Protein Interactions

EVE was found to repress all promoters that contained EVE-binding sites, including both TATA-containing and TATA-lacking minimal promoters, as well as promoters activated by several different classes of activator proteins. In addition to the DNA-binding domain, a potent repressor requires a repression domain, which can be as small as 27 residues. The minimal 57-residue EVE repression domain is proline rich and contains a high percentage of hydrophobic residues, especially alanine (Han, 1993).

C-terminal 236 amino acids of the Even-skipped protein (region CD) repress transcription. The CD region acts by inhibiting TFIID binding. Occupancy of secondary sites within promoters by EVE protein inhibits subsequent TFIID binding resulting in transciptional repression (Austin, 1995).

There is a direct interaction between EVE and the TATA-binding protein (TBP). EVE can efficiently either block or squelch promoters whose activity can be enhanced by coexpression of TBP. Squelching does not require EVE DNA-binding sites but is dependent on the presence of the EVE repression domain. Blocking requires an intact EVE repression domain and the conserved C-terminus of TBP. The EVE homeodomain is also required for these associations, suggesting that it may function in protein-protein interactions (Um, 1995).

The Drosophila homeodomain protein Even-skipped (Eve) is a transcriptional repressor. The mechanism of Eve repression involves a direct interaction with the TATA binding protein (TBP) which blocks binding of TBP-TFIID to the promoter. Eve activities were compared in in vitro transcription systems reconstituted with either all the general transcription factors or only TBP, TFIIB, TFIIF30, and RNA polymerase II. In each case, equivalent and very efficient levels of repression are observed, indicating that no factors other than those in the minimal system are required for repression. Eve can function efficiently when its recognition sites are far from the promoter; the same regions of Eve required for repression in vivo are necessary and sufficient for in vitro repression. This includes, in addition to an Ala-Pro-rich region, residues within the homeodomain. Eve-ABCD, in which the C-terminal 130 residues of Eve (Eve-EF) are deleted, retains full repressive activity. This derivative functions like full-length Eve in transfection and TBP binding assays. In sharp contrast, two other Eve derivatives, Eve-ABF and Eve-ABEF, which lack the defined repression domain, are completely inactive, indicating that the Ala-Pro-rich region is essential for transcriptional repression in vitro. In previous studies, the Eve repression domain CD was further subdivided to produce fragments that have various degrees of repressive activity in transfection assays. C2D2, called the minimal repression domain, contains the majority of Ala-Pro residues and imparts to the homeodomain nearly full repression and TBP binding activities. Another His-tagged Eve derivative, Eve-BC2D2, was prepared that contains only the homeodomain plus the minimal repression domain, and its repressive activity was tested. The 135-residue Eve-BC2D2 protein functions as a strong binding site-dependent repressor, with activity comparable to that of full-length Eve. As expected, gel shift assays indicated that BC2D2 binds DNA with an affinity similar to that of wild-type Eve. Taken together, these data show that Eve's ability to repress in vitro transcription correlates completely both with repression activity in vivo and with TBP binding. Using GAL4-Eve fusion proteins, it has been demonstrated that in addition to DNA binding, the homeodomain plays a role in repression. This additional role is to facilitate interaction with TBP. Single-round transcription experiments indicate that Eve must function prior to TBP binding to the promoter, suggesting a mechanism whereby Eve represses by competing with the TATA box for TBP binding. Consistent with this, excess TATA box-containing oligonucleotide is shown to specifically and efficiently disrupt the TBP-Eve interaction. Importantly, it has been shown that Eve binds directly to TFIID and that this interaction can also be disrupted by the TATA oligonucleotide. It is concluded that Eve represses transcription via a direct interaction with TBP, which blocks TFIID binding to the promoter (Li, 1998).

Prospero is a sequence-specific DNA-binding protein with novel sequence preferences that can act as a transcription factor. The consensus binding site for Pros protein is C A/C c/t N N C T/c. Pros binds to a 21-bp fragement of the asense promoter, which contains a CATTTCT sequence, resembling the consensus sequence. Pros binds to a synthetic oligomer containing multiple consensus sequences and activates transcription when this sequence is used as a promoter. The nervous system expression of even-skipped and fushi tarazu requires both these genes in addition to pros for normal function. Prospero can interact with homeodomain proteins to differentially modulate their DNA-binding properties (Hassan, 1997).

The relevance of functional interactions between Prospero and homeodomain proteins is supported by the observation that Prospero, together with the homeodomain protein Deformed, is required for proper regulation of a Deformed-dependent neural-specific transcriptional enhancer. Deformed and mouse Hoxa-5 binding to this neuronal enhancer is increased more than 10 fold by Pros. Pros reduces Eve's DNA binding to this enhancer, but does not modulate the binding of Engrailed. This interaction is unidirectional and specific, since neither Dfd, Eve nor En has an effect on Pros binding. The modulation by Pros does not require Pros binding to DNA. Pros protein modifies the trypsin sensitivity of Dfd protein, suggesting that Pros binds Dfd and is able to induce a conformation change in Dfd. Nevertheless, Pros is able to bind the Dfd neuronal autoregulatory enhancer and enhances Dfd binding to this DNA sequence. The DNA-binding and homeodomain protein-interacting activities of Prospero are localized to its highly conserved C-terminal region, and the two regulatory capacities are independent (Hassan, 1997).

Working out the effects of Rpd3 on segmentation gene expression requires the scientific equivalent of investigative journalism. The Berkeley Drosophila Genome Project identified a P-induced lethal mutation (l(3)04556) that maps 47 bp downstream of the Rpd3 putative transcription start site. Previous work by Perrimon (1996) has shown that embryos derived from l(3)04556 homozygous germline clones exhibit pair-rule patterning defects that are similar to those observed in fushi tarazu mutants. It was first established that most repressors are active in Rpd3 mutant embryos. These results suggest that the Rpd3 mutation might impair expression of Fushi tarazu or Ftz-F1 proteins, known to be gene activators, because these are required for the expression of the even-numbered engrailed stripes. Alternatively, the loss of Rpd3 might lead to a change in the expression pattern of a repressor, which in turn inhibits Ftz activity (Mannervik, 1999).

To distinguish between these possibilities, an examination was made of the expression of odd-skipped, a known repressor of en. odd is initially expressed in a pair-rule pattern of seven stripes, but during gastrulation seven additional secondary stripes are formed to generate a 14-stripe expression pattern. In normal embryos, these stripes are evenly spaced, whereas in Rpd3 mutants they are not. In the mutant embryos there is a partial pair-wise alignment of adjacent odd stripes. A similar change is observed in eve embryos. Previous studies suggest that both ftz and odd stripes are under the control of the Eve repressor. Differential repression of ftz and odd resolves the two patterns, so that each ftz stripe is normally shifted anterior to each odd-numbered odd stripe. In Rpd3 mutants, the ftz and odd patterns fail to resolve, so that odd-numbered odd stripes mostly coincide with the ftz stripes. It is suggested that this failure in ftz-odd resolution is responsible for the pair-rule phenotype observed in Rpd3 mutant embryos. A prediction of this proposal is that eve mutants should exhibit similar alterations in the ftz and odd expression patterns. Double staining assays reveal that eve mutant embryos exhibit a similar failure to resolve the ftz and odd expression patterns (Mannervik, 1999).

There are several possible explanations for impaired Ftz function in Rpd3 mutants. It is conceivable that the Rpd3 mutation disrupts Ftz-mediated activation. However, the idea that Rpd3 functions as a corepressor of Eve is favored. The similarities in the Rpd3 and ftz mutant phenotypes may be caused by the coincident odd and ftz expression patterns observed in embryos derived from l(3)04556 germline clones. The Odd repressor is thought to block Ftz-mediated activation of en. Evidence is presented that this expansion in Odd might result from an inability of Eve to repress odd expression in Rpd3 mutant embryos. Consistent with this proposal, in vitro translated Eve is shown to interact with a glutathione S-transferase-Rpd3 fusion protein. Because the Eve repressor is required for both the odd- and even-numbered en stripes, it would appear that the Rpd3 mutation does not cause a general loss of Eve function. For example, eve hypomorphs cause the loss of odd-numbered en stripes, whereas null mutations cause a loss of all en stripes. It would therefore appear that Eve fails to repress certain promoters (e.g., odd and possibly ftz) in Rpd3 mutant embryos, but retains repressor function on other promoters (e.g., paired and sloppy-paired). This selectivity in the regulation of different target promoters is consistent with the notion that Eve mediates repression through multiple mechanisms, including the recruitment of corepressors and direct interactions with TBP. Multiple modes of repression may be mediated by other transcriptional repressors, such as Hairy, which appears to interact with different classes of corepressors (Mannervik, 1999 and references).

Protein Interactions: Eve and Grunge

As a dedicated repressor, the mechanism of transcriptional repression by eve has been extensively studied. Previous studies have shown that Eve can interact with the TATA binding protein (TBP) to prevent the binding of TBP to TATA elements, suggesting that it represses transcription by disrupting the formation of a preinitiation complex at the promoter (Li, 1998). However, TBP binding alone can not account for all of Eve's repressive activity, since Eve does not disrupt a preformed TFIID-TATA complex to prevent transcription (Li, 1998). Furthermore, Eve can inhibit transcription from a TATA-less promoter, which does not require the presence of TBP. These findings suggest that Eve might utilize additional mechanisms to carry out its repressive function. In addition, Eve has been shown to interact with Rpd3, a histone deacetylase, to regulate gene expression (Zhang, 2001).

The data suggests that Eve requires Atrophin/Grunge to repress transcription during embryogenesis. Atro is a ubiquitously expressed nuclear protein with strong maternal contribution. Strong dosage-sensitive genetic interactions are observed between eve and maternal Atro. The expression of eve's target genes wg and en is derepressed in the eve and Atro double heterozygous embryos with reduced Atro maternal contribution, a phenotype reminiscent of mutants with reduced eve activity. Using in vitro binding assays, it was found that Eve can interact with Atro protein via Eve's minimal repression domain. Finally, using the in vivo repression assay, it was shown that Atro can directly repress transcription when tethered to DNA via the Gal4 DNA binding domain. Together, these data support the notion that Atro/Grunge functions as a corepressor for Eve during embryogenesis (Zhang, 2001).

To define the region in Eve responsible for its interaction with Atro, a series of GST-Eve deletions were generated. Previous work has divided the Eve protein into six regions (regions A-F). It was found that neither Eve's homeodomain (region B) nor the EF region shows significant interaction with Atro. Instead, only the CD region of Eve binds to the Atro protein. Further deletion of either region C or D significantly reduces its binding ability to Atro. This CD region has been previously defined as Eve's minimal repressor domain, suggesting that the minimal repression domain in Eve functions to bind Atro (Zhang, 2001).

Previous study has demonstrated that the CD region in Eve protein, a small alanine- and proline-rich region, functions as Eve's minimal repression domain, although the mechanism of repression mediated by this small domain is not clear. The genetic and biochemical data suggest that the function of this minimal repression domain is to bind to the Atro protein. Furthermore, it seems likely that Eve can bind to multiple corepressors at the same time, because another region of Eve has been shown to interact with Gro. Thus, in addition to its ability to interfere with TBP function and interact with Rpd3, Eve might recruit a battery of corepressors to achieve its potent repressive power (Zhang, 2001).

fushi tarazu is expressed at the blastoderm stage in seven stripes that serve to define the even-numbered parasegments. ftz encodes a DNA-binding homeodomain protein and is known to regulate genes of the segment polarity, homeotic, and pair-rule classes. Despite intensive analysis in a number of laboratories, how ftz is regulated and how it controls its targets are still poorly understood. To help understand these processes, a screen was conducted to identify dominant mutations that enhance the lethality of a ftz temperature-sensitive mutant. Twenty-six enhancers were isolated, which define 21 genes. All but one of the mutations recovered show a maternal effect in their interaction with ftz. Three of the enhancers proved to be alleles of the known ftz protein cofactor gene ftz-f1, demonstrating the efficacy of the screen. Four enhancers are alleles of Atrophin (Atro), the Drosophila homolog of the human gene responsible for the neurodegenerative disease dentatorubral-pallidoluysian atrophy. Embryos from Atro mutant germ-line mothers lack the even-numbered (ftz-dependent) engrailed stripes and show strong ftz-like segmentation defects. These defects likely result from a reduction in Even-skipped (Eve) repression ability, since Atro has been shown to function as a corepressor for Eve. In this study, evidence is presented that Atro is also a member of the trithorax group (trxG) of Hox gene regulators. Atro appears to be particularly closely related in function to the trxG gene osa, which encodes a component of the brahma chromatin remodeling complex. One additional gene was identified that causes pair-rule segmentation defects in embryos from homozygous mutant germ-line mothers. The single allele of this gene, called bek, also causes nuclear abnormalities similar to those caused by alleles of the Trithorax-like gene, which encodes the GAGA factor (Kankel, 2004).

Four of the ftz enhancers isolated in the screen proved to be alleles of Atrophin (Atro). Polyglutamine tract expansion within one of the human homologs of Atro (Atrophin-1) causes the neurodegenerative disease dentatorubral-pallidoluysian atrophy. Humans possess at least one additional Atrophin family member, Atrophin-2, which encodes a protein that can heterodimerize with Atr1. The functions of the mammalian Atrophin proteins are not well characterized. However, a role in gene repression seems likely, because Atr1 binds Eto1, a corepressor complex component, and overexpression of Atr1 can repress transcription of a reporter gene in tissue culture cells. In addition, Atr2 has been shown to interact with the histone deacetylase Hdac1. Compelling evidence has been presented that Atro also functions as a corepressor in Drosophila. eve mutations show strong dominant lethality when crossed to mothers heterozygous for Atro alleles. In the eve/+; Atro/+ embryos produced in this cross, odd-numbered en stripes are expanded, suggesting a weakening in the ability of Eve to repress paired, runt, or sloppy-paired (other pair-rule genes involved in specifying these stripes). Atro binds to the minimal repression domain of Eve, and artificial recruitment of Atro to a target gene can cause repression in vivo. A failure in the repressive activity of Eve may account for the absence of even-numbered en stripes described for embryos from Atro mutant germ-line mothers. In normal development, the even-numbered en stripes form as a result of differential repression of ftz and odd-skipped (odd) by Eve. Ftz is an activator of en, whereas Odd is a repressor. The even-numbered en stripes form where odd, but not ftz, has been repressed by Eve. If there were a failure of Eve to repress odd, zones expressing ftz but not odd would not form, and the even-numbered en stripes would not be established. Exactly this mechanism appears to be responsible for a reduction in even-numbered en stripes in mutants for the Rpd3 histone deacetylase. However, it is also possible that the even-numbered en stripes fail to appear in Atro- embryos because of a defect in the ability of Ftz to activate en. It is important to note that the odd-numbered en stripes are established almost normally in Atro mutant embryos (although they are wider than normal). These stripes are thought to be defined by differential repression of sloppy-paired, runt, and paired by Eve; the presence of these stripes in Atro- embryos indicates that Atro is not required for all repressive activities of Eve (Kankel, 2004).

Protein Interactions: Eve and Groucho

Groucho acts as a co-repressor for several Drosophila DNA binding transcriptional repressors. Several of these proteins have been found to contain both Groucho-dependent and -independent repression domains, but the extent to which this distinction has functional consequences for the regulation of different target genes is not known. The product of the pair-rule gene even skipped contains a Groucho-independent repression activity. Outside the Groucho-independent repression domain of Eve, a conserved C-terminal motif (LFKPY), similar to motifs that mediate Groucho interaction in Hairy, Runt and Hückebein, has been identified. Eve interacts with Groucho in yeast and in vitro, and groucho and even skipped genetically interact in vivo. Eve with a mutated Groucho interaction motif, which abolishes binding to Groucho, shows a significantly reduced ability to rescue the eve null phenotype when driven by the complete eve regulatory region. Replacing this motif with a heterologous Groucho interaction motif restores the rescuing function of Eve in segmentation. Further functional assays demonstrate that the Eve C terminus acts as a Groucho-dependent repression domain in early Drosophila embryos. This novel repression domain is active on two target genes that are normally repressed by Eve at different concentrations: paired and sloppy paired. When the Groucho interaction motif is mutated, repression of each target gene is reduced to a similar extent, with some activity remaining. Thus, the ability of Eve to repress different target genes at different concentrations does not appear to involve differential recruitment or function of Groucho. The accumulation of multiple domains of similar function within a single protein may be a common evolutionary mechanism that fine-tunes the level of activity for different regulatory functions (Kobayashi, 2001).

When comparing the activities of altered proteins in their normal context in vivo, it is desirable to have a reliable dose-sensitive assay for function. eve is weakly haplo-insufficient, showing a reduced viability when only one copy is present. Although most single copy embryos have no apparent defects at the end of embryogenesis, most of them do have abnormally narrow odd-numbered parasegments at early stages of development. Conversely, introducing extra copies of eve using the rescuing transgene results in abnormally wide odd-numbered parasegments. The hypomorphic (pair-rule) eve phenotype arises from odd-numbered parasegments that are severely reduced in width, and are unstable, so that at later stages they are eliminated by processes that repair patterning defects. Thus, one copy of the endogenous gene is near a threshold of sufficiency for generating stable parasegments, and the spacing of parasegments is a dose-dependent assay for early eve function (Kobayashi, 2001).

Two copies of the wild-type eve transgene, inserted at various chromosomal locations, do not completely rescue the parasegment spacing defects of eve null mutants. Rather, a homozygous transgene phenocopies heterozygous eve, while a single (heterozygous) eve transgene produces a hypomorphic phenotype. This provides a means with which to compare the activities of altered Eve proteins expressed in their normal pattern, in either one or two copies. When the early parasegment spacing was examined in eve mutant embryos rescued by a homozygous transgene with a point mutation in the Groucho interaction domain (GID), it was found that odd-numbered parasegments were reduced, closely resembling an eve hypomorph. This was consistently observed with each of the GID mutants that abolished the Gro interaction. In contrast, in transgenic flies in which the wild-type Eve motif was replaced with the Hairy GID, odd-numbered parasegments were normal, or sometimes even slightly increased relative to even-numbered parasegments. Thus, the parasegment spacing parallels the relative strength of the in vitro interaction with Gro. The similarity in phenotype produced by one copy of the wild-type transgene and two copies of the GID-mutant transgene suggests that the activity of Eve is reduced about twofold by mutation of the GID. The more complete rescue by the Hairy GID-containing protein suggests that it has a stronger activity than does wild-type Eve, consistent with in vitro interaction data and the embryonic pattern rescue. However, the apparently more complete rescue of embryonic pattern was not fully reflected in the ability to rescue to adulthood (Kobayashi, 2001).

What is the significance of the two distinct Eve repression activities, only one of which is dependent on Gro? Gro could be required to repress a subset of Eve targets, whereas repression of other target genes might be Gro independent. Alternatively, the two repression activities might function cooperatively, in which case both activities might be required for repression of each target gene. Extensive molecular and genetic studies have identified several target genes that are likely to be directly repressed by Eve. The best characterized of these genes are sloppy paired (slp), paired (prd) and odd skipped (odd). The posterior boundaries of expression of slp and prd correspond to the anterior and posterior borders, respectively, of the odd-numbered en stripes. As these en stripes shift posteriorly, both when the dose of gro is reduced and when the GID is mutated, the boundaries of slp and prd may be coordinately shifted. slp and prd expression were examined in embryos rescued with a GID-mutated transgene. Both slp and prd expression were expanded in the eve domains, relative to wild-type embryos. The degree of expansion of each gene correlates with the shift of en stripes. Furthermore, both the width of individual en stripes and their spacing are very similar to those in eve hypomorphs. Thus, the removal of the GID has an effect that is similar to that of a general reduction of eve activity on both targets, slp and prd. This expansion of slp and prd expression is reversed, in each case, when the Eve GID motif is replaced by that of Hairy. These results suggest that Gro is required by Eve to a similar degree for its repression activity on each of these genes (Kobayashi, 2001).

Repression of another Eve target gene (odd) is required for the establishment of the even-numbered (ftz-dependent) en stripes. Intriguingly, these are established more or less normally in embryos rescued by the GID-mutated transgene, and examination of odd expression in those embryos has showen it to be normal in the even-numbered parasegments. However, repression of odd and the establishment of even-numbered en stripes are also normal when eve function is reduced in other ways (e.g. in the hypomorph), suggesting that a lower threshold of Eve activity is required for this eve function than for proper repression of slp and prd. Therefore, this assay did not allow for a full assessment of the contribution of Gro to odd repression by Eve (Kobayashi, 2001).

Initially two repressors were characterized in transient-transfection assays: Eve and Engrailed (En). Although both contain Ala-rich motifs and display some similarities in activity in these assays, it is now clear that their mechanisms are distinct. This was suggested initially by one difference in their behavior in transfection assays: Eve can repress basal (i.e., non-activated) expression whereas En cannot. Using in vitro binding assays, an interaction between En and TBP could not be detected. More recently, genetic and biochemical experiments have shown that an interaction between the En repression domain and the corepressor Groucho can be required for En-mediated repression in vivo. In contrast, the Eve repression domain does not interact with Groucho and Eve-mediated repression is Groucho independent. The global repressor Dr1 also contains an Ala-rich domain that is essential for repression, and recent studies have shown that this region also interacts with TBP. However, since Dr1 blocks a step subsequent to DNA binding, the Eve-TBP and Dr1-TBP interactions are functionally distinct. The Drosophila Kruppel protein likewise contains an Ala-rich repression domain, which is located at its N terminus. Although its precise mode of action is not known, it is likely distinct from those just described and may involve a quenching mechanism. (Note that Kruppel also contains a C-terminal repression domain, one not Ala-rich, that appears to function by interacting with the small subunit of TFIIE). Thus, while the presence of an Ala-rich region in a transcription factor may be diagnostic of a repression domain, it does not suggest a clear mechanism, since four Ala-rich repression domains appear to employ four distinct mechanisms. Many other repressors, of course, contain repression domains that are not Ala-rich, and it appears that repression motifs are as diverse in composition and function as are activation domains (Li, 1998 and references).

Interactive Fly, Drosophila even-skipped: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Post-transcriptional regulation | Targets of activity | Developmental Biology | Effects of Mutation | References

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