InteractiveFly: GeneBrief

shutdown : Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

In animal gonads, PIWI proteins and their bound 23-30 nt piRNAs guard genome integrity by the sequence specific silencing of transposons. Two branches of piRNA biogenesis, namely primary processing and ping-pong amplification, have been proposed. Despite an overall conceptual understanding of piRNA biogenesis, identity and/or function of the involved players are largely unknown. This study demonstrates an essential role for the female sterility gene shutdown in piRNA biology. Shutdown, an evolutionarily conserved cochaperone collaborates with Hsp90 during piRNA biogenesis, potentially at the loading step of RNAs into PIWI proteins. Shutdown is shown to be essential for both primary and secondary piRNA populations in Drosophila. An extension of this study to previously described piRNA pathway members revealed three distinct groups of biogenesis factors. Together with data on how PIWI proteins are wired into primary and secondary processing, a unified model for piRNA biogenesis is proposed (Olivieri, 2012).

PIWI interacting RNAs (piRNAs) are a class of animal small RNAs. They are bound by PIWI family proteins and guide the sequence specific silencing of selfish genetic elements such as transposable elements (TEs). Defects in the piRNA pathway lead to TE derepression, genomic instability and ultimately sterility (Olivieri, 2012).

In Drosophila, most piRNAs are generated from two sources; on the one hand, these are piRNA cluster transcripts that originate from discrete genomic loci and serve as reservoirs of TE sequences; on the other hand, these are RNAs derived from active TEs that engage - together with cluster transcripts - in a piRNA amplification loop called the ping-pong cycle (Olivieri, 2012).

Two modes of piRNA biogenesis exist: (1) during primary piRNA biogenesis, a single stranded RNA is processed into pre-piRNAs, which are loaded onto PIWI proteins and are subsequently 3' trimmed and methylated, yielding mature piRNA induced silencing complexes (piRISCs). (2) piRISCs with active slicer activity can trigger secondary piRNA biogenesis, where a new piRNA is formed out of the sliced target RNA. In the presence of corresponding sense and antisense precursor RNAs, secondary piRNA biogenesis acts as the ping-pong amplification loop. The two piRNAs engaged in ping-pong have opposite orientation and exhibit a characteristic ten nucleotide 5' overlap (ping-pong signature) (Olivieri, 2012).

Primary and secondary piRNA biogenesis co-occur in germline cells, complicating the genetic and mechanistic dissection of these processes. However, somatic cells of the gonad also harbor a piRNA pathway and this is based exclusively on primary piRNA biogenesis. The Drosophila ovary is therefore ideally suited to identify and characterize factors required for either primary or secondary piRNA biogenesis or both (Olivieri, 2012).

Somatic support cells of the Drosophila ovary express Piwi as the only PIWI family protein. Primary piRNA biogenesis is thought to take place in peri-nuclear Yb-bodies, where the RNA helicases Armitage (Armi) and Yb as well as the TUDOR domain protein Vreteno (Vret) accumulate. In addition to these three factors, the putative mitochondria-localized nuclease Zucchini (Zuc) and the RNA helicase Sister of Yb (SoYb) are essential for piRNA biogenesis in the soma. Formation of mature Piwi-RISC triggers its nuclear import, while failure in piRNA biogenesis results in destabilization of presumably unloaded Piwi. Mature Piwi-RISC triggers TE silencing by an unknown mechanism that requires Piwi's nuclear localization but not its slicer activity (Olivieri, 2012).

With the exception of Yb, the above mentioned biogenesis factors are also essential in germline cells for the formation of Piwi-RISC. Germline cells, however, express two additional PIWI proteins, Aubergine (Aub) and Argonaute 3 (AGO3), which localize to the cytoplasm and are enriched in peri-nuclear nuage. Aub and AGO3 are the main players in the ping-pong cycle. Several factors with essential or modulatory roles in the ping-pong cycle have been identified. These are the RNA helicases Spindle-E and Vasa and the TUDOR domain proteins Krimper, Tejas (Tej), Qin and Tudor (Olivieri, 2012).

The analysis of piRNA populations from wild-type and piRNA pathway mutant ovaries indicated that Piwi is mainly a recipient of primary piRNAs, while Aub and AGO3 are predominantly or exclusively recipients of secondary piRNA biogenesis. Given this, three major questions arise: (1) Are primary and secondary piRNA biogenesis processes genetically and mechanistically separate or do common factors act in both processes? (2) In which processing step do identified piRNA biogenesis factors act? (3) How are the three PIWI family proteins wired into piRNA biogenesis? In other words, are certain PIWI proteins only receiving primary or only secondary piRNAs (Olivieri, 2012)?

This study shows that the female sterility gene shutdown (Munn, 2000; Schüpbach and Wieschaus, 1991) encodes a piRNA biogenesis factor. Shu is required for all piRNA populations in ovaries and it acts downstream of known piRNA biogenesis factors. A comparison of Shu to several other pathway factors led to the definition of three major groups of piRNA biogenesis factors. In combination with data on how PIWI proteins are wired into piRNA biogenesis, a model is proposed that accounts for the distinct association of piRNA subpopulations with specific PIWI proteins in Drosophila (Olivieri, 2012).

The outcome of this work is threefold: (1) The cochaperone Shutdown is essential for the biogenesis of all Drosophila piRNA populations. (2) Three major groups of piRNA biogenesis factors can be distinguished. (3) Piwi and Aub but not AGO3 are loaded with primary piRNAs, explaining how the cell maintains highly specific piRNA populations in the three PIWI proteins (Olivieri, 2012).

A remarkable feature of the shu mutant phenotype is that piRNA populations for every TE collapse. This already points to a common piRNA biogenesis step downstream of the primary and secondary branches. Epistatic analysis in somatic follicle cells is consistent with Shu acting at a late step in piRNA biogenesis: Shu is not required for the localization of any known biogenesis factor to Yb-bodies. On the other hand, Shu's localization to Yb-bodies depends on all other biogenesis factors and even on Piwi, arguing that unloaded Piwi recruits Shu to the Yb-body. Similarly, Shu colocalizes with nonloadable AGO3 in OSCs as well as in ovaries defective of ping-pong in discrete foci that also contain and are dependent on Krimp. Thus, in wild-type and in biogenesis mutants, Shu appears to colocalize with unloaded PIWI proteins (Olivieri, 2012).

Shu's C-terminal TPR domain falls into the class of Hsp90 binders and Hsp90 is important for small RNA loading into Argonaute proteins (Iki, 2010; Iwasaki, 2010; Miyoshi, 2010). In addition, the plant cochaperone Cyp40 interacts with Hsp90 via its TPR domain and is a critical cofactor for small RNA loading into AGO1 (Iki, 2012). The genetic and localization data support an analogous role for Shu and Hsp90 during small RNA loading into PIWI proteins. Clearly, in vitro assays will be crucial to dissect the precise order of events and the molecular role of Shu, especially its PPIase domain (Olivieri, 2012).

A major challenge in the field is to assemble piRNA biogenesis factors into pathways that explain the stereotypic populations of piRNAs in vivo. Advantage was taken of efficient germline specific knockdowns to study the impact of several factors on piRNA populations. Based on levels and localization of PIWI proteins as well as on piRNA populations obtained from several pathway factor knockdowns, three major groups of piRNA biogenesis factors are proposed (Olivieri, 2012).

Group I factors are required for primary piRNA biogenesis but dispensable for secondary biogenesis. In fact, piRNAs that initiated ping-pong in group I knockdowns were amplified and ping-pong signatures of such TEs were strongly increased, presumably as primary piRNAs that do not feed into ping-pong were absent (Olivieri, 2012).

Group II factors are specific for ping-pong, as primary piRNA biogenesis feeding into Piwi was unaffected. An alternative explanation that cannot be excluded is that some or all group II genes are required specifically for Aub biology (primary and secondary) per se. This would similarly leave Piwi bound piRNAs intact and would lead to a collapse in ping-pong. Given the data on Aub loading in OSCs, a model is favored however where the primary biogenesis machineries that feed Aub and Piwi are very similar (Olivieri, 2012).

Finally, group III factors are required for the biogenesis of Piwi/Aub/AGO3 bound piRNAs. The prototypic member of this group is Shu. Loss of Shu affects essentially all piRNA populations to the same extent. It is noted that analysis of piRNA populations from vret mutants indicated a role for this group III factor in primary biogenesis but not ping-pong (Handler, 2011; Zamparini, 2011). The distorted tissue composition of vret mutant ovaries coupled with perdurance of maternal Vret protein or RNA may underlie this discrepancy. The existence of group III factors predicts that primary and secondary piRNA biogenesis feed into a final piRISC maturation step that requires a set of common factors for all PIWI proteins. Given that piRNA biogenesis -- irrespective of the source of the precursor RNA -- requires an RNA loading step as well as a 3' trimming step, the existence of group III factors suggests itself (Olivieri, 2012).

The three proposed groups serve as a rough classification of biogenesis factors. Clearly, at a molecular level, the precise role of each factor within the biogenesis process will vary considerably. Of note, the classification of group I and group II genes extends to the mouse piRNA pathway. The Armi and Zuc orthologs MOV10L1 and PLD6 are required for primary piRNA biogenesis, whereas mouse VAS and TDRD9 (mouse Spn-E) were reported to be dispensable for primary biogenesis but are required for secondary biogenesis pathway (Olivieri, 2012).

The data indicate that Aub is not only loaded via ping-pong, but also via primary piRNA biogenesis. It is also postulate that Aub and Piwi proteins are wired into primary piRNA biogenesis processes in a very similar manner, meaning that they require the same or highly overlapping core factors (e.g., Armi or Zuc). In agreement with this, ectopically expressed Aub is loaded in OSCs that harbor a fully functional primary pathway but lack critical ping-pong factors such as Vas. The genetic requirements for Aub loading in OSCs are identical to those for Piwi. It is extrapolated from this that the core primary biogenesis machinery that loads Piwi in the soma also loads Piwi and Aub in the germline. Analyses of piRNA populations from armi versus piwi or aub-GLKDs support a model where Armi and Zuc are required for the biogenesis of both Piwi and Aub bound primary piRNAs. The possibility is not excluded that - despite a similar biogenesis machinery - populations of primary piRNAs in Aub and Piwi are different. For example, differences in subcellular localizations of PIWI proteins as well as piRNA precursor RNAs might result in such differences (Olivieri, 2012).

In contrast to Aub, AGO3 was unstable in OSCs. Coexpression of Aub or simultaneous knockdown of krimp had no impact on AGO3 stability. It is therefore concluded that primary piRNA biogenesis is incompatible with AGO3. In fact, also in the germline genetic data indicated that AGO3 depends on secondary piRNA biogenesis for being loaded. Blocking AGO3's access to the primary biogenesis machinery would allow the cell to load AGO3 with a unique class of piRNAs if it couples AGO3 loading to a precursor RNA originating from Aub-slicer mediated cleavage of an active TE. This would explain the remarkable bias of AGO3 bound piRNAs being sense and carrying an Adenosine at position ten (Olivieri, 2012).

Interestingly, on a primary sequence level Aub -- despite its significantly different biology -- is more closely related to Piwi than to AGO3. A critical question emanating from this is to which extent Piwi is participating in ping-pong, and if it does not, why. A weak, yet statistically significant, ping-pong signature has been observed between Piwi and AGO3 bound piRNAs. This could mean that there is indeed low level of Piwi-AGO3 ping-pong. An alternative explanation is that the Piwi-AGO3 signal is a misleading computational signal: If Piwi and Aub are loaded via the same primary biogenesis machinery, initiator piRNAs for ping-pong that end up in Aub also end up in Piwi. As primary piRNA biogenesis appears to be nonrandom and preferentially processed piRNAs likely trigger ping-pong more robustly, an 'artificial' AGO3/Piwi ping-pong signature might result (Olivieri, 2012).

What could be the molecular basis of why Piwi does not or only moderately participate in ping-pong? Either, specific features of Aub (e.g., symmetric Arginine methylation) are funneling this protein into ping-pong and similar features are absent on Piwi. Or, the mere sequestration of Piwi into the nucleus prevents Piwi from participating in ping-pong. Notably, N-terminally truncated Piwi that is still loaded but that cannot translocate into the nucleus is enriched in nuage the proposed site of secondary piRNA biogenesis. A simple difference in the subcellular localization of Aub and Piwi might thus contribute to the dramatic differences of piRNA populations residing in Aub or Piwi (Olivieri, 2012).

shutdown encodes a novel FK506-binding protein essential for the formation of germline cysts during oogenesis

In Drosophila, the process of oogenesis is initiated with the asymmetric division of a germline stem cell. This division results in the self-renewal of the stem cell and the generation of a daughter cell that undergoes four successive mitotic divisions to produce a germline cyst of 16 cells. shutdown is essential for the normal function of the germline stem cells. Analysis of weak loss-of-function alleles confirms that shutdown is also required at later stages of oogenesis. Clonal analysis indicates that shutdown functions autonomously in the germline. shutdown was identified using a positional cloning approach. Consistent with its function, the RNA and protein are strongly expressed in the germline stem cells and in 16-cell cysts. The RNA is also present in the germ cells throughout embryogenesis. shutdown encodes a novel Drosophila protein similar to the heat-shock protein-binding immunophilins. Like immunophilins, Shutdown contains an FK506-binding protein domain and a tetratricopeptide repeat. In plants, high-molecular-weight immunophilins have been shown to regulate cell divisions in the root meristem in response to extracellular signals. These results suggest that shutdown may regulate germ cell divisions in the germarium (Munn, 2000).

In contrast to wild-type ovaries, 40%-60% of the ovarioles from newly eclosed shu mutant females do not contain any developing egg chambers. Usually, the other ovarioles contain only one to three egg chambers and their numbers fail to increase as the females age. None of the egg chambers develop into normal eggs but instead degenerate by mid- to late-oogenesis. Those egg chambers with the correct number of germ cells may have arisen through the differentiation of prestem cells, i.e., germ cells that developed as cystoblasts without first being established as stem cells. The strong cytoplasmic staining of germ cells with antibodies recognizing the Sex Lethal protein indicates that shu function is dispensable for the establishment of the female mode of sexual differentiation of the germ cells. To determine the developmental stage of shu mutant germ cells, they were analyzed using an antibody specific for the cytoplasmic form of the Bam protein. In wild-type germaria, the cytoplasmic form of Bam accumulates in mitotic cystoblasts and cystocytes. However, in newly eclosed shu mutant females no staining is observed. This suggests that, after the first few egg chambers are formed, no further mitotic cystoblasts develop. Instead, these germ cells appear to redivide to form clusters of abnormal germ cells that degenerate or occasionally produce tumorous cysts. Collectively, these phenotypes suggest that shu's function is essential for the normal activity of the stem cell. The absence of wild-type function could affect the first asymmetric division of the stem cell, resulting in an abnormal cystoblast that is compromised in its ability to undergo the normal four rounds of mitotic division. In addition, this abnormal asymmetric division would affect stem cell renewal, resulting in an abnormal stem cell that divides several times to produce ill-fated germ cell clusters (Munn, 2000).

Analysis of the shu phenotype, using a complete loss-of-function allele, has revealed requirements for shu in the normal behavior and differentiation of the germline. The few developing egg chambers with 16 germ cells present in newly eclosed shu^WQ41 females have presumably arisen through the differentiation of prestem cells, i.e., germ cells that developed as cystoblasts without first being established as stem cells. These early egg chambers develop until midoogenesis and then degenerate. Germline stem cells are clearly established in the absence of shu function, because they express Sex Lethal protein and have spectrosomes. Although the stem cells appear to have undergone several divisions, their progeny do not become committed to the normal pathway of germline differentiation. Instead, they presumably redivide to produce clusters of ill-fated germ cells. This phenotype contrasts with that of mutants such as bam and bgcn, which specifically block cystoblast differentiation and result in tumors of mitotically active cells that continue to behave like stem cells. As shu females age, a loss of germ cells is observed, resulting in agametic ovarioles. This indicates that stem cell renewal is also affected by the loss of shu function. One way in which these phenotypes could have arisen is through an aberrant asymmetric division of the stem cell (Munn, 2000).

A similar asymmetric division controls the production of primary spermatocytes from male germline stem cells during spermatogenesis. Strong loss-of-function alleles of shu also result in male sterility. The mutant testes contain fewer than normal elongating sperm bundles: the apical tip of the testes, where the germline stem cells divide, appears reduced compared to wild type, and many of the cells are degenerating (Tirronen, 1993). These observations indicate that shu also has an essential function in germline stem cell regulation in males (Munn, 2000).

Mutations in decapentaplegic (dpp), pumilio (pum), nanos (nos), piwi, and fs(1)Yb that affect stem cell maintenance and asymmetric division have been described. While the source of the dpp signal remains unknown, the function of both piwi and fs(1)Yb is thought to be required in the somatic cells of the terminal filament. Both piwi and fs(1)Yb have been proposed to be required for the production of a somatic signal that regulates the germline stem cells. In contrast, pum and nos have been reported to function in the germline. The analysis of ovarian germline clones indicates that shu also functions in the germline autonomously. This is supported by the observation that both male and female sterility can be rescued using a transgene whose expression is controlled by the germline-specific otu promoter. As some of the phenotypes observed in shu are similar to those described for piwi and fs(1)Yb, it is possible that shu is required for the response of the germline cells to these somatic signals. No effects on fertility have been observed in transheterozygous combinations of shu with piwi, pum, or nos. In contrast to shu, mutations in dpp, piwi, pum, and nos allow the production of mature eggs. The other phenotypes observed in shu egg chambers, including a loss of oocyte identity and occasionally mispositioned oocytes, are therefore likely due to later requirements for shu in egg chamber development. Consistent with this possibility, shu expression increases in newly formed 16-cell cysts (Munn, 2000).

The expression pattern of the SHU mRNA suggests that shu may function in germline development during embryogenesis. The mRNA is incorporated into the pole cells and can be detected in the germ cells throughout their migration into the embryonic gonad. A number of RNAs have been identified that are incorporated into the pole cells including cyclin B, germ cell less, hsp83, nos, orb, oskar, pum, tudor, and vasa. Mutations in some of these genes also affect early germline development. Since the existing alleles of shu fail to produce normal eggs that are fertilized, the possible effects of zygotic shu function on germ cell migration or proliferation could not be studied (Munn, 2000).

The Shu protein shows significant homology to an evolutionarily conserved class of proteins, the immunophilins. Although these proteins have been shown, in vitro, to catalyze changes in protein folding, their function in vivo remains unclear. The best characterized of the FK506BPs is the low-molecular-weight immunophilin human FKBP12. It is a cytosolic protein that has been implicated in signal transduction (reviewed by Marks, 1996). In vertebrates, FKBP12 has also been proposed to regulate translation through its association with the FKBP12-associated rapamycin-binding protein (reviewed by Brown, 1996). This complex regulates the binding of translation initiation factors to the 5' end of the mRNA. The Drosophila gene vasa encodes a germline-specific homologue of the translation initiation factor eIF4A. Null alleles of vasa produce a variety of phenotypes that include atrophied germaria containing reduced numbers of developing germline cysts. No genetic evidence has been found that shu functions in this particular pathway, because females trans-heterozygous for a null allele of shu and a deficiency that removes vasa show no defects in oogenesis (Munn, 2000).

In addition to its FK506-binding domain, Shu contains a predicted TPR motif. This is a protein-protein interaction motif that was originally identified in cell cycle regulatory proteins but has since been found in a number of different proteins with no common biochemical function (Lamb, 1995). A mutation in an allele of shu within the TPR has been identified indicating the importance of this motif for shu function. This allele does not cause male sterility, suggesting that the protein might function differently in spermatogenesis, perhaps through interacting with different partners. Alternatively, it is also possible that spermatogenesis is less sensitive to reductions in levels of wild-type function (Munn, 2000).

The presence of a TPR motif, in addition to the FK506-binding domain, is characteristic of the high-molecular-weight immunophilins, an evolutionarily conserved family of proteins whose function remains uncharacterized. Interestingly, there are no examples of this type of immunophilin in yeast or Caenorhabditis elegans. Mammalian immunophilins were originally identified in complexes of HSP90 with unliganded steroid hormone receptors. It has been proposed that immunophilins function in the cytoplasmic to nuclear targeting of these complexes. Interestingly, the germline expression pattern of the Drosophila homolog of HSP90, Hsp83, is strikingly similar to that of shu (Ding, 1993). Biochemical experiments will confirm whether Shu interacts with HSP83 in the germline or with alternative partners (Munn, 2000).

Unfortunately, the lack of mutants in the mammalian immunophilins has prevented the identification of their in vivo functions. Human FKBP6 maps to a common 1-Mb deletion in patients with William's syndrome, a developmental disorder associated with a haploinsufficiency at chromosome 7q11.23. William's syndrome has multiple associated phenotypes. Together with the large size of the deletions, this has made it difficult to correlate specific gene functions with a particular aspect of the disease. Human FKBP6 and rodent FKBP52 are both expressed at particularly high levels in testes, suggesting that immunophilins may have a conserved function in germline development. Interestingly, germline stem cells of the mammalian testes, like Drosophila germline stem cells, undergo asymmetric, self-renewing divisions (Munn, 2000 and references therein).

Some insight into the function of immunophilins was recently obtained through the analysis of the pasticcino-1 (pas-1) mutant in A. thaliana. The pas-1 mutant was isolated in a screen for mutants that showed an abnormal response to the cell division-promoting plant hormones, cytokinins. The mutants have defects in cell division and elongation in the cotyledons and the apical root meristem. Cloning of pas-1 reveals that it is a homolog of mammalian FKBP52. FKBP52 has been shown to co-localize with the mitotic apparatus and to copurify with cytoplasmic dynein, suggesting that it too may be required for cell divisions (Munn, 2000 and references therein).

The shu phenotypes support the possibility that shu may also function during germline cell divisions. Specifically, its function seems to be important for the divisions of the germline stem cells. In support of this, expression of the protein can be detected in the stem cells while the levels are greatly reduced in the dividing cystocytes. The future identification of Shu-interacting proteins, coupled with the potential of genetic analysis in Drosophila, should greatly increase understanding of how germline stem cells are regulated and provide valuable information about the function of the immunophilins (Munn, 2000).

DEVELOPMENTAL BIOLOGY

The expression pattern of shu during oogenesis, spermatogenesis, and embryogenesis was examined using whole mount in situ hybridization with RNA probes made from the cDNA clone corresponding to its transcript. The mRNA can first be detected at the apical tip of the germarium in the germline stem cells and cystoblasts. The level of expression decreases in the remainder of region 1, where the cystocytes are dividing to produce 2-, 4-, 8-, and 16-cell cysts. However, in region 2b, where 16-cell cysts are present, strong levels of expression are again seen. In region 3, equivalent levels of expression can be seen in all 16 germ cells of the stage 1 egg chamber. This pattern of expression corresponds well with the earliest phenotypes of shu and confirms the results of the clonal analysis that indicate that shu function is required in the germline. Subsequent to stage 4 of oogenesis, there appears to be an abrupt downregulation of shu expression until stage 10, where the transcript can be detected at high levels in the nurse cells. In early cleavage-stage embryos uniform levels of SHU mRNA are detected, suggesting a possible maternal function for shu. Interestingly, by the cellular blastoderm-stage shu mRNA is exclusively found in the pole cells. The mRNA can be detected in the germ cells throughout their migration through the midgut and into the gonadal mesoderm and is present in the embryonic gonad of stage 15 embryos. No staining above background level can be detected in any tissues other than the gonads. The shu mRNA can also be detected at the apical tip of the testes where the stem cells are dividing to produce cysts of primary spermatocytes (Munn, 2000).

Rat antisera were raised against a 6x His-tagged protein containing amino acids 31-392 of Shu. Western analysis reveals that the antisera specifically recognize a 52-kD band in wild-type ovary extracts. This protein is not detected in ovary extracts from hemizygous shu^WQ41 females, confirming the specificity of the antisera. To determine the subcellular localization of the Shu protein, affinity-purified antibodies were used for immunofluorescent staining of ovaries. The Shu protein is expressed in the germline stem cells and cystoblasts. Low levels of staining were observed in dividing cystocytes in the posterior of region 1. In region 2b, staining is evident in all cells of the newly formed 16-cell cysts. Like the shu mRNA, this persists until stage 4-5 egg chambers. The staining is present in the cytoplasm of the germ cells. Only background levels are detected in the follicle cells. Germline staining is eliminated in shu^WQ41 ovaries. Cytoplasmic staining is also seen later in the nurse cells of stage 10 and older egg chambers. However, since the strong loss-of-function alleles of shu do not develop normal eggs at this stage it is difficult to ascertain if this later staining is specific. Staining in the germ cells of embryos could not be detected with this antiserum. This could be due to technical reasons, such as the antibody sensitivity or specificity; only very low levels of the protein can be detected on immunoblots of embryonic extracts (Munn, 2000).

EFFECTS OF MUTATION

All three shu alleles cause recessive female sterility with no effects on zygotic viability (Schupbach, 1991). The two strong loss-of-function alleles, shu^WQ41 and shu^WM40, also result in male sterility while the weaker allele, PB70, does not affect male fertility. Comparison of the phenotypes of shu^WQ41 and shu^WM40 as homozygotes and hemizygotes [over Df(2R)b23] reveals that shu^WQ41 is likely to be a null allele while shu^WM40 is a very strong loss-of-function allele. Due to the presence of a closely linked recessive lethal mutation on the shu^WQ41 and shu^PB70 chromosomes, few homozygous adults could be isolated. The phenotype of shu^WQ41 was therefore studied in hemizygous flies [over Df(2R)b23], is referred to here as shu^WQ41, while that of shu^PB70 was analyzed in hemizygotes or in the heteroallelic combination with shu^WM40 (Munn, 2000).

To analyze the shu^WQ41 ovarian phenotype, ovaries were dissected from 0- to 1- and 2- to 4-day-old females and stained with the nuclear stain Hoechst and with antibodies recognizing several different markers of germ cell differentiation. The severity of the phenotypes observed in shu^WQ41 can vary between females, and even between ovarioles, suggesting that there may be some functional redundancy for shu. In contrast to wild-type ovaries, 40%-60% of the ovarioles from newly eclosed shu^WQ41 females do not contain any developing egg chambers. Usually, the other ovarioles contain only one to three egg chambers and their numbers fail to increase as the females age. Although some egg chambers with 15 nurse cells and an apparently normal oocyte nucleus are observed, subsequent egg chambers have fewer than 16 germ cells, e.g., 2, 4, 8, 10, or 12. None of the egg chambers develop into normal eggs but instead degenerate by mid- to late oogenesis. Those egg chambers with the correct number of germ cells may have arisen through the differentiation of prestem cells, i.e., germ cells that developed as cystoblasts without first being established as stem cells. The remainder of the shu^WQ41 ovarioles contain no developing egg chambers, only germaria-containing clusters of germline cells that can sometimes appear pycnotic, indicating that they may be dying (Munn, 2000).

The strong cytoplasmic staining of these germ cells with antibodies recognizing the Sex Lethal protein indicates that shu function is dispensable for the establishment of the female mode of sexual differentiation of the germ cells. Staining of these germ cell clusters with antibodies that recognize spectrosomes and fusomes reveals that they do not develop branched fusomes characteristic of older cysts. To determine the developmental stage of these shu germ cells, they were further analyzed using an antibody specific for the cytoplasmic form of the Bam protein. In wild-type germaria, the cytoplasmic form of Bam accumulates in mitotic cystoblasts and cystocytes. However, in newly eclosed shu^WQ41 females no staining was observed in any of the germaria analyzed. This suggests that, after the first few egg chambers are formed, no further mitotic cystoblasts develop. Instead, these germ cells appear to redivide to form clusters of abnormal germ cells that degenerate or occasionally produce tumorous cysts. In older females (>4 days), ovarioles in which the cells at the tip do not express Vasa, apparently failing to maintain germline cells at the tip, are frequently observed consistent with defects in stem cell renewal or maintenance. In addition, some mispackaged egg chambers have also been observed (Munn, 2000).

Females heteroallelic for the weak loss-of-function allele PB70 and the strong loss-of-function allele WM40 lay only ~30% as many eggs as wild-type females, none of which develop. Typically, these eggs are abnormally shaped and dorsal appendages, if present, are reduced or fused. Inspection of the ovaries of these females reveals at least five developing egg chambers in the majority of ovarioles. About 80% of these egg chambers contain the correct 15:1 ratio of nurse cells to oocyte but never develop into wild-type eggs. Although an oocyte is established, as seen by the localization of Bic-D and Orb proteins to the posterior-most cell in the cyst, its identity is not maintained and the oocyte appears to fail in its further development. Subsequent to stage 2 or 3, the distribution of the two proteins becomes diffuse and accumulation of the proteins is observed in nurse cells. This pattern is particularly striking for the Bic-D protein and suggests that the transport system from the nurse cells to the oocyte has collapsed in these egg chambers. Subsequently, oocyte development fails and the egg chambers degenerate. These observations corroborate the results of Tirronen (1993). At low frequency (5%-10%) egg chambers were observed in which the oocyte is not correctly positioned and egg chambers in which the oocyte nucleus appears polyploid rather than diploid, although not to the same extent as the nurse cells. This is similar to phenotypes observed in Bic-D partial loss-of-function mutations. These observations suggest that there may be a later requirement for shu function during egg chamber development (Munn, 2000).

The phenotype of shu indicates that its function is required for the normal activity of the germline stem cell. To address whether shu functions autonomously in the germline, use was made of the FRT-mediated, dominant female sterile (DFS) technique and mutant clones were generated in the germline by mitotic recombination. Females heterozygous for autosomal insertions of the DFS mutation ovo^D1 fail to lay eggs and their ovaries contain no egg chambers that have developed beyond stage 4-5 of oogenesis. In contrast, females of the genotype y w P[FLP]/w; FRT ovo^D1/FRT shu^PB70, which were subjected to heat shock, lay abnormal eggs. These eggs are similar to those laid by females hemizygous or heteroallelic for the PB70 allele. Inspection of ovaries from females in which germline clones of the strong loss-of-function alleles WQ41 and WM40 were induced reveals the presence of abnormal egg chambers that appear, by Hoechst staining, to be degenerating by mid- to late-oogenesis. These results indicate that shu functions in the germline. They do not, however, exclude the possibility of an additional somatic function (Munn, 2000).

To investigate the nature of the shu mutations, the genomic DNA from flies hemizygous for each allele was sequenced. The WQ41 allele has a C to T transversion at position 88 that creates a premature stop codon (Q11 to STOP). This confirms the prediction, based on genetic analysis, that WQ41 is a complete loss-of-function allele. The WM40 allele has a G to A transversion at position 1142 that creates a premature termination of translation 31 amino acids before the end of the protein (W342 to STOP). The weakest loss-of-function allele of shu, PB70, is due to a G to A transversion at position 1051 that results in an alanine (332) to threonine substitution within the TPR motif. This result suggests that the TPR motif is likely to be important for shu function. Interestingly, this mutation does not affect male fertility (Munn, 2000).

EVOLUTIONARY HOMOLOGS

Williams syndrome (WS) is a developmental disorder caused by haploinsufficiency of genes at 7q11.23. Hemizygosity of elastin is responsible for one feature of WS, supravalvular aortic stenosis. LIM-kinase 1 hemizygosity has been implicated as a contributing factor to impaired visual-spatial constructive cognition in WS. A novel gene, FKBP6, has been identified and characterized within the common WS deletion region. FKBP6 shows homology to the FK-506 binding protein (FKBP) class of immunophilins. FKBP6 has a putative N-terminal FK-506 binding and peptidylproyl isomerase (rotamase) domain and, like known high-molecular-weight FKBPs, an imperfect C-terminal tetratricopeptide repeat domain. FKBP6 is expressed in testis, heart, skeletal muscle, liver, and kidney. FKBP6 consists of nine exons and is completely contained within a 35-kb cosmid clone. Fluorescence in situ hybridization experiments show that the FKBP6 gene is deleted in 40/40 WS individuals. Hemizygous deletion of FKBP6 may contribute to certain defects such as hypercalcemia and growth delay in WS (Meng, 1998).

The structurally related immunophilins cyclophilin 40 (CyP-40) and FKBP52 have been identified as components of the unactivated estrogen receptor. Both immunophilins have a similar molecular architecture that includes a C-terminal segment with a tetratricopeptide repeat (TPR) domain predicted to mediate protein interaction. hsp90 is a common cellular target for CyP-40 and FKBP52. Deletion mutants of CyP-40 fused to glutathione S-transferase were immobilized on glutathione-agarose and then used in a rapid hsp90 retention assay to define regions of the CyP-40 C terminus that are important for hsp90 binding. The evidence suggests that the TPR domain is not sufficient for stable association of CyP-40 with hsp90 and requires the participation of flanking acidic and basic residues clustered at the N- and C-terminal ends, respectively. Both microdomains are characterized by alpha-helical structures with segregated hydrophobic and charged residues. Corresponding regions were identified in FKBP52. By preincubating myometrial cytosol with lysates containing bacterially expressed FKBP52, it has been shown that FKBP52 competes with CyP-40 for hsp90 binding. These results raise the possibility of a mutually exclusive association of CyP-40 and FKBP52 with hsp90. This would lead to separate immunophilin-hsp90-receptor complexes and place the estrogen receptor under the control of distinct immunophilin signaling pathways (Ratajczak, 1996).

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor-hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). By cross-linking of the purified proteins it has been demonstrated that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, it has been shown that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins (Silverstein, 1999).

Interferon regulatory factor-4 (IRF-4) plays an important role in immunoregulatory gene expression in B and T lymphocytes and is also highly expressed in human T cell leukemia virus type 1 infected cells. A novel interaction has been characterized between IRF-4 and the FK506-binding protein 52 (FKBP52), a 59 kDa member of the immunophilin family with peptidyl-prolyl isomerase activity (PPIase). IRF-4-FKBP52 association inhibits IRF4-PU.1 binding to the immunoglobulin light chain enhancer E(lambda2-4) as well as IRF-4-PU.1 transactivation, effects that are dependent on functional PPIase activity. FKBP52 association also results in a structural modification of IRF-4, detectable by immunoblot analysis and by IRF-4 partial proteolysis. These results demonstrate a novel posttranslational mechanism of transcriptional control, mediated through the interaction of an immunophilin with a transcriptional regulator (Mamane, 2000).

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