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., ëXX pseudomalesí. 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 20-25ƒC, 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).
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).
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