BIOLOGICAL OVERVIEW

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).

GENE STRUCTURE

cDNA clone length - 1682

PROTEIN STRUCTURE

Amino Acids - 392

Structural Domains

A search for recognized protein motifs, using the Prosite database, revealed a peptidyl-prolyl cis-trans isomerase (PPIASE) domain at amino acids 103-193 and one tetratricopeptide repeat (TPR) at amino acids 303-334. PPIASE domains are protein-protein interaction motifs that have been shown, in vitro, to catalyze the cis-trans isomerization of the peptide bond of proline residues, resulting in changes in protein folding (Fischer, 1990). Their activity is inhibited by binding of the drugs FK506 and rapamycin. Hence, proteins containing these domains are commonly referred to as FK506-binding proteins (FK506BP). The TPR motif is a degenerate 34-amino-acid sequence, which is also thought to mediate protein-protein interactions (Lamb, 1995). In addition, the Shu sequence contains a putative nuclear localization signal (NLS), and 18 predicted phosphorylation sites, although the functional significance of these, if any, remains unknown (Munn, 2000).

Comparison of the predicted amino acid sequence of Shu with other proteins in the database, using the Gapped BLAST search program, reveals that it is a novel Drosophila protein that shows similarity to proteins of the high-molecular-weight immunophilin family. This class of protein typically contains both PPIASE and TPR motifs. They are evolutionarily conserved, multifunctional proteins, examples of which have been identified in mammals and plants. Human FKBP6 (p36) (Meng, 1998) shows 28% identity and 46% similarity (probability 10^-24 with 3% gaps) to Shu along the length of the entire protein sequence. A similar level of homology is also observed with human FKBP4 (Sanchez, 1990), FKBP52 (the mammalian p59 heat-shock protein-binding immunophilin; Peattie, 1992), and several plant immunophilins. Functional studies of human FKBP12, the best-characterized FKBP, have identified 14 residues important for enzymatic activity and drug binding (Kay, 1996). Shu retains 8 of these residues and a further amino acid is also conserved, suggesting that this domain is functionally important. Similarly, comparison of the Shu TPR motif with the consensus TPR sequence reveals that 5 of 6 consensus residues are conserved (Munn, 2000).

date revised: 15 November 2000

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