ovo
ovo sequences (from the start of the ovarian transcript of ovo to 1.9 kb downstream of the start site) are sufficient for germ-line-specific sex-biased ovo transcription. In the germarium, staining for a reporter gene attached to these promoter sequences is seen in stem cells, cystoblasts and young cysts (Oliver, 1994).
The ovo+ and ovarian tumor+ (otu) genes function in the germline sex determination pathway in Drosophila,
but the hierarchical relationship between them is unknown. Increased ovo+ copy number
results in increased ovarian tumor expression in the female germline and increased ovo expression in
the male germline. Males with two or three copies of ovo+ show increased staining activity in the apex of the testis. The zone of expression does not extend into the region of advanced primary spermatocytes, suggesting that the regulation of germline OVO mRNA abundance and the regulation of stage-specific expression are distinct. The correlation between ovo+ copy number and the degree of transactivation of a reporter, strongly suggests that ovo+ is autoregulated in the male germline, either directly or indirectly (Lu, 1998).
The bacterially expressed Ovo
zinc-finger domain binds to multiple sites at or near the ovo and ovarian tumor promoters. This strongly
suggests that Ovo is directly autoregulatory and that ovarian tumor is a direct downstream target of
ovo in the germline sex determination hierarchy. Both positive and negative regulation by Ovo
proteins appear likely, depending on promoter context and on the sex of the fly. The most striking observation is the presence in females of protected regions overlapping the ovo-B transcription initiation site, and near the major start sites for the otu promoter. In the case of the ovo-B promoter, the protected region is no more than about 10 base pairs from the three principle start sites and extends about 23 bp downstream of these start sites. Both appear to be high affinity sites based on DNase protection and gel shift assays. There are three binding sites located between the ovo-A and ovo-B transcription start sites. Additional binding sites are found upstream from the ovo-A promoter and downstream of the ovo-B promoter. Two sites between the ovo-A and the ovo-B promoters show high binding activity. Two Ovo-binding sites upstream of the otu promoters are found at positions 370-389 and 401-422 in a region required for otu+ function in vivo (Lu, 1998).
Sequence alignment reveal an 11-bp consensus sequence located centrally within each of the nine binding sites. The strong binding site at the ovo-B promoter has a direct repeat separated by a single G residue. The first A residue in this sequece is the Ovo-B transcripiton start site.
The observation that
two strong Ovo-binding sites are at the initiator of the TATA-less ovo-B and ovarian tumor promoters
raises the possibility that Ovo proteins influence the nucleation of transcriptional pre-initiation
complexes (Lu, 1998).
Utilization of tightly linked ovo-A vs. ovo-B germline promoters results in the expression of OVO-A and OVO-B, C2H2 transcription factors with different N –termini, and different effects on target gene transcription and on female germline development. Two sex-determination signals, the X chromosome number within the germ cells and a female soma, differentially regulate ovo-B and ovo-A. Ovo regulates ovarian tumor transcription by binding the transcription start site. The regulation of the ovo-B promoter was explored using an extensive series of transgenic reporter gene constructs to delimit cis-regulatory sequences as assayed in wild-type and sex-transformed flies and flies with altered ovo dose. Minimum regulated expression of ovo-B requires a short region flanking the transcription start site, suggesting that the ovo-B core promoter bears regulatory information in addition to a "basal" activity. In support of this idea, the core promoter region binds distinct factors in ovary and testis extracts, but not in soma extracts, suggesting that regulatory complexes form at the start site. This idea is further supported by the evolutionarily conserved organization of Ovo binding sites at or near the start sites of ovo loci in other flies (Bielinska, 2005).
A reasonable understanding exists of the germline pathway centered on ovo. OVO-A and OVO-B functions are in a delicate balance in the female germline. OVO-B is absolutely required for oogenesis and is downregulated by OVO-A. An excess OVO-A results in defective oogenesis and subsequent embryogenesis, while too little results in defective germline function in progeny. Having the female soma repress ovo-A function in the germline may prevent damage to developing eggs, while the positive effect of a 2X karyotype may ensure that OVO-A protein is ultimately deposited in those eggs. OVO-B can have a positive effect on the ovo-B promoter following the deletion of some promoter-proximal sequences, but negative autoregulation occurs in all reporters. This difference between response to OVO-A vs. OVO-B does not appear to be due to different inherent strengths of the two transcription factors, since the otu promoter, a direct target of ovo, is strongly positively regulated by OVO-B in addition to being negatively regulated by OVO-A. Further, this difference in response dose not appear to be due to the ovo-B core promoter sequence, since in the otu sequence milieu, the ovo-B promoter is also strongly positively regulated by OVO-B. Thus, the ovo context is likely to specifically dampen the trans effect of OVO-B, but not OVO-A, on ovo-B promoter activity (Bielinska, 2005).
The ovo-B promoter encodes the OVO-B isoforms required and sufficient for female germline development and is regulated by the number of X chromosomes in the germline cells, and the sex of the surrounding soma positively regulates ovo-B, even though neither signal is absolutely required. For example, only 1X males fail to robustly express ovo-B in the germline, suggesting that both the intrinsic 2X signal and the extrinsic female somatic signal can upregulate ovo-B independently. Also it is known that somatic signaling is not required for ovo genetic function, because 2X males have germline cells, while 2X males lacking ovo do not. This dual regulatory input ensures that ovo-B is most highly expressed in the cells that require ovo activity—wild-type female germ cells. ovo-A expression is more dynamically regulated. The highest ovo-A promoter activity is in 2X males, followed by 2X females, 1X males, and 1X females. This pattern suggests that a 2X karyotype activates ovo-A, while a female soma inhibits ovo-A activity within the germline (Bielinska, 2005).
The combination of negative and positive autoregulation adds considerable complexity to the regulatory circuit. For example, the positive effect of a female soma on the expression of ovo-B in the working model could be due to repression of ovo-A expression by a female soma, followed by derepression of ovo-B because of lowered OVO-A levels, or a more direct positive effect of the female soma on ovo-B (Bielinska, 2005).
Analysis of promoters active in the germline of Drosophila suggests that they are often more compact than many of the promoters studied in somatic cells. This may be the case for ovo-B. While the ovo-B core promoter alone is insufficient for transcription, transcriptional activity from ovo-B is remarkably resistant to deletions from either the 5' or the 3' direction. The lacZDeltaapDelta6 reporter has only 268 bp of ovo sequence but is expressed in the female germline. The overlap between the lacZDeltaapDelta6 and lacZDeltaapDelta8 reporters, both of which are expressed, is only 73 bp. This is unusually close to the transcription start site. The OVO binding site footprints overlap the transcriptional start sites of both otu and ovo-B , and there are proteins or complexes in gonad extracts that bind to this core sequence. It is therefore suggested that OVO alters the structure of the core promoter and promotes preinitiation complex formation. The highly conserved position of OVO binding sites at ovo-B in multiple species of flies supports the idea that OVO functions at the transcription start site. A recent study of human promoters suggests that the binding of transcription factors within 100 bp of the transcription start site may be more common than previously thought (Bielinska, 2005).
The importance of the core promoter raises some interesting questions about how ovo interprets the number of X chromosomes in the germline and the sex of the surrounding soma. For example, the Sex-lethal gene counts X chromosomes in the soma by binding several transcription factors, encoded on the X chromosome, to a region rich in the corresponding binding sites. The balance toward expression of Sxl is thus tipped by a graded occupancy at a complex cis-regulatory module. There does not appear to be an extended cis-regulatory module that is essential for the qualitative expression of ovo. Perhaps sex-determination signals indirectly regulate ovo. The molecular nature of the karyotype and somatic signals to the germline is a major unresolved problem in germline sex determination (Bielinska, 2005).
In Drosophila, compatibility between the sexually differentiated state of the soma and the constitution of the sex chromosome in the germline is required for normal gametogenesis. In this study, important aspects of the soma-germline interactions controlling early oogenesis are defined. In particular, the sex-specific germline activity of the ovarian tumor (otu) promoter has been demonstrated to be dependent on somatic factors controlled by the somatic sex differentiation gene transformer. This regulation defines whether there is sufficient ovarian tumor expression in adult XX germ cells to support oogenesis. In addition, the ovarian tumor function required for female germline differentiation is dependent on the activity of another germline gene, ovo, whose regulation is transformer-independent. These and other data indicate that ovarian tumor plays a central role in coordinating regulatory inputs from the soma (as regulated by transformer) with those from the germline (involving ovo). transformer-dependent interactions influence whether XX germ cells require ovarian tumor or ovo functions to undergo early gametogenic differentiation. These results are incorporated into a model that hypothesizes that the functions of ovarian tumor and ovo are dependent on an early sex determination decision in the XX germline -- a decision that is at least partially controlled by somatic transformer activity (Hinson, 1999 and references).
With respect to interactions with the germline, transformer (tra) is the most extensively studied of the somatic sex regulatory genes. The masculinization of XX soma due to loss-of-function tra mutations causes germ cell aberrations during first instar larval stages and misregulates sex-specific germline gene expression in the embryo. Furthermore, when XY soma is feminized by ectopic tra expression (to form 'pseudofemales', the somatic components of the ovaries are sufficiently 'female' so that they can support the maturation of transplanted XX germ cells. The pseudofemale soma also appears to partially feminize the XY germline, since these cells now require the normally female-specific otu function for optimal proliferation. These observations indicate that tra controls a substantial portion of the somatic-germline interactions affecting early gametogenic differentiation (Hinson, 1999 and references).
In Drosophila, the sexual differentiation of the germline requires a complex interplay between cell autonomous factors controlled by the X:A ratio of the germ cells and sex-specific somatic functions. For example, certain allele combinations of transformer, transformer-2 and doublesex can cause chromosomally female (XX) flies to develop with most of their somatic tissues having a male identity, i.e., ‘XX pseudomales’. In these flies, oogenesis is aborted and there is even occasionally what appears to be early spermatogenic development. Since the germline expressions of these sex regulatory genes are not required for early stages of gametogenesis, the aberrant germline phenotypes must result from the male transformation of the soma (Hinson, 1999 and references).
It is not clear which germline genes are influenced by the proposed somatic interactions. Three possible candidates based on their early and sex-specific roles in female germline differentiation are ovarian tumor, ovo and Sex-lethal (Sxl). During oogenesis, the expression of otu is required in the germline at several stages, if not continually. The null mutant phenotype is characterized by the absence of egg chambers in an otherwise normal ovary, denoted as the quiescent phenotype, although substantial numbers of germ cells are still present in the germarium. Null and severe loss-of-function mutations can also produce 'ovarian tumors', a phenotype characterized by egg chambers containing hundreds of seemingly undifferentiated germ cells. Both the quiescent and tumorous cells are aborted at early oogenic stages, during the cystocyte divisions prior to cyst formation. Mutations in otu have no significant effect on spermatogenesis, although some aberrations in male courtship behavior have been reported. The ovo gene has been implicated in regulating sex determination and dosage compensation in the germline. This is based primarily on observations that ovo null XX germ cells are typically not found in the adult ovary, presumably because of reduced cell viability. In addition, certain ovo allele combinations produce tumorous germ cells that morphologically resemble primary spermatocytes. These phenotypes make ovo a candidate target for a somatic signal regulating early oogenesis, although the expression of ovo in adult germ cells does not appear to be responsive to somatic influences. ovo might directly regulate otu. The Ovo protein can bind to sites in the otu promoter, which displays sensitivity to changes in the dosage of ovo + function. It is not known when this putative regulation of otu occurs nor what role it plays in oogenesis (Hinson, 1999 and references).
The effects of an ovo null mutation on XX germ cells developing in pseudomale testes and female ovaries were examined. In females, ovo mutant XX germ cells typically arrest beginning at larval gonial stages. Occasionally, mutant germ cells survived to the adult stage. However, these cells generally failed to undergo gametogenic differentiation as seen by the absence of spectrosomes, fusomes or ring canals. It was reasoned that, if the requirement for ovo is solely dependent on the X:A ratio, then the phenotype of ovo mutant germ cells in pseudomales should be at least as severe. In this case, the ovo mutant XX pseudomale gonads should be either atrophic or contain a few clusters of mostly undifferentiated germ cells. There is an increase in the frequency of atrophic gonads (82%) compared to normal pseudomales (48%), many of the non-oogenic type. The non-oogenic gonads contained VASA-positive germ cells. This indicates that not only are a substantial fraction of the mutant germ cells viable in adults, but gametogenic differentiation occurs as well. The frequency of the non-oogenic gonads in ovo mutant pseudomales is essentially unchanged from that observed in normal pseudomales. This suggests that the observed increase in the atrophic category is due primarily to the loss of the oogenic class. Mutations in otu gave results similar to those described for ovo. This suggests that otu and ovo mutations specifically disrupt only those germ cells attempting female differentiation, rather than the indiscriminate elimination of the entire XX germline (Hinson, 1999).
Heat shock-otu can alter the XX pseudomale gonadal phenotype; to examine whether and to what degree otu expression could induce oogenic development in pseudomales, immunohistochemical studies were performed. When continually cultured at 20-25°C, hs-otu pseudomale gonads are as much as two to three times longer than normal. In addition, 88% of the hs-otu gonads examined show extensive Hu-li tai shao-labeling of ring canals (Hts is an adducin-like protein). These feminized gonads display a developmental progression of gametogenic stages. In section III of the gonad, the pseudomale germ cells have differentiated to postgermarial stages as defined by the expression of kelch. Kelch, an actin binding protein, is localized to female ring canals after the ring canal deposition of Hts and f-actin . Kelch is first detected in female ring canals in stage 1 egg chambers, but is not seen in all ring canals until stage 4. In hs-otu XX pseudomales, the germ cell clusters in section III contain thick ring canals, with virtually all of them showing Kelch deposition along the inner surface of the f-actin layer. In comparison, no Kelch-labeled ring canals are observed in XX pseudomales without hs-otu, indicating that oogenesis is not only less frequent, but also more limited. Taken together, these results indicate the masculinizing effect of male soma (or the absence of female soma) on XX germ cells can be partially, but consistently, overridden by the expression of otu from a heterologous promoter. The resulting fusome and ring canal development follows the same sequence of events as occurs in normal oogenesis. Therefore, pseudomale germ cells are competent to both initiate and undergo substantial oogenesis if provided with adequate levels of otu. Both ovo and Sxl were shown to be required for otu induced oogenic differentiation in XX pseudomales. However, an additional role for otu in some process affecting germline viability and/or proliferation can be identified that is separable from oogenic differentiation and independent of ovo and, possibly, Sxl functions (Hinson, 1999).
The finding that hs-otu can feminize XX pseudomale germ cells suggests oogenesis is blocked because of insufficient otu levels. Therefore, an examination was carried out to see whether tra-induced sexual transformation affects the level of otu gene expression. otu-lacZ is expressed in most, if not all, larval and pupal germ cells in both female and male gonads. Sex-specific regulation only becomes apparent in the adult testis where male germline expression become restricted to a few cells at the apical tip. As with otu, the ovo promoter is initially active in both male and female larval gonads. However, ovo-lacZ becomes sex-specific at an earlier stage, showing restricted expression in male gonads during the third instar larval and pupal periods. These results demonstrate that the otu and ovo promoters are under different regulatory control in the pre-adult germline. However, otu, but not ovo, promoter activity is influenced by tra-induced sexual transformation. These data demonstrate that the tra-induced sexual transformation specifically inhibits otu promoter activity. Also carried out was the reciprocal experiment, in which otu-lacZ activity was examined in XY germ cells developing in a female somatic background. XY pseudofemales produced by the ectopic expression of tra result in ovaries containing tumorous egg chambers. Because XY pseudofemale germ cells become sufficiently 'feminized' so that they acquire a need for otu function for optimal proliferation, it was anticipated they would also be permissive for otu promoter activity. This is in fact the case. Even in the absence of ovo function, XY pseudofemale germ cells consistently express otu-lacZ. This indicates that the feminizing effects of tra, but not ovo, are necessary for otu transcription. In comparison, the ovo promoter is not detectably active in XY pseudofemales, again illustrating differential regulation of ovo and otu (Hinson, 1999).
It is thought that during the
pupal and adult stages, two critical events occur in the female
germarium: (1) ovo activity allows XX germ cells
to become receptive to the otu function controlling oogenic
differentiation, and (2) tra-dependent somatic signals allow
continued expression of otu in the female germline by
maintaining otu promoter activity. The combination of these events constitutes a mechanism by which the otu gene serves to link the somatic sex
differentiation pathway controlled by tra with a female
germline developmental pathway controlled by ovo (Hinson, 1999).
One central, and yet unsolved, question in evolutionary biology is the relationship between the genetic variants segregating within species and the causes of morphological differences between species. The classic neo-darwinian view postulates that species differences result from the accumulation of small-effect changes at multiple loci. However, many examples support the possible role of larger abrupt changes in the expression of developmental genes in morphological evolution. Although this evidence might be considered a challenge to a neo-darwinian micromutationist view of evolution, there are currently few examples of the actual genes causing morphological differences between species. This study examined the genetic basis of a trichome pattern difference between Drosophila species, previously shown to result from the evolution of a single gene, shavenbaby (svb), probably through cis-regulatory changes (Sucena, 2000). Three distinct svb enhancers were identified from D. melanogaster driving reporter gene expression in partly overlapping patterns that together recapitulate endogenous svb expression. All three homologous enhancers from D. sechellia drive expression in modified patterns, in a direction consistent with the evolved svb expression pattern. To test the influence of these enhancers on the actual phenotypic difference, interspecific genetic mapping was conducted at a resolution sufficient to recover multiple intragenic recombinants. This functional analysis revealed that independent genetic regions upstream of svb that overlap the three identified enhancers are collectively required to generate the D. sechellia trichome pattern. The results demonstrate that the accumulation of multiple small-effect changes at a single locus underlies the evolution of a morphological difference between species. These data support the view that alleles of large effect that distinguish species may sometimes reflect the accumulation of multiple mutations of small effect at select genes (McGregor, 2008).
Differences in larval trichome pattern between Drosophila species offer an attractive model of morphological evolution. Over the past 30 years, numerous studies have identified upstream patterning and downstream effector genes regulating trichome development in D. melanogaster. Questions about the evolution of trichome patterns can therefore be formulated explicitly within a developmental framework (McGregor, 2008).
Although the pattern of ventral trichomes has been conserved for more than 60 Myr, new dorsal trichome patterns have evolved repeatedly. In most species of the D. melanogaster subgroup, the dorsal and lateral surface displays stout trichomes on 1° and 3° cells and naked 2° cells, and a lawn of fine trichomes on 4° cells. D. sechellia has evolved a trichome pattern in which 4° trichomes were replaced by naked cuticle. Interspecific whole-genome genetic mapping demonstrated that the D. sechellia 'naked' phenotype is recessive to the 'hairy' phenotype of other species and mapped this evolutionary change to a single X-linked gene, shavenbaby/ovo (svb). Svb is required cell-autonomously for trichome formation and encodes a transcription factor regulating several classes of effector genes, which collectively build trichomes (McGregor, 2008).
In D. melanogaster, D. simulans and D. mauritiana, svb is expressed in 1° and 3° dorsal cells and 4° dorsal and lateral cells. In D. sechellia, svb is expressed in 1° and 3° dorsal cells but not in the 4° cells, where trichomes are absent. Together with previous genetic analyses, these expression patterns suggest that changes in the cis-regulatory region of svb underlie this evolved morphological pattern (McGregor, 2008).
This study sought to identify enhancers that drive svb expression. A systematic series of D. melanogaster reporter constructs, from 50 kilobases (kb) upstream to 20 kb downstream of the first exon of svb was used. Reporter expression was precisely mapped by double-staining for Miniature, the product of a cell-autonomous target of svb that accumulates in trichomes (McGregor, 2008).
Three genomic regions were found to drive expression in the epidermis, just before trichome differentiation. Each element contributes to both evolutionarily conserved and evolutionarily derived expression patterns. Dorsal expression of the 'proximal' enhancer started in stage 13 embryos, in 1° and 3° cells. Beginning at stage 15, expression was observed in some dorsal, but not dorsolateral, 4° cell. The 'medial' enhancer drove expression in the dorsal 4° cells at stage 13 and later expanded into dorsolateral 4° cells. The 'distal' enhancer drove expression in thoracic dorsal stripes and lateral 4° cells, starting at stage 14 and strengthening later. In ventral trichome-producing cells, the proximal and medial enhancers drove strong expression and the distal enhancer drove weak expression. The epidermal expression of svb therefore seems to be regulated in a complex manner by three separable cis-regulatory elements spread over 50 kb (McGregor, 2008).
To determine whether these enhancers have evolved in D. sechellia, orthologous D. sechellia regions (which differ by 3%-5% from the D. melanogaster sequences) were identified, and their activity was assayed as transgenes in D. melanogaster. The D. sechellia 'proximal' enhancer drove expression in 1° and 3° dorsal cells in a pattern similar to that of the D. melanogaster 'proximal' enhancer. However, unlike the D. melanogaster enhancer, expression from the D. sechellia proximal enhancer was never observed in dorsal 4° cells. Expression of the D. sechellia 'medial' enhancer was restricted to dorsal 4° cells. In contrast with the D. melanogaster medial enhancer, expression of the D. sechellia enhancer started later and did not extend to the lateral region. The D. sechellia 'distal' enhancer drove expression in thoracic stripes, in a similar manner to the D. melanogaster enhancer, but expression was observed only in restricted lateral spots. At the time of trichome formation, each D. sechellia enhancer drove a ventral expression pattern similar to that of its D. melanogaster counterpart (McGregor, 2008).
These results show that all three svb enhancers have evolved in D. sechellia and that these changes reflect a precise loss of expression in 4° cells. In addition, the D. sechellia medial and distal enhancers retain some activity in 4° cells, indicating that sites outside these regions might be required to repress this activity. Finally, minor changes were observed in the conserved 1° and 3° dorsal cells, and in ventral cells. These results suggest that evolution of the D. sechellia svb expression pattern was caused by multiple changes of limited effect rather than by drastic elimination of entire enhancers (McGregor, 2008).
To test the actual function of these enhancers within their native genomic locations for patterning trichomes, high-resolution interspecific recombination mapping was performed. A two-step screen was designed to maximize the probability of identifying recombinants within the svb gene. First, a screen was performed for recombinants between visible markers that flanked svb by about 1.2 × 106 base pairs (bp) and then these selected individuals were scored with molecular markers to identify 50 individuals with recombination breakpoints within the svb locus. This experiment provided a resolution of about one recombination breakpoint every 2 kb (McGregor, 2008).
Recombinants that included the entire region upstream of the first svb exon from D. mauritiana produced trichome patterns indistinguishable from those of D. mauritiana. Conversely, chromosomes with the upstream svb region from D. sechellia produced a D. sechellia-like trichome pattern. These results demonstrate that the change(s) responsible for evolution of the D. sechellia phenotype are restricted to the genomic region that contains the three identified enhancers (McGregor, 2008).
If the D. sechellia trichome pattern resulted from the evolution of a single site, then only sechellia-like and mauritiana-like phenotypes would have been observed. Instead, three additional phenotypic classes were observed. First, recombinants that included only the proximal enhancer from D. mauritiana produced a few dorsal 4° trichomes (intermediate type 1). Second, a chromosome including the medial and proximal enhancers from D. mauritiana produced a dense pattern of 4° trichomes in the dorsal and dorsolateral region (intermediate type 2). Last, chromosomes that included only the distal enhancer from D. mauritiana produced a patch of dorsolateral and a few dorsal 4° trichomes (intermediate type 3). Backcrossing of all viable recombinant lines further ruled out any detectable influence of genomic regions outside svb on trichome patterns (McGregor, 2008).
These genetic results prove that at least three separate changes have evolved in the svb upstream region to cause trichome loss in D. sechellia. Furthermore, the recombination breakpoints localize functionally evolved sites to genomic positions containing enhancers defined by reporter constructs. The distal svb enhancer element includes CG12680, which has the potential to encode a short peptide. However, this gene is unlikely to contribute to the evolved difference because CG12680 is not expressed in embryos, and complementation assays implicate svb alone as the causal determinant. Finally, the recombinant intermediate phenotypes are similar to the expression patterns of the three individual enhancers. The combined results therefore imply that each enhancer contains at least one genetic change. These changes may have occurred sequentially by loss of expression from the distal, medial and proximal enhancers, or in any other order (McGregor, 2008).
Given that laboratory-induced mutations in dozens of genes alter trichome patterns, it is striking that multiple mutations at a single locus account for the entire evolved difference. Svb seems peculiar in the network of genetic interactions that establish the trichome pattern, because it sits at the nexus of the upstream patterning genes and the downstream effector genes. Although trichome pattern could be changed by altering any of several upstream genes, these changes would probably produce pleiotropic effects on other developmental processes. In contrast, none of the known downstream genes is sufficient on its own to prevent or promote trichome formation. Thus, changes at svb enhancers may provide the only available genetic mechanism to evolve trichome patterns without pleiotropic consequences (McGregor, 2008).
The results provide experimental evidence that the conflicting views of micromutationism and macromutationism can actually reflect observations of the same molecular mechanisms at different levels of resolution. Specifically, genes at integrative positions in developmental networks may be genetic 'hotspots' for evolutionary changes that differentiate species, although the individual mutations contributing to this change may be of smaller effect. Although results recently obtained from a broad range of species are consistent with this interpretation, only additional fine-scale functional analyses of morphological differences between species will allow a robust test of this hypothesis (McGregor, 2008).
Genes include cis-regulatory regions that contain transcriptional enhancers. Recent reports have shown that developmental genes often possess multiple discrete enhancer modules that drive transcription in similar spatio-temporal patterns: primary enhancers located near the basal promoter and secondary, or ‘shadow’, enhancers located at more remote positions. It has been proposed that the seemingly redundant activity of primary and secondary enhancers contributes to phenotypic robustness. This hypothesis was tested by generating a deficiency that removes two newly discovered enhancers of shavenbaby (svb, a transcript of the ovo locus), a gene encoding a transcription factor that directs development of Drosophila larval trichomes. At optimal temperatures for embryonic development, this deficiency causes minor defects in trichome patterning. In embryos that develop at both low and high extreme temperatures, however, absence of these secondary enhancers leads to extensive loss of trichomes. These temperature-dependent defects can be rescued by a transgene carrying a secondary enhancer driving transcription of the svb cDNA. Finally, removal of one copy of wingless, a gene required for normal trichome patterning, causes a similar loss of trichomes only in flies lacking the secondary enhancers. These results support the hypothesis that secondary enhancers contribute to phenotypic robustness in the face of environmental and genetic variability (Frankel, 2010).
The cis-regulatory region of the svb gene integrates inputs from multiple gene regulatory networks to generate a complex pattern of expression in the embryonic epidermis of insect species. SVB protein then activates many downstream genes, ultimately resulting in trichome morphogenesis. Three enhancer modules located in a 50 kilobase (kb) region upstream of the svb transcription start site (called 7, E and A) together recapitulate the complete svb epidermal expression pattern. Partial loss of function of all three enhancers led to the evolutionary loss of the long, thin quaternary trichomes on first-instar larvae of It was noticed that a 41 kb region upstream of the three known svb enhancers displays high conservation among drosophilids, but contains only one small gene named SIP3. To test whether this region contained additional svb enhancers, reporter constructs were assayed encompassing the entire region. Two constructs drove expression in the dorso-lateral epidermis in patterns that reproduced part of the native svb expression pattern. To characterize the precise expression domains driven by these newly discovered enhancers, co-immunodetection was performed of the β-galactosidase reporter and of the Dusky-like protein, an early component of developing trichomes (Frankel, 2010).
The Z enhancer drove expression in many cells that produce quaternary trichomes. This expression overlaps the patterns driven by the three enhancers identified previously: 7, E and A. The DG2 enhancer drove expression in a more restricted region that overlaps the domain of expression driven by the E enhancer. Both Z and DG2 drive expression starting at stage 14 of embryogenesis, which is similar to the time when svb mRNA can be detected in epidermal cells (Frankel, 2010).
Given the redundant expression patterns of Z and DG2 with the three enhancers identified previously, further evidence was sought that Z and DG2 encode functional svb enhancers. It was reasoned that if the Z and DG2 enhancers contribute to trichome patterning, then they should have evolved in a similar way to the previously discovered 7, E and A enhancers; they should retain expression in species that also produce quaternary trichomes (such as Drosophila simulans), and show reduced expression in D. sechellia, which has lost quaternary trichomes. Therefore Z and DG2 enhancer constructs made with orthologous regions from D. simulans and D. sechellia were assayed. These regions were straightforward to identify because the genomes of these species are 3-5% divergent from D. melanogaster. The D. simulans Z and DG2 enhancers drove an expression pattern similar to that of the orthologous D. melanogaster enhancers, which indicates that Z and DG2 contribute to the production of quaternary trichomes both in D. melanogaster and in D. simulans. In contrast, the Z and DG2 enhancers from D. sechellia drove low levels of expression in only a few cells. The weak expression driven by the D. sechellia Z and DG2 constructs is consistent with the partial loss of expression driven by the D. sechellia A, E and 7 enhancers and with the loss of quaternary trichomes in this species (Frankel, 2010).
To further assess the functional importance of the Z and DG2 enhancers, a 32 kb chromosomal deficiency was generated on the X chromosome that removes both enhancers, called Df(X)svb108. As a control, strain C108 was used, which carries both of the parental transposable elements that were used to generate the deletion. Df(X)svb108 flies are viable and display no gross abnormalities. First-instar larvae were examined in detail and it was found that, when Df(X)svb108 embryos developed at the optimal temperature for development (25°C), larvae exhibited slightly fewer quaternary trichomes and a reduction in the size of the lateral sensory bristles. These results indicate that, under optimal conditions, Z and DG2 are functional enhancers of the svb gene that contribute to fine details of trichome patterning and perhaps to bristle morphogenesis. Despite this evidence that the Z and DG2 enhancers contribute to svb activity, their loss-of-function phenotype was considerably weaker than one would have expected, given the strong expression driven by these enhancers. It was reasoned that this resulted from the fact that the Z and DG2 enhancers drive overlapping expression with the enhancers 7, E and A, and that the latter three enhancers drive expression levels that are sufficient to generate most larval trichomes when embryos develop under optimal conditions (Frankel, 2010).
Therefore the hypothesis that Z and DG2 contribute to phenotypic robustness was considered. Natural populations experience repeated stresses over evolutionary time, including variable temperatures. Temperature influences membrane fluidity, enzymatic activity, protein folding, protein-protein interactions, and protein-DNA interactions. Organisms have evolved developmental mechanisms to buffer the phenotype in the face of temperature-induced cellular changes. It esd reasoned that sub-optimal temperatures might destabilize the transcriptional output of genes during embryogenesis and that secondary enhancers may confer a selective advantage by maintaining transcription above a required minimum threshold. Therefore the effect of Df(X)svb108 was tested in embryos that had developed at 17 and 32°C, temperatures close to the extremes at which Drosophila embryos survive. The number of quaternary trichomes were counted in the regions where Z and DG2 are expressed strongly. The svb gene is an ideal target for this analysis, because quantitative changes in SVB level influence trichome density, size and shape (Frankel, 2010).
Control embryos reared at all temperatures produced similar numbers of trichomes, implying that the number of trichomes is canalized against temperature variation. The number of trichomes on Df(X)svb108 larvae reared at 25° C was similar to the number on control C108 larvae at all temperatures. In contrast, Df(X)svb108 larvae displayed a highly significant decrease in trichome numbers when reared at extreme temperatures. The primary and tertiary trichomes look normal on Df(X)svb108 larvae at all temperatures, which is expected, because the Z and DG2 enhancers do not drive expression in cells producing primary and tertiary trichomes (Frankel, 2010).
In principle, the loss of trichomes observed on Df(X)svb108 larvae reared at extreme temperatures may have resulted from mechanisms acting independently of the Z and DG2 enhancers. If the effects observed with Df(X)svb108 resulted from loss of the Z and DG2 enhancers, then reintroducing a functional Z or DG2 enhancer into a Df(X)svb108 background should rescue some trichomes. This hypothesis was tested for the Z enhancer. A transgene was generated carrying the svb cDNA under the transcriptional control of the Z enhancer and introduced onto the third chromosome of Df(X)svb108 flies. At extreme temperatures, the Z::svb cDNA transgene completely rescued wild-type trichome numbers in the lateral patch. However, in the region dorsal to the lateral patch, the rescue is very weak or absent. This is consistent with the fact that Z drives expression at high levels in the lateral region, where rescue is observed, and only weakly in a small number of cells of the dorsal region. The loss of canalization in the dorsal region of Df(X)svb108 larvae may be caused by loss of DG2, which drives expression mainly in this dorsal region. These results demonstrate that Z contributes to phenotypic robustness. Moreover, the rescue of trichome numbers by a transgene introduced onto a different chromosome from the svb locus indicates that Z does not need to be in intimate contact with other svb enhancers or with the svb basal promoter to buffer svb function. Instead, it is propose that Z contributes to phenotypic robustness simply by boosting levels of svb transcription in the cells in which Z drives expression (Frankel, 2010).
These results indicate that the production of larval trichomes is normally canalized and that this is accomplished, at least in part, through transcriptional activation mediated by the svb secondary enhancers that are removed in Df(X)svb108 (Frankel, 2010).
The svb locus contains multiple enhancers with overlapping expression patterns. Similar patterns of overlapping enhancer activity have been found for the cis-regulatory regions of the Drosophila genes sog and for the cis-regulatory regions of the mouse genes sonic hedgehog. A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers. Moreover, it has been estimated that 50% of the target genes of the transcription factor Dorsal contain shadow enhancers. Therefore, the presence of additional enhancers in cis-regulatory regions may be a common signature of developmental regulators. This may explain why, in previous reports, animals carrying deletions of highly conserved enhancers have not displayed observable phenotypic defects when reared in standard laboratory conditions (Frankel, 2010).
Developmental buffering is likely to result from many molecular mechanisms. For example, deletion of the conserved miRNA miR7 in D. melanogaster has no obvious phenotypic effect in normal laboratory conditions, but it is required to canalize the expression of the gene atonal under fluctuating temperatures. Similarly, the results indicate that svb secondary enhancers have a minimal role at optimal conditions for development, but that they are essential to buffer the trichome phenotype under genetic or environmental variability. Secondary enhancers are likely to be evolutionarily maintained by selection for robustness against temperature fluctuation, genetic background effects and expression noise (Frankel, 2010).
The Drosophila ovo locus codes for several tissue- and stage-specific proteins that all possess a common C-terminal array of four C2H2 zinc fingers. Three fingers conform to the motif framework and are evolutionarily conserved; the fourth diverges considerably. The ovo genetic function affects germ cell viability, sex identity and oogenesis, while the overlapping svb function is a key selector for epidermal structures under the control of wnt and EGF receptor signaling. Synthetic DNA oligomers bound by the OVO zinc finger array have been isolated from a highly complexity starting population, and a statistically significant 9 bp long DNA consensus sequence has been derived that is nearly identical to a consensus derived from several Drosophila genes known or suspected of being regulated by the ovo function in vivo. The DNA consensus recognized by Drosophila Ovo protein is atypical for zinc finger proteins in that it does not conform to many of the 'rules' for the interaction of amino acid contact residues and DNA bases. Additionally, these results suggest that only three of the Ovo zinc fingers contribute to DNA-binding specificity (Lee, 2000).
The function of four ovarian tumor genes (ovarian tumor, ovo, fused and snf) are required for the female-specific splicing of SXL pre-messenger RNA. A further ordering of the sex determination genes that function within the germ line can be inferred from the difference between the phenotypes produced by Sxl and snf mutations, versus otu and ovo mutations. Mutations of either otu or ovo result in the absence of 2X germ-line cells and sexual transformations of 2X germ-line cells. While Sxl or snf also result in sexual transformations, they do not have any effect on germ cell viability. A branched pathway provides the simple explanation for the differences found between these two groups of genes. Genes that function prior to the branch would be required for both germ cell viability and sex determation, whereas sex determination genes functioning after viability affects would be required only for sex determination roles (Oliver, 1993).
Three Ovo-binding sites exist in a compact
regulatory region that controls germline expression of the
otu gene. Interestingly, the strongest Ovo-binding site
is very near the otu transcription start, where basal
transcriptional complexes must function. Loss-of-function,
gain-of-function and promoter swapping constructs
demonstrate that Ovo binding near the transcription start
site is required for Ovo-dependent otu transcription in
vivo. These data unambiguously identify otu as a direct
Ovo target gene and raise the tantalizing possibility that
an Ovo site, at the location normally occupied by basal
components, functions as part of a specialized core
promoter (Lu, 2001).
An extensive set of transgenes have been prepared
with deleted and reconstituted Ovo-binding sites, which were
tested in females with differing doses of ovo+ . Removal of
Ovo-binding sites reduces or eliminates the response to ovo+
activity in trans, while reconstituting Ovo-binding sites
confers activity. These data indicate that Ovo protein
directly regulates otu transcription (Lu, 2001).
Surprisingly, Ovo functions very close to the transcription
start site of otu. Ovo footprints within 20 bp of the
transcription start sites of all but one of the reporter genes that
respond to ovo dose in trans. Indeed, in the case of the ovo-B
promoter, the transcription start site is in the middle of the
region protected by Ovo. It is a reasonable assumption that
RNA Polymerase II and basal transcription complex
components also bind this region. For example, the
TFIID complex protects about 60 bp, centered on the core
promoter. Certainly, RNA Polymerase II must contact the +1
position in the ovo-B promoter that is covered by Ovo protein
in vitro (Lu, 2001).
A standard model for transcriptional regulation holds that
the binding of regulatory factors at control regions modulates
the transcriptional activity of a variety of core promoters. In
this model, core promoters (where the start site is +1 and the
core promoter is from -35 to +35) can have different basal
strengths, but they have little regulatory information. While in
many cases different core promoters respond similarly to a
given enhancer, there is some evidence supporting the idea that
core promoters can bear important regulatory information. These
data suggest that the ovo-B and otu core promoters have a
regulatory function. Several possible mechanistic
explanations for the promoter proximal binding of Ovo to
these core promoters has been explored (Lu, 2001).
Binding of a regulatory protein to the transcription start site
is unusual. There are only a few core-promoter binding
proteins, such as AEF1 and YY1, that function in tissue or
promoter-specific transcriptional control. Binding a short distance away from the
transcription start is more common. Start sites are not often
mapped to the base. Thus, a trivial explanation of the effect of
promoter proximal binding of Ovo is that it binds near the start
sites, but not at them. This is unlikely. For example, two groups have mapped
the ovo-B transcription start site to the same location. Full-length
cDNAs, showing evidence of 5' caps, also end at this
site. The sequenced RACE product from the otu::lacZ swb transgene ends precisely at the same site. Similarly, the otu transcription start sites have been mapped by primer extension and by RACE. The otu start sites in reporter genes are within 20 bp upstream of the Ovo footprints, well within the
region expected to bind basal factors. Thus, Ovo and basal
transcription factors occupy the same region of the otu core
promoter, concurrently or in series (Lu, 2001).
The concurrent occupancy model for Ovo function at the
core promoter places Ovo in the basal transcriptional
apparatus. Core promoters typically have binding sites for
basal factors at characteristic locations. The best-studied site is
the TATA element at about -30 to -25, but about half of
Drosophila genes are TATA-less. In addition, Initiator elements (Inr) at the transcription start site, and downstream promoter elements (DPE) at about +28 to +34
have been described. The proteins that bind core promoter sites
are components of the enormous pre-initiation complex,
TFIID, which protects the entire 60 bp core promoter region. The combinatorial binding of TFIID components to characteristically spaced sequence elements provides enhanced specificity and binding strength. Ovo could function as a
tissue-specific core element to augment TFIID binding, but this
seems unlikely for three reasons. (1) The Ovo-binding site
is slightly downstream of the otu start site, but overlaps the
ovo-B initiation site. A more constrained
position relative to the start site would be expected. (2) The promoter proximal
Ovo binding sites at otu and ovo-B are in opposite orientation.
Transcription is certainly directional. If Ovo serves to orient
the complex at the transcription start site in a manner analogous
to TATA, Inr and DPE elements, then directionality would be
expected. To account for function in each orientation, Ovo
would need a flexible domain between the DNA binding and
complex contact domains, or a highly symmetrical structure
outside the DNA-binding domain. (3) Tests were performed for
dose-dependent genetic interactions between ovoD and
mutations in the Drosophila TBP associated factors (TAFs)
that are components of TFIID. Mutations in any of several TAFs fail to interact with ovoD. This is a circumstantial argument against an
intimate relationship between Ovo and TFIID (Lu, 2001).
If Ovo and basal factors occupy the otu core promoter
serially, orientation and spacing issues are less important. Ovo
binding might alter the structure of the core promoter to make
it more accessible to transcription initiation complexes. There
is precedent for preconditioning a core promoter. For example,
a bent configuration can enhance the binding of TBP to
the TATA element. Similarly, RNA Polymerase II can initiate from a melted or negatively supercoiled core promoter in the absence of the normal stable
of transcription factors. Thus, Ovo could precondition the core promoter to allow stronger and/or more precise subsequent binding by the transcriptional apparatus, by
generating or stabilizing bends or single stranded regions (Lu, 2001).
Indeed, retrotransposon targeting suggests that the ovo-B
promoter has an unusual structure. The ovo-B promoter region,
and the Ovo-binding sites in particular, are preferred targets
for de-novo gypsy-transposon insertion. Transposable element targeting is
believed to be sensitive to chromatin structure in many systems. It is thus possible that Ovo binding makes the chromatin especially available for
gypsy insertion. Such accessibility could also promote the entry
of transcriptional machinery. Finally, the presence of bound
Ovo might even circumvent the need for TFIID. The YY-1
protein, also a C2H2-zinc-finger protein that binds core
promoters, binds double-stranded DNA and a single-stranded bubble in the direction of transcription. YY-1 binding and RNA Polymerase II, but not TFIID, are sufficient
for transcription from those core promoters in vitro. In summary, while there is no
mechanistic understanding of Ovo function at the core promoter, it seems likely that Ovo and components of the machinery performing the work of transcription bind to the
same sequence, but not at the same time (Lu, 2001).
The gypsy retrovirus of Drosophila is quite unique among retroviruses in that it shows a strong preference for integration into specific sites in the genome. In particular, gypsy integrates with a frequency of >10% into the regulatory region of the ovo gene. In vivo transgenic assays were used to dissect the role of Ovo proteins and the gypsy insulator during the process of gypsy site-specific integration. DNA containing binding sites for the Ovo protein are required to promote site-specific gypsy integration into the regulatory region of the ovo gene. Using a synthetic sequence, it was found that Ovo binding sites alone are also sufficient to promote gypsy site-specific integration into transgenes. These results indicate that Ovo proteins can determine the specificity of gypsy insertion. In addition, interactions between a gypsy provirus and the gypsy preintegration complex may also participate in the process leading to the selection of gypsy integration sites. Finally, the results suggest that the relative orientation of two integrated gypsy sequences has an important role in the enhancer-blocking activity of the gypsy insulator (Labrador, 2008).
Retroviral DNA integration into the host genome is an essential step for production and replication of viral RNA. It has been traditionally difficult to study the factors controlling selection of integration sites, since most retroviruses integrate throughout the genome with no apparent DNA sequence specificity. However, detailed analysis of multiple genomic integration sites in vivo has revealed that retroviruses have a strong preference for certain genomic regions. In particular, retroviruses integrate preferentially into actively transcribed DNA, which will thereafter facilitate transcription of the provirus. The distribution of retroviral integration sites along chromosomes suggests that open chromatin favors retroviral insertion, since integration events are favored in transcriptionally active chromatin and are rare in DNA sequences associated with heterochromatin. However, chromatin state or DNA accessibility could not be the only factor influencing integration, since different retroviruses manifest preferences for integration that are unlikely to be only the result of chromatin organization. For example, both HIV and murine leukemia virus (MLV) integrate in actively transcribing DNA, but HIV integrates with equal frequency throughout all transcribed DNA, whereas MLV integrates preferentially into transcription start sites (Labrador, 2008).
The mechanism of integration of retrotransposons is fundamentally identical to that of retroviruses. However, constraints imposed by small genome sizes have led some retrotransposons to the acquisition of mechanisms for site-specific integration. The best examples of site-specific integration are found in non-LTR retrotransposons such as the Drosophila telomeric elements HeT-A, TART, and TAHRE or rDNA elements such as the Drosophila R1 and R2 non-LTR retrotransposons. A number of examples involving integrases from yeast LTR retrotransposons have also shown that retroviral-like integrases have evolved to acquire strong site-specific integration properties. For example, the integration of Saccharomyces cerevisiae retrotransposons Ty1 and Ty3 is associated with RNA polymerase III transcription and the Tf1 retrotransposon from Schizosaccharomyces pombe integrates near RNA polymerase II promoters. In other examples, transposable elements are targeted to heterochromatic sites by tethering mechanisms involving interactions between the integrase and DNA binding proteins. Targeting of the Ty5 retrotransposon from S. cerevisiae to heterochromatin, for example, requires a six-amino-acid motif at the C terminus of the Ty5 integrase that interacts with the heterochromatin protein Sir4. It has also been proposed that the chromodomain (CHD domain) from certain transposable element chromointegrases targets the retrotransposon for insertion into sites bearing the specific epigenetic marks recognized by the CHD domain (Labrador, 2008).
In contrast to mammalian retroviruses, Drosophila retroviruses such as gypsy, ZAM, or Idefix, display a high rate of site-specific integration into certain regions of the genome. The mechanisms governing this specificity however are poorly understood, but the genetic tools available in Drosophila provide a unique opportunity to analyze retroviral site integration specificity in higher eukaryotes. In particular, gypsy insertions into the ovo locus occur in the germ line of ~10% of the female offspring from mothers carrying permissive mutations in the flamenco (flam) locus (Prud'homme, 1995; Dej, 1998). Gypsy integrations take place specifically into a sequence of ~1.3 kb spanning the 5' regulatory region of the ovo gene (Dej, 1998). The flam locus is located in the heterochromatin of the X chromosome (Prud'homme, 1995) and produces a long noncoding RNA that controls transcription of the gypsy retrovirus through the piwiRNA pathway (Labrador, 2008).
The process of gypsy transposition is maternally regulated, involving maternally inherited gypsy particles that originate in the developing oocyte of flam mutant females. These females fail to produce the flam RNA, allowing the transcription of euchromatic gypsy elements in the follicle cells surrounding the oocyte during oogenesis. Transcription of gypsy in follicle cells leads to the formation of virus particles that infect the oocyte and subsequently participate in the integration of gypsy in the germ line of the resulting embryo after fertilization (Song, 1994: Song, 1997). These integration events take place preferentially in the ovo gene, whose product is necessary for the development of the female germ line and the normal progression of oogenesis. The ovo gene encodes two isoform proteins, Ovo-A and Ovo-B, which have a common DNA-binding domain but different N-terminal domains. Ovo-B positively regulates the ovo promoter, whereas Ovo-A functions as a negative regulator of the ovo promoter. Adult females homozygous for a null mutation of the ovo gene do not develop germ line cells. The ovoD1 allele is caused by a point mutation that creates a new in-frame methionine codon in the 5' region of ovo, adding an extra amino terminus domain to Ovo-B that is normally only present in the wild-type Ovo-A protein. The ovoD1 allele is dominant negative and causes female sterility even when heterozygous. The sterility is due to the expression of OvoD1B protein, which is made at the same time of development as Ovo-B but has the repressor activity of Ovo-A; the presence of OvoD1B is sufficient to arrest oogenesis at stage 4 (Labrador, 2008).
Insertion of gypsy into the ovoD1 allele in a heterozygous female reverts to fertility by preventing the expression of the OvoD1B protein, although the reversion occurs only in those germ cells in which gypsy is inserted into the ovoD1 sequence. The ability of gypsy to integrate specifically into ovo sequences was analyzed by Dej (1998). These studies concluded that gypsy integrates in at least seven different target sites localized within a 200-bp sequence present in the promoter region of the ovo gene. Close analysis of these sites reveals a very relaxed consensus sequence consisting of six alternating pyrimidines and purines. The weak conservation of the observed target sequence suggests that gypsy site-specific integration is not due to a direct interaction of the gypsy integrase with these sequences. Instead, it has been proposed that Ovo proteins may mediate gypsy insertion specificity by promoting protein-protein interactions between Ovo or an associated protein and the gypsy preintegration complex (Labrador, 2001; Labrador, 2008 and references therein).
The gypsy retrovirus of Drosophila also exhibits the interesting property of blocking enhancers from activating promoters when gypsy is inserted between them. This property is referred to as insulator activity and resides in the Suppressor of Hairy wing [Su(Hw)] binding sites present in a 350-bp sequence located in the 5'-UTR of the gypsy retroviral genome. In addition, gypsy insulators are also able to buffer transgenes from position effects by preventing heterochromatin from spreading through the chromatin fiber. There is mounting evidence suggesting that gypsy insulators function by creating chromatin domains most probably defined by the interaction between adjacent insulator sites in chromosomes. Molecular evidence for such interactions has been obtained by measuring the distance between adjacent gypsy insertions in wild-type and in su(Hw) mutant cells (Gerasimova, 2000). These experiments revealed that the two gypsy sequences were significantly closer during interphase when the Su(Hw) protein was present. It has been proposed that such interactions might create chromatin domains by looping out the DNA contained between two interacting insulators. Additional evidence in support of this model has been provided by showing the presence of DNA loops attached at their base to the nuclear matrix by the gypsy insulator in the nucleus of Drosophila imaginal disc cells (Byrd; 2003). Interaction between gypsy insulators is also supported by data showing that two adjacent insulators were able to cancel each other, no longer exerting their enhancer blocking effect when located between the enhancer and the promoter of a reporter gene (Cai, 2001; Muravyova, 2001; Kuhn, 2003; Labrador, 2008 and references therein).
The molecular basis for interactions between individual insulators is not well understood but it has been suggested that Modifier of mdg4 [Mod(mdg4)] and CP190, both components of the insulator complex, might facilitate such interactions by mediating protein-protein contacts between the BTB domains present in the two proteins. These two properties of gypsy, site-specific integration and insulator activity, have been the subject of intense but unrelated studies during the past two decades. This study attempts to analyze these two properties simultaneously in an effort to understand how insulators might mediate genome organization and how this organization may influence retroviral selection of integration sites through the genome. An assay has been developed to show that the 5' regulatory region of ovo is able to recruit gypsy insertions independently of its position in the genome (Labrador, 2001). Genetic evidence has been provided suggesting that the Ovo protein is directly implicated in such recruitment. This study takes advantage of the ability of inducing two consecutive gypsy insertions into a yellow reporter gene to analyze the role that interactions between a gypsy provirus and the gypsy preintegration complex may play in the selection of retrovirus integration sites and the effect of the relative orientation of interacting proviruses on the enhancer-blocking activity of the gypsy retrovirus (Labrador, 2008).
The gypsy retrovirus of Drosophila may offer valuable clues as to how retroviruses develop strategies to specifically select integration sites into the genome. Results shown in this study suggest that interactions between the gypsy preintegration complex and, most likely, Ovo proteins are sufficient to promote site-specific integration of gypsy into the ovo locus of Drosophila. Alteration of Ovo binding sites from a wild-type ovo gene fragment abolishes the ability of gypsy to specifically integrate into adjacent sequences. In addition, a synthetic DNA sequence carrying eight Ovo binding sites flanked by random DNA sequences is sufficient to function as a highly specific target for integration of the gypsy retrovirus. Although direct interactions between Ovo proteins and the gypsy preintegration complex have not been substantiated, the data point to a mechanism by which Ovo proteins may tether the gypsy preintegration complexes to their binding sites (Labrador, 2008).
It is tempting to speculate that the gypsy integrase may actually interact with the Ovo proteins and that such interaction may target integration to genome sites enriched in Ovo. Evidence demonstrating tethering of integrases as a mechanism capable of targeting retroviruses to specific DNA binding sites has been shown in experiments using fusion proteins in which the DNA binding domain of phage lambda-repressor was fused to the integrase of the HIV retrovirus and successfully showed preferential integration into target DNA near lambda-repressor-binding sites. Similarly, experiments with yeast retrotransposons have shown that such interactions may occur between the retrotransposon integrase and proteins that target the integration to their cognate chromosomal DNA binding sites (Labrador, 2008).
It has been reasoned that retrotransposons in small genomes such as that of yeast may develop tethering mechanisms of site-specific integration by stimulating interactions between the integrase encoded by the retrotransposon and endogenous proteins, thus minimizing the chances of deleterious mutations induced by retrotransposon integration events. In larger genomes such mechanisms appear infrequently, probably due to the lack of selective pressure from the host genome. In humans for example, sequences related to interspersed retroviruses occupy >50% of the genome and only relatively low frequencies of integration events in specific target spots have been reported. In Drosophila only a number of specialized non-LTR retrotransposons have acquired specificity of integration associated with specialized chromosomal regions such as telomeres or ribosomal DNA. Nonvertebrate retroviruses such as ZAM, Idefix, and gypsy appear to be an exception when compared with their vertebrate counterparts, since some degree of sequence specificity and targeted site integration has been described in all three. The high rate of insertion of gypsy into the ovo gene and the role apparently played by the Ovo proteins provide an excellent tool to study the integration mechanism and how retroviruses may acquire integration site specificity in vivo (Labrador, 2008).
Interestingly, even though Ovo proteins appear to have a role in targeting gypsy to their binding sites, gypsy insertion sites do not necessarily occur into the Ovo binding sequences themselves. From a total of 85 sequenced insertion sites, only 13 (15%) occurred into the DNA fragment containing Ovo binding sites; the remaining integration sites fall within an interval of >1300 bp flanking the DNA containing the Ovo binding sites. The analysis of insertion sites suggests that the targeting and the integration mechanisms are uncoupled, with the precise integration sites distributed in a nonrandom manner. Results shown in this study confirm previous observations suggesting that gypsy has a preference for integration into YRYRYR sequences. However, a variety of other sequences appear to be able to function as integration sites. This disparity makes it difficult to draw a clear conclusion as to what is the mechanism ultimately involved in selecting target sites; however, it is tempting to speculate a role for nucleosome positioning as one of the factors determining the selection of insertion sites by the gypsy retrovirus (Labrador, 2008).
Several indirect lines of evidence suggest such a role. For example, integration frequencies are significantly higher between the promoter of the yellow gene and the Ovo binding sites, indicating a preference that probably reflects a difference in chromatin structure. Forty-two independent integration events occurred into a fragment of 441 bp located between Ovo binding sequences and the promoter of the yellow gene, whereas only 30 integrations occurred in a 869-bp DNA fragment located distal to the promoter and upstream of the Ovo binding sites. This asymmetry does not appear to reflect sequence differences or viability effects, suggesting an epigenetic basis for integration site selection. In addition, insertion sites found distally to the yellow promoter and upstream of the Ovo binding DNA sequences appear to be spatially distributed in four intervals >140 bp, whereas insertions proximal to the promoter are distributed in a random manner, with only one large gap of 145 bp and apparently lacking meaningful spacing intervals. A possible interpretation of these results is that gypsy integration preferentially occurs at specific points of either the nucleosome or the linker DNA. In the distal interval, one spacing >140 bp could reflect nucleosome positioning, whereas nucleosomes may be absent or not positioned in the promoter proximal region of the transgene (Labrador, 2008).
Results presented in this study also suggest that a preexisting gypsy insertion significantly increases the chances of new gypsy insertions into adjacent sequences by more than twofold. It is speculated that such enhancement of insertion frequency might be the result of a tethering mechanism mediated by protein-protein interactions between the gypsy element located in the chromosomal DNA and the gypsy preintegration complex during the normal process by which a new copy of the retrovirus is inserted into the chromosome. Since two copies of the gypsy insulator have been shown to be able to interact with each other, it is tempting to speculate that interactions between gypsy insulators are responsible for the increased frequency of gypsy insertions. Nevertheless, the possibility that gypsy sequences other than the insulator or proteins associated with the gypsy element itself are responsible for the observed interactions cannot be ruled out. If the high frequency of secondary insertions is due to interactions between gypsy insulator proteins present in the provirus and in the preintegration complex, the results would lend support to proposed models suggesting that individual insulators located in different regions of a chromosome can interact to form chromatin loops (Labrador, 2008).
The analysis of phenotypes resulting from double insertions allows further elaboration of this model and offers additional insights into the mechanisms by which insulators affect enhancer-promoter interactions. For example, it has been shown that a wing enhancer distal to two adjacent gypsy insertions is capable of bypassing the activity of the two insulators when the two copies of the gypsy provirus are inserted in opposite orientation. These results demonstrate that interactions such as the ones determined genetically in transgenes, involving pairs of 400-bp gypsy insulators, also occur between pairs of gypsy insulators embedded in the gypsy provirus and suggest that establishing such interactions is part of the normal life cycle strategy used by the retrovirus. Interestingly, when two gypsy insertions occur in the same orientation, distal enhancers are unable to bypass the two insulators and are blocked from activating the promoter, contrary to what it has been observed with direct repeats of insulator sequences. The main difference between the two sets of experiments is the presence of additional DNA sequences in the gypsy provirus. These sequences may be able to form a stem-loop structure when the two copies of gypsy are arranged in opposite, but not when they are in the same, orientation. A similar role has been suggested for the relative orientation of insulator sequences between interacting Mcp insulators. The stem-loop structure would allow interactions between insulator proteins present in the two copies of gypsy with opposite orientations but a direct tandem arrangement of the two copies of the provirus would preclude such interactions. These observations support the hypothesis that interactions between paired insulators are required to bypass insulator function and allow enhancer-promoter communication (Labrador, 2008).
Low stringency genomic library screens with genomic fragments from the sex determination gene doublesex identified the Drosophila secreted cuticle protein 73 (dsc73; doublesex cognate 73A or dsx-c73A) gene, which encodes an 852-residue protein with an N-terminal signal sequence. In embryos, dsc73 RNA and protein are expressed to high levels in the epidermal cells that secrete the larval cuticle as well as in other cuticle-secreting tissues such as the trachea and salivary duct. Embryonic expression of dsc73 requires Shavenbaby, a transcription factor regulating cuticle formation. Double-labeling experiments with alphaCrb and alphaSAS reveal that, as with chitin and other known cuticle proteins, Dsc73 is secreted apically. Zygotic loss of dsc73 results in larval lethality but loss does not result in overt patterning defects or overt morphological defects in the embryonic tissues in which it is expressed. Thus, dsc73 encodes a novel secreted protein, and it is conserved within the Drosophila group. dsc73 may serve as a useful embryonic marker for cuticular patterning (Andrew, 2008; Full text of article).
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