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