ovarian tumor


Promoter Structure

A genomic DNA fragment bearing the otu promoter restores fertility to mutants of the ovarian tumor locus (otu) of Drosophila. Germ-line transformants bearing this fragment express OTU mRNA with the same tissue specificity as the wild-type otu gene, and at comparable levels. Transcription from the otu promoter, P(otu), which lacks a TATA element, appears to be initiated at multiple transcription start points (tsp) within an 80-bp region. Deletion of sequences upstream of the tsp indicates that a region between nucleotides -190 and -310 is required for proper expression of the otu gene. A DNA fragment containing 452 bp upstream and 126 bp downstream from the tsp is able to direct expression of the Escherichia coli lacZ gene in the germ cells of the ovary and testis, indicating that cis-acting regulatory elements governing these expression patterns are located in a 578-bp region surrounding the multiple tsp (Comer, 1992).

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).

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). Increased ovo+ dose results in increased ovo mRNA and genetic activity. This important control means that an increased ovo+ copy number translates into increased functional Ovo protein. In those flies with increased Ovo activity, endogenous otu transcripts were present in greater quantity than wild type. Transgenes driven by the otu promoter respond positively to increased Ovo activity. This response is not limited to late stages. otu reporters respond to increased Ovo activity in larval gonads and in the stem cells and cystocytes of the adult ovary. Cells expressing otu reporters also express ovo reporters, suggesting that Ovo is at the scene of otu promoter activity. These data suggest that Ovo controls otu expression in early stages of oogenesis (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).

Transcriptional Regulation

The Drosophila gene stand still (stil) encodes a novel protein required for survival, sexual identity and differentiation of female germ cells. The Stil protein accumulates in the nucleus of all female germ cells throughout development, and is transiently expressed during early stages of male germline differentiation. Changes of Stil subnuclear localization during oogenesis suggest an association with chromatin. Several mutant alleles, which are point mutations in the Stil N-terminal domain, encode proteins that no longer co-localize with chromatin. Stil binds to many sites on polytene chromosomes with strong preference for decondensed chromatin. This localization is very similar to that of RNA polymerase II. Stil is required for high levels of transcription of the ovarian tumor gene in germ cells. Expression of ovarian tumor in somatic cells can be induced by ectopic expression of Stil. Ubiquitous somatic expression of Stil results in lethality of the fly at all stages of development (Sahut-Barnola, 1999).

Germline sex determination is controlled by both cell-autonomous (germ cell intrinsic components) and non-cell-autonomous factors (somatic signals). Candidate genes involved in germline sex determination might be identified on the basis of two mutant phenotypes: the disappearance of germ cells in a sex-specific manner and the differentiation of chromosomally female (2X/2A) germ cells as male germ cells. Genetic analyses have shown that the three genes ovo, ovarian tumor (otu), and stand still (stil) meet both criteria. They are required only in females for survival of the germ cells, establishment of their sexual identity, and oogenesis. In addition to the similar phenotypes shown by mutations in these three genes, genetic interactions between them have been demonstrated. The phenotype of females bearing dominant female-sterile alleles of ovo, ovoD, is sensitive to the gene dosage of otu and stil, such that decreased amounts of these genes lead to enhancement and increased gene doses to partial suppression of the phenotype. These genetic interactions suggest that the otu, ovo and stil genes function in a common pathway, or in closely linked parallel pathways, involved in the survival and differentiation of the female germline (Sahut-Barnola, 1999 and references).

During embryogenesis, the Stil protein is first detectable at stage 11 in the nucleus of germ cells, soon after their migration through the midgut epithelium, but before their separation into two groups. This is very soon after germ cells becomes competent for RNA polymerase II-dependent transcription. At stage 11, the staining is very faint, but becomes strong by stage 13. Double staining with antibodies against the Vasa and Stil proteins shows that all germ cells express Stil. No evidence has been found for either sex-specific expression during embryogenesis or expression in somatic tissues. During the first and second larval instar, Stil protein is found in the nucleus of all germ cells in both sexes. A difference between males and females first becomes apparent during the third larval stage. All female germ cells, which are located in the middle part of larval ovaries, show strong staining. In adult ovaries, Stil expression is maintained in all germ cells. By contrast, Stil is present only during some stages of male germline differentiation. At the testis apex, Stil expression is low in stem cells and dividing spermatogonia. In newly formed 16-cell cysts of primary spermatocytes, Stil staining is as strong as that found in female germ cells, but then quickly vanishes during the spermatocyte growth phase. This pattern of expression is also found in adult testes, which have the same apical-distal organization. Larval fat bodies show low levels of Stil expression (Sahut-Barnola, 1999).

An adult ovary is composed of about 16 egg assembly lines called ovarioles. A special region, the germarium, lies near the anterior end of an ovariole. Two to three germline stem cells are located at the anterior end of a germarium. A germline stem cell divides asymmetrically to produce a new stem cell and a cystoblast, which then undergoes four rounds of synchronous incomplete mitotic divisions forming a cluster of 16 interconnected cystocytes. One of the 16 cells becomes the oocyte and the 15 others differentiate into nurse cells. The 16-cell cyst, surrounded by a layer of somatic follicle cells, leaves the germarium and undergoes final differentiation. In adults, Stil is present in the nucleus of all female germline-derived cells, including the oocyte. Staining of the oocyte nucleus is detected transiently, from stage 1 to 10 of oogenesis, and is weak compared to staining in nurse cell nuclei. After stage 10, staining is observed only in nurse cell nuclei and disappears when they degenerate. The oocyte cytoplasm presents no detectable level of Stil protein, irrespective of the developmental stage, indicating that most of the Stil protein is not transferred from the nurse cells to the oocyte, and that what is transferred is trapped in the oocyte nucleus (Sahut-Barnola, 1999).

Given the similar phenotypes exhibited by mutations in stil, ovo and otu, as well as the genetic interactions between them, it is likely that these genes function in a common pathway, or in parallel pathways, to determine germline survival and sexual identity. A common pathway is supported by the finding that high levels of otu expression requires ovo gene activity. An investigation was therefore carried out to see whether Stil controls the expression of the ovo and otu genes. The expression of three constructs containing parts of the ovo gene fused to a lacZ reporter gene was analyzed in gonads of wild-type, stil heterozygous and stil homozygous or hemizygous flies. No effect of stil gene dosage is found on expression of the ovo::lacZ reporter genes. To determine the effect of stil mutations on otu expression, antibodies against the Otu proteins were used as well as an otu::lacZ reporter, which contains the 5' region and the first untranslated exon of the otu gene. Using Otu antibodies in wild-type or in stil heterozygous backgrounds, a cytoplasmic staining was observed in all the germ cells in ovaries and in stem cells and dividing spermatogonia at the apex of testes. In stil mutant ovaries, in which only a few cells are present, a strong reduction in the intensity of Otu antibody staining was seen. However, it is difficult to be sure that the reduction of staining is due to decreased expression of otu and not to the fact that the mutant cells are unhealthy and dying. In males, stil mutant germ cells are completely healthy. A strong reduction of the intensity of Otu antibody staining has been found at the apex of stil mutant testes. Experiments using the otu::lacZ transgene give the same results. In wild-type and in stil heterozygotes, expression of otu::lacZ is restricted to the apex of testes. In stil mutant testes, beta-galactosidase expression is almost undetectable. These results show that transcription of the otu gene in male and female germ cells is regulated by Stil (Sahut-Barnola, 1999).

The ovo gene products are positive regulators of otu expression and the Ovo zinc-finger domain binds specifically to several sites at the otu promoter. Thus it is possible that Ovo and Stil work together at the otu promoter. One way to get a hint of such a cooperation is to combine ovo and stil mutations and to test the consequence on otu expression. An effect of stil+ gene dosage is observed in a background in which otu transcription is strongly impaired by a dominant antimorphic mutation of ovo. In ovaries heterozygous for ovoD2, expression of otu::lacZ is almost completely abolished. Expression of the otu::lacZ transgene is partially restored by the addition of an extra wild-type copy of the stil gene. Phenotypic analysis has shown that the ovoD2 mutation can be partially suppressed by increasing the dosage of stil+. These results suggest that this phenotypic improvement is in part due to restoration of otu expression. Ectopic Stil can induce the transcription of otu in tissues other than the germline. However, this ectopic activation is rather inefficient, despite the fact that the amount of ectopic Stil artificially induced in follicle cells is similar to the amount present in germ cells. This suggests that the somatic follicle cells may not contain all the components needed for full activity of Stil (Sahut-Barnola, 1999).

One striking observation described in this article is the widespread association of the Stil protein to chromatin. Analysis of polytene chromosomes, either after ectopic expression in salivary glands or in nurse cells that normally express stil, reveals that Stil is preferentially associated with regions of decondensed chromatin (interbands), including puffs. This localization is very similar to that found for RNA polymerase II (Pol II), in particular the form of the enzyme in which its largest subunit is in a hypophosphorylated state. A co-localization of Stil and Pol II on polytene chromosomes is consistent with the observation that Stil controls the transcription of the otu gene. The numerous binding sites suggest that Stil probably contributes to the regulation of many genes in the germline (Sahut-Barnola, 1999).

The importance for germ cell development of proteins that show broad association with chromatin and may exert a general effect on Pol II activity has recently be documented, particularly in Caenorhabditis elegans. For example, the product of the pie gene contains two putative zinc-finger motifs and is required during early embryogenesis to repress overall Pol II transcription in germline precursor cells and to prevent them from differentiating as somatic cells. The effect of PIE-1 appears to be mediated by prevention of phosphorylation of the carboxy-terminal repeat domain of the largest Pol II subunit. Even more striking is the case of the mes-2, mes-3, mes-4 and mes-6 genes. Maternal-effect mutations in these four genes lead to healthy but sterile offspring characterized by post-embryonic degeneration of the germline. The sex-karyotype of the germ cells is critical for the expressivity of the phenotype, since worms bearing 2 X chromosomes are much more sensitive to these mutations than worms bearing a single X chromosome, regardless of the sexual identity of the soma or of the germline. This karyotype-dependent requirement is very similar to that found for ovo in Drosophila, except that ovo shows zygotic, not maternal, requirement. A link to gene regulation at the level of chromatin structure became plausible when MES-2 and MES-6 were found to be homologous (respectively) to two members of the Polycomb group proteins, Enhancer of zeste and Extra sex combs. Polycomb group proteins are involved in gene silencing in various situations (Sahut-Barnola, 1999).

In C. elegans, transgenes arranged in large repetitive arrays are usually active in somatic cells, but silenced in germ cells, a phenomenon that is attributed to some unknown particularity of chromatin organization in the germline. This hypothesis received strong support with the recent observation that silenced transgenes can be reactivated by mutations in the four mes genes listed above. Finally, proteins involved in higher-order chromatin structure are also important in the process of sex determination in mammals: male-to-female sex reversal phenotype has been observed in mice mutant for the M33 gene, a homolog of Polycomb. These examples are meant to stress the likely importance of global gene regulation and higher-order chromatin structure in the development of germ cells, as well as in the establishment of particular cell fate such as sexual identity. In the case of Drosophila, genetic interactions between some alleles of ovo and mutations of either Polycomb or polycomb-like also point to a role of higher-order chromatin structure in germline sexual identity and differentiation. Given the evidence of a genetic interaction between ovo and stil, and the extensive chromatin association described here, it is plausible that Stil has a rather general effect on transcription. The difference between female and male germ cells would be determined by the interaction with sex-specific partners or by post-translational modifications possibly controlled by the feminizing somatic signals (Sahut-Barnola, 1999).

Ovo controls germline and epidermis differentiation in flies and mice. In the Drosophila germline, alternative Ovo-B and Ovo-A isoforms have a common DNA-binding domain, but different N-termini. These isoforms are transcription factors with opposite regulatory activities. Using yeast one-hybrid assays, a strong activation domain was identified within a common region and a counteracting repression domain within the Ovo-A-specific region. To identify and map Ovo transcriptional effector domains, the effect of fusion proteins on reporter gene expression was monitored. In flies, Ovo-B positively regulates the ovarian tumor promoter, while Ovo-A is a negative regulator of the ovarian tumor and ovo promoters. Ovo-B isoforms supply ovo+ function in the female germline and epidermis, while Ovo-A isoforms have dominant-negative activity in both tissues. Moreover, elevated expression of Ovo-A results in maternal-effect lethality while the absence of Ovo-A results in maternal-effect sterility. These data indicate that tight regulation of antagonistic Ovo-B and Ovo-A isoforms is critical for germline formation and differentiation (Andrews, 2000).

The function of ovo in, and for, female germline development appears to be relatively straight forward. The data indicate that Ovo-B supplies the essential ovo + function in the female germline. This is fully consistent with the expression of Ovo-B isoforms early in oogenesis where ovo + activity is required. Genetic and molecular data indicate that ovo plus acts upstream of otu + and ultimately Sxl +. Ovo-B positively regulates otu transcription in the female germline, suggesting that part of the function of Ovo-B is to up-regulate Otu production. There are at least three known positive regulators of otu promoter activity: ovo; somatic signals, and stand still (stil). While it is not known how Ovo-B activates otu in conjunction with these other regulators, Stil is a germline restricted chromatin associated protein that is present at cytological sites of transcription. Thus, Stil and Ovo-B may act directly at the promoter. The somatic sex determination signals that influence otu expression are undefined. Whereas maternal Ovo-A is required for the germline of the progeny, it is extremely toxic when produced during early oogenesis (Andrews, 2000).

Alternative splicing of otu mRNA

Alternative splicing is used by metazoans to increase protein diversity and to alter gene expression during development. However, few factors that control splice site choice in vivo have been identified. Half pint (Hfp; FlyBase designation Poly-U-binding splicing factor) regulates RNA splicing in Drosophila. Females harboring hypomorphic mutations in hfp lay short eggs and show defects in germline mitosis, nuclear morphology, and RNA localization during oogenesis. hfp encodes the Drosophila ortholog of human PUF60 and functions in both constitutive and alternative splicing in vivo. In particular, hfp mutants display striking defects in the developmentally regulated splicing of ovarian tumor (otu). Furthermore, transgenic expression of the missing otu splice form can rescue the ovarian phenotypes of hfp. hfp is also required for efficient splicing of gurken mRNA and in the splicing of eukaryotic initiation factor 4E (eIF4E), which binds to the seven-methylguanosine cap at the 5' end of messenger RNAs and is a limiting factor in translation initiation (Van Buskirk, 2002).

The polytene nurse cell chromosome morphology of hfp mutants is strikingly similar to that observed in females mutant for certain alleles of ovarian tumor (otu). otu also plays a role in germline cell division, as otu egg chambers can have too few or too many germline cells. The otu transcript is subject to alternative splicing, resulting in the production of a 98 kDa protein and a less abundant 104 kDa protein (Otu104), depending on the incorporation of a 126 bp alternatively spliced exon. The biochemical functions of the Otu isoforms are unknown, but they share an N terminus that contains a cysteine protease-like domain and a C terminus that possesses weak similarity to microtubule-associated proteins. Intriguingly, the alternatively spliced exon encodes a tudor domain, a sequence element found multiply repeated in the Drosophila Tudor protein and also present in proteins with putative RNA binding functions in several species. Genetic and molecular analysis reveal distinct requirements and expression patterns for the two Otu isoforms, suggesting that the splicing of the otu transcript may be developmentally regulated. Indeed, RT-PCR shows that expression of the transcript encoding the 104 kDa isoform is restricted to the early stages of oogenesis (Van Buskirk, 2002).

In order to determine whether the alternative splicing of otu is dependent on hfp function, the expression of otu was examined in hfp mutants. RT-PCR analysis of RNA isolated from early stage egg chambers shows that the relative abundance of the larger otu transcript is decreased in hfp, and Western analysis shows that the levels of the corresponding 104 kDa Otu isoform are significantly decreased. Severe loss of Otu104 activity results in the production of tumorous egg chambers, which are not observed in hfp. Therefore, some residual Otu104 protein must be present, possibly because the hfp mutants represent partial loss-of-function alleles. To determine which, if any, of the hfp phenotypes are due to an effect on otu splicing regulation, a hybrid genomic-cDNA transgene encoding exclusively the 104 kDa Otu isoform was introduced into an hfp mutant background. hfp mutants expressing two copies of the Otu104 transgene produce egg chambers with predominantly wild-type nurse cell nuclear morphology and expression of four copies of the transgene completely rescues the nurse cell and oocyte nuclear morphology as well as the germline division defect. Expression of Otu104 also restores normal grk mRNA localization. This was unexpected, since defects in grk RNA localization have not been previously described in otu mutants, and no grk RNA localization defects are observed in otu7 homozygotes or otu7/otu11 mutants, which give rise to differentiated egg chambers. However, otu11 females, though often producing tumorous germaria, will under certain conditions produce rare eggs, and these show defects, including the expansion of dorsal appendages associated with mislocalization of grk RNA, very similar to those of hfp9 mutant eggs. Interestingly, otu11 is a point mutation in the alternatively spliced exon and hence specifically affects the activity of the 104 kDa Otu isoform (Van Buskirk, 2002).

Hrb27C, Sqd and Otu cooperatively regulate gurken RNA localization and mediate nurse cell chromosome dispersion in Drosophila oogenesis

Heterogeneous nuclear ribonucleoproteins, hnRNPs, are RNA-binding proteins that play crucial roles in controlling gene expression. In Drosophila oogenesis, the hnRNP Squid (Sqd) functions in the localization and translational regulation of gurken (grk) mRNA. Sqd interacts with Hrb27C, an hnRNP previously implicated in splicing. Like sqd, hrb27C mutants lay eggs with dorsoventral defects and Hrb27C can directly bind to grk RNA. These data demonstrate a novel role for Hrb27C in promoting grk localization. A direct physical interaction is also observed between Hrb27C and Ovarian tumor (Otu), a cytoplasmic protein implicated in RNA localization. Some otu alleles produce dorsalized eggs and it appears that Otu cooperates with Hrb27C and Sqd in the oocyte to mediate proper grk localization. All three mutants share another phenotype, persistent polytene nurse cell chromosomes. These analyses support dual cooperative roles for Sqd, Hrb27C and Otu during Drosophila oogenesis (Goodrich, 2004).

This study identifies a physical and genetic interaction between Hrb27C and Sqd. The two proteins were previously biochemically purified as part of an hnRNP complex from Drosophila cells, but it was not known if they interact directly nor had their RNA targets been isolated. The interaction was originally detected in a two-hybrid screen and it has been confirmed biochemically by co-immunoprecipitation. In vivo, this interaction requires the presence of RNA; this result was unexpected because the yeast two-hybrid constructs that originally revealed the interaction did not contain the RRMs and presumably cannot bind RNA. The interaction may require the full-length proteins to be in unique conformations that are achieved only when they are bound to RNA. The truncated proteins used in the yeast two-hybrid screen may be folded in such a manner that the protein-protein interaction domains are exposed even in the absence of RNA binding, making it possible for the interaction to occur in yeast (Goodrich, 2004).

A striking genetic interaction was detected between Sqd and Hrb27C; weak hrb27C mutants can strongly enhance the DV defects of weak sqd mutants. This suggests that the physical interaction has in vivo significance. The phenotypes of sqd and hrb27C place both proteins in a pathway required for proper grk localization and suggest a previously undetected role for Hrb27C. hrb27C mutants lay variably dorsalized eggs, and in situ hybridization analysis reveals that grk RNA is mislocalized. Since the mislocalized RNA in hrb27C mutants is translated, causing a dorsalized egg phenotype, it is likely that Hrb27C also functions with Sqd in regulating Grk protein accumulation. A role in the regulation of RNA localization and translation is novel for Hrb27C/Hrp48; it has been previously described as functioning in the inhibition of IVS3 splicing of P-element encoded transposase (Siebel, 1994; Hammond, 1997). Hrb27C has nuclear functions and has been observed in the nucleus and cytoplasm of somatic and germline cells in embryos (Siebel, 1995). Because Sqd is localized to the oocyte nucleus where it is thought to bind grk RNA, a model is favored where Hrb27C and Sqd bind grk RNA together and are exported possibly as a complex into the cytoplasm (Goodrich, 2004).

Two unpublished studies have implicated Hrb27C in osk RNA localization (J. Huynh, T. Munro, K. Litière-Smith and D. St Johnston, communication to Goodrich, 2004) and translational regulation (T. Yano, S. Lopez de Quinto, A. Shevenchenko, A. Shevenchenko, T. Matsui and A. Ephrussi, communication to Goodrich, 2004). Translational repression of osk RNA until it is properly localized is essential to prevent disruptions in the posterior patterning of the embryo. An analogous mechanism of co-regulation of mRNA localization and translation appears to control Grk expression. Certain parallels between osk and grk regulation are very striking. Both RNAs are tightly localized and subject to complex translational regulation; they also share certain factors that mediate this regulation. In addition to Hrb27C, the translational repressor Bruno appears to be a part of the regulatory complexes that are required for the proper expression of both RNAs. It will be interesting to determine if there are other shared partners that function in the regulation of both RNAs, as well as identify the factors that give each complex its localization specificity (Goodrich, 2004).

Otu is also involved in the localization of grk mRNA and interacts physically with Hrb27C. Though a definitive role of Otu in regulating Grk expression has not been previously described, Van Buskirk (2002) found that four copies of Otu104 (flies carry a transgene expressing only the 104 kDa isoform of Otu under the control of the otu promoter) are able to rescue the grk RNA mislocalization defect of half pint (hfp; poly U binding factor 68kD) mutants. Analysis of the hypomorphic alleles, otu7 and otu11, reveals a requirement for Otu in localizing grk RNA for proper DV patterning. Alternative splicing of the otu transcript produces two protein isoforms: a 98 kDa isoform and a 104 kDa isoform that differ by the inclusion of a 126 bp alternatively spliced exon (6a) in the 104 kDa isoform. This alternatively spliced exon encodes a tudor domain, a sequence element present in proteins with putative RNA-binding abilities. Interestingly, the otu11 allele, which contains a missense mutation in exon 6a, shows the grk localization and dorsalization defect, strongly suggesting that the tudor domain of Otu plays an important function in grk localization. Since otu mutants also show defects in osk localization, it appears that like several other factors, Otu is required for both grk and osk localization. Additionally, Otu has been isolated from cytoplasmic mRNP complexes (Goodrich, 2004).

Surprisingly, an additional shared phenotype of hrb27C, sqd and otu mutants was found. During early oogenesis, the endoreplicated nurse cell chromosomes are polytene. As they begin to disperse, the chromosomes are visible as distinct masses of blob-like chromatin that completely disperse by stage 6. The formation of polytene nurse cell chromosomes is due to the endocycling that occurs during early oogenesis. The mechanism of chromosome dispersal is hypothesized to involve the degradation of securin by the anaphase-promoting complex/cyclosome as a result of separase activity that cleaves cohesin. The significance of chromosome dispersion is not clear, but it has been suggested that it could facilitate rapid ribosome synthesis. A defect in the dispersal of polytene chromosomes has been previously described for otu mutants, and both sqd and hrb27C mutants also have nurse cell chromosomes that fail to disperse. Weak double transheterozygous allele combinations of sqd and hrb27C produce the persistent polytene nurse cell chromosome phenotype that is not observed in either of the weak mutants alone. Thus, genetic and physical interactions between these gene products suggest that they function together in regulation of this process (Goodrich, 2004).

The failure to disperse nurse cell chromosomes has also been observed in mutants of hfp where the nurse cell nuclear morphology defect is due to improper splicing of Otu104 (Van Buskirk, 2002). As was shown for hfp, expressing Otu104 in a sqd mutant background is able to rescue the polytene nurse cell chromosome phenotype. Although Hfp affects otu splicing, it is not clear how Sqd functions to mediate this phenotype, but evidence supports a role in Otu104 protein accumulation. The transcript that encodes Otu104 is clearly present in sqd mutants, as assayed by RT-PCR, but the level of Otu104 protein is decreased. These results suggest that the level of Otu104 is crucial to maintaining proper nurse cell chromosome morphology because the polytene phenotype of sqd mutants can be rescued by expressing only one extra copy of Otu104 and the majority of egg chambers from females heterozygous for otu13, otu11, or a deficiency lacking otu, have nurse cell chromosomes that fail to disperse (Goodrich, 2004).

The reduced level of Otu104 in sqd mutants raises the possibility that Sqd could translationally regulate otu RNA and that the polytene nurse cell phenotype may be a result of the decreased level of Otu104. Alternatively, Sqd and Hrb27C could form a complex that recruits and stabilizes Otu104 protein, and this complex in turn functions directly or indirectly to regulate chromosome dispersion. An indirect role seems more likely, given that Otu has never been observed in the nucleus. Since Otu104 can rescue the phenotype of sqd mutants and Otu and Hrb27C co-precipitate, it seems plausible that Sqd and Hrb27C interact with Otu in the nurse cell cytoplasm to affect an RNA target that could then mediate chromosome dispersion (Goodrich, 2004).

The genetic data support a model in which Sqd, Hrb27C, and Otu function together in a complex that affects at least two processes during oogenesis: DV patterning within the oocyte and mediation of nurse cell chromosome dispersion. Although the biochemical data are consistent with this model, it is also possible that Hrb27C and Sqd could form a complex that is distinct from a complex containing Hrb27C and Otu. However, the in vivo genetic interactions and mutant phenotypes reveal that all three proteins affect both processes and favor a model in which all three proteins function in one complex (Goodrich, 2004).

These results allow expansion upon the model proposed by Norvell (1999) for the regulation of grk RNA expression. Sqd and Hrb27C associate with grk RNA in the oocyte nucleus. Hrb27C and Sqd remain associated with grk in the cytoplasm where Otu and possibly other unidentified proteins associate with the complex as necessary to properly localize, anchor and translationally regulate grk RNA. A likely candidate to be recruited to the complex is Bruno, which interacts with Sqd (Norvell, 1999). Although the RNA target of Sqd, Hrb27C and Otu in the nurse cells is not known, the proteins may form a complex composed of different accessory factors to regulate localization and/or translation of RNAs encoding proteins that affect chromosome morphology (Goodrich, 2004).

Although Hrb27C, Sqd and Otu function together to affect different processes in the oocyte and the nurse cells, they probably function to mediate the precise spatial and temporal regulation of a target RNA that is unique to each cell type where the presence of additional factors would provide specificity to each complex. The importance of the precise localization and translational regulation of grk is well defined, and Hrb27C and Otu have not been identified as additional proteins that facilitate these processes. The transition from polytene to dispersed chromosomes in the nurse cells is less well characterized, but most probably requires precise spatial and temporal regulation as well. A complex containing Sqd, Hrb27C and Otu could function to ensure that the target RNA is properly localized and functional in the nurse cells only at the proper stage of development to promote chromosome dispersal (Goodrich, 2004).

Otu in germ-line sex determination

Continued: see Regulation part 2/2

ovarian tumor: Biological Overview | Developmental Biology | Effects of Mutation | References

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