the lab and Edg84A genes, with their respective cis-regulatory elements narrowly partitioned on either side. The adjacent regulatory elements and promoters of zen and bcd are similarly insulator segregated (Négre, 2010).

Consistent with their observed regulatory boundary functions, Class I insulators are embedded within local regions of active chromatin and are frequently associated with syntenic breakpoints between species. Previous work has demonstrated that active promoters in yeast and Drosophila are associated with reduced nucleosome occupancy and low-salt soluble and high-salt insoluble chromatin. Therefore, surprisingly, dynamic chromatin is a shared feature between promoters and most classes of insulators. It is notable however that some studies have revealed functional similarities between insulators and promoters in transgenic assays. These results have been described as paradoxical, as insulators can negatively affect promoters by blocking communication between enhancers and promoters. One proposed model for insulator function is that they act as promoter 'decoys' by recruiting away factors necessary for transcriptional initiation. Alternatively, insulators and promoters might require common chromatin features to function by mechanisms that are still unknown. One potential interpretation is that the dynamic chromatin at insulators forms a flexible chromatin joint that would affect the probability of productive contact between separated regulatory elements. In this way, the similarity between promoters and insulators would be a consequence of their common requirement for dynamic chromatin, although with very different consequences. This model may explain why promoters are so frequently scored as insulators in the classical insulator assay, when an element is placed between an enhancer and a promoter (Négre, 2010).

Protein Interactions

The Drosophila protein Chip potentiates activation by several enhancers and is required for embryonic segmentation. Chip and its mammalian homologs interact with and promote dimerization of nuclear LIM proteins. No known Drosophila LIM proteins, however, are required for segmentation, nor for expression of most genes known to be regulated by Chip. Chip also interacts with diverse homeodomain proteins using residues distinct from those that interact with LIM proteins, and Chip potentiates activity of one of these homeodomain proteins in Drosophila embryos and in yeast. These and other observations help explain the roles of Chip in segmentation and suggest a model to explain how Chip potentiates activation by diverse enhancers (Torigoi, 2000).

Full-length Chip interacts with the HD proteins Bicoid (Bcd) and Ftz, and with a fragment of the Su(Hw) insulator protein. The HD protein Otd binds almost as efficiently as does Bcd and Ftz to Chip, but the Eve HD protein binds poorly, a result possibly attributable to improper folding of the in vitro-translated protein. The domains of Chip involved in homotypic and heterotypic interactions include the LIM interaction domain (LID) and the self-interaction domain (SID). Deletion of the LID reduces interaction with Apterous. That deletion, however, has no effect on interaction with Bcd, Ftz, Su(Hw)DeltaCTD, or Chip. In contrast, two other deletion mutants, ChipDelta404-465 and ChipDelta441-454, reduce binding to Bcd, Ftz, Su(Hw)DeltaCTD, and Chip but have little effect on binding to Apterous. On the basis of this and additional deletion mutants, Chip residues 439-456 are identified as the region that interacts with the HD proteins, Su(Hw), and with Chip itself. This region is termed the other interaction domain (OID) (Torigoi, 2000).

It is suggested that Chip plays two roles in the regulation of gene expression: (1) Chip is likely to aid binding of proteins to enhancers, and (2) Chip is also likely to function between enhancers and promoters to support enhancer-promoter communication. The in vitro interaction between Chip and the Su(Hw) insulator protein shown here is consistent with the notion that Su(Hw) is directly antagonistic to Chip activity as previously demonstrated genetically at the cut locus. It remains to be seen how, if these speculations are correct, Chip facilitates enhancer-promoter communication and how that communication is disrupted by Su(Hw). It is believed that Su(Hw) blocks activation not by reducing the binding of proteins to enhancers, but rather by hindering enhancer-promoter communication. For instance, an enhancer blocked in its interaction with one promoter by Su(Hw) can nevertheless activate a second promoter located on the opposite side of the enhancer from Su(Hw). Thus, although Su(Hw) is antagonistic to Chip, it is unlikely to affect binding of proteins to enhancers. It is also unlikely that Chip functions merely by preventing binding of Su(Hw) to gypsy because Chip is also important for the expression of several genes, e.g., cut and eve, in the absence of gypsy and Su(Hw) (Torigoi, 2000).

Function of su(Hw) as an insulator (part 1/2)

Enhancers are able to activate promoters located several kilobases away, but how this is accomplished is not known. Activation by the wing margin enhancer in the cut gene, located 85 kb from the promoter, requires several genes that participate in the Notch receptor pathway in the wing margin, including scalloped, vestigial, mastermind, Chip, and the Nipped locus. Nipped mutations disrupt one or more of four essential complementation groups: l(2)41Ae, l(2)41Af, Nipped-A, and Nipped-B. Heterozygous Nipped mutations modify Notch mutant phenotypes in the wing margin and other tissues, and magnify the effects that mutations in the cis regulatory region of cut have on cut expression. Nipped-A and l(2)41Af mutations further diminish activation of a wing margin enhancer that has been partly impaired due to a small deletion. In contrast, Nipped-B mutations do not diminish activation by the impaired enhancer, but increase the inhibitory effect of a gypsy transposon insertion between the enhancer and promoter. Nipped-B mutations also magnify the effect of a gypsy insertion in the Ultrabithorax gene. Gypsy binds the Suppressor of Hairy-wing insulator protein [Su(Hw)] that blocks enhancer-promoter communication. Increased insulation by Su(Hw) in Nipped-B mutants suggests that Nipped-B products structurally facilitate enhancer-promoter communication. Compatible with this idea, Nipped-B protein is homologous to a family of chromosomal adherins with broad roles in sister chromatid cohesion, chromosome condensation, and DNA repair (Rollins, 1999).

Database searches reveal homologs of the Nipped-B protein in fungi, worms, and mammals. Only short expressed-sequence tags (ESTs) of Caenorhabditis elegans, mouse, and humans were identified. The human ESTs are from a variety of tissue-specific libraries, suggesting that the human homologs are widely expressed. The combined human ESTs, which do not represent a complete sequence, encode 411 amino acids. Residues 2-232 of the partial human protein overlap Nipped-B residues 1744-1994 with 34% identity and 52% similarity. In order of decreasing homology, the 2157-amino acid Rad9 protein of Coprinus cinereus, the 1583-amino acid Mis4 protein of Schizosaccharomyces pombe, and the 1493-amino acid Scc2 protein of Saccharomyces cerevisiae, are more distantly related. Rad9 residues 669-2071 display 21% identity and 41% similarity to Nipped-B residues 576-1887; Mis4 residues 780-1492 have 19% identity and 41% similarity to Nipped-B residues 1110-1818, and Scc2 residues 697-1291 display 19% identity with 39% similarity to Nipped-B residues 1103-1704. The three fungal homologs show similar levels of homology among themselves, but it is evident that there is a large conserved domain shared by all three proteins. Consistent with the idea that Nipped-B plays an architectural role in enhancer-promoter communication, the fungal homologs of Nipped-B all participate in regulating chromosome structure, with roles in DNA repair, meiotic chromosome condensation, or sister chromatid cohesion. It has been proposed that these three fungal proteins define a new class of chromosomal proteins and have been named adherins to distinguish them from the cohesins that have similar functions (Rollins, 1999 and references).

Although the data do not yet distinguish whether the heterochromatic Nipped locus is a single complex transcription unit or a cluster of distinct genes, several conclusions may be drawn about Nipped functions and their roles relative to the other cut regulators. To summarize, Nipped mutations define three separable essential functions that regulate cut in the wing margin, provided by the Nipped-A, Nipped-B, and l(2)41Af lethal complementation groups. Dosage-sensitive genetic interactions indicate that Nipped-A and l(2)41Af cooperate closely with mam and vg in the regulation of cut. Similar to mam and unlike sd and vg, Nipped-A and l(2)41Af also modulate Notch receptor signaling or expression in multiple tissues. Nipped-B has the most unique function. Like Chip, Nipped-B regulates both cut and Ubx and is antagonistic to insulation by Su(Hw). Together, the antagonism to Su(Hw) and the homology to chromosomal adherins lead to a proposal that Nipped-B protein performs an architectural role in enhancer-promoter communication (Rollins, 1999).

The primary evidence that Nipped-B is antagonistic to Su(Hw) insulator activity is that Nipped-B activity is only strongly limiting for cut expression when there is a gypsy insertion between the wing margin enhancer and promoter. Strikingly, in contrast to mutations disrupting any of the other cut regulators (including sd, mam, Chip, vg, Nipped-A, and l(2)41Af), heterozygous Nipped-B mutations do not detectably reduce activation by the partially crippled wing margin enhancer in ct53d. Compared with sd, mam, or Nipped-A mutations, heterozygous Nipped-B mutations also only slightly reduce activation of cut expression by the solo wild-type wing margin enhancer present in ct2s heterozygotes. Therefore, with both ct53d and ct2s, Nipped-B products are less limiting for wing margin enhancer activity than are Nipped-A products. Remarkably, the opposite is true when there is a gypsy insulator insertion in cut. Heterozygous Nipped-B mutations are severalfold more effective than Nipped-A mutations in magnifying the effect of the Su(Hw) insulator in ctL-32; su(Hw)e2 flies. Furthermore, of the known cut regulators, only Chip and Nipped-B mutations magnify the effect of the Su(Hw) insulator in su(Hw)e2 bx34e flies. The antagonism between Nipped-B and Su(Hw) is unlikely to be specific to the Su(Hw)e2 protein. Su(Hw)e2 has an amino acid substitution in a zinc finger that reduces DNA-binding activity but contains a wild-type enhancer-blocking domain. Moreover, Nipped-B mutations also reduce cut expression in the absence of a gypsy insertion, indicating that the increased effectiveness of Su(Hw)e2 in Nipped-B mutants reflects a change in cut regulation rather than a change in Su(Hw)e2 protein activity (Rollins, 1999).

The available data are insufficient to determine with absolute certainty whether or not Nipped-B directly regulates cut. However, direct regulation provides the simplest explanation for several observations. The ability of Nipped-B mutations to exacerbate different cut mutant phenotypes differs from all other cut regulators such as sd, vg, and mam. Therefore, Nipped-B does not regulate cut indirectly by altering expression of any of the other known cut regulators. Moreover, the effects of the Nipped-B407 mutation on cut and Ubx mutant phenotypes are dominant, although Nipped-B407 only partially reduces Nipped-B mRNA levels. A partial loss of Nipped-B activity is unlikely to cause a similar or greater loss of activity of another cut regulator. Therefore, in light of the observation that Nipped-B mutations magnify insulation by gypsy insertions in both cut and Ubx, the idea that Nipped-B products directly support enhancer-promoter communication in cut and Ubx is strongly favored. Because Nipped-B is essential and Nipped-B mRNA is expressed at all developmental stages, it may play a similar role in other genes (Rollins, 1999).

The hypothesis that Nipped-B protein plays an architectural role to facilitate enhancer-promoter interactions in cut and Ubx is supported by the diverse effects that the fungal adherin homologs of the Nipped-B protein have on chromosome structure and function. The Rad9 protein of Coprinus was identified in a screen for radiation-sensitive mutants. rad9 mutants were subsequently observed to display defects in synaptonemal complex formation and chromosome condensation during meiosis. Mutations in the Scc2 gene of budding yeast were identified as lethal temperature-sensitive mutants that display defects in sister chromatid cohesion during mitosis. In scc2 mutants, sister chromatids separate prematurely, just after formation of the bipolar spindle. Mutations in the Mis4 gene of fission yeast were identified as temperature-sensitive lethal mutants that missegregate minichromosomes. mis4 mutants also missegregate regular chromosomes and are radiation sensitive. The Mis4 protein is required during S phase and associates with chromosomes during the entire cell cycle. These diverse mutant phenotypes indicate that adherins play fundamental roles in chromosome structure. Although it is not yet known if Nipped-B also participates in mitotic or meiotic chromosome structure, its homology to adherins suggests explanations for how Nipped-B could architecturally facilitate enhancer-promoter communication. It is tempting to speculate, for example, that the biochemical activity of Nipped-B is to recognize and stabilize chromatin loops that hold distant chromosomal sites closer together. The chromatin loops could be created by other factors involved in enhancer-promoter interactions (Rollins, 1999 and references).

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

Continued: see suppressor of Hairy wing Regulation part 2/2

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

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