Protein competition switches the function of COP9 from self-renewal to differentiation.

The balance between stem cell self-renewal and differentiation is controlled by intrinsic factors and niche signals. In the Drosophila melanogaster ovary, some intrinsic factors promote germline stem cell (GSC) self-renewal, whereas others stimulate differentiation. However, it remains poorly understood how the balance between self-renewal and differentiation is controlled. Here we use D. melanogaster ovarian GSCs to demonstrate that the differentiation factor Bam controls the functional switch of the COP9 complex from self-renewal to differentiation via protein competition. The COP9 complex is composed of eight Csn subunits, Csn1-8, and removes Nedd8 modifications from target proteins. Genetic results indicated that the COP9 complex is required intrinsically for GSC self-renewal, whereas other Csn proteins, with the exception of Csn4, were also required for GSC progeny differentiation. Bam-mediated Csn4 sequestration from the COP9 complex via protein competition inactivated the self-renewing function of COP9 and allowed other Csn proteins to promote GSC differentiation. Therefore, this study reveals a protein-competition-based mechanism for controlling the balance between stem cell self-renewal and differentiation. Because numerous self-renewal factors are ubiquitously expressed throughout the stem cell lineage in various systems, protein competition may function as an important mechanism for controlling the self-renewal-to-differentiation switch.

Mei-p26 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary.

In the Drosophila female germline, spatially and temporally specific translation of mRNAs governs both stem cell maintenance and the differentiation of their progeny. However, the mechanisms that control and coordinate different modes of translational repression within this lineage remain incompletely understood. Here we present data showing that Mei-P26 associates with Bam, Bgcn and Sxl and nanos mRNA during early cyst development, suggesting that this protein helps to repress the translation of nanos mRNA. Together with recently published studies, these data suggest that Mei-P26 mediates both GSC self-renewal and germline differentiation through distinct modes of translational repression depending on the presence of Bam.

Mei-P26 regulates the maintenance of ovarian germline stem cells by promoting BMP signaling.

In the Drosophila ovary, bone morphogenetic protein (BMP) ligands maintain germline stem cells (GSCs) in an undifferentiated state. The activation of the BMP pathway within GSCs results in the transcriptional repression of the differentiation factor bag of marbles (bam). The Nanos-Pumilio translational repressor complex and the miRNA pathway also help to promote GSC self-renewal. How the activities of different transcriptional and translational regulators are coordinated to keep the GSC in an undifferentiated state remains uncertain. Data presented here show that Mei-P26 cell-autonomously regulates GSC maintenance in addition to its previously described role of promoting germline cyst development. Within undifferentiated germ cells, Mei-P26 associates with miRNA pathway components and represses the translation of a shared target mRNA, suggesting that Mei-P26 can enhance miRNA-mediated silencing in specific contexts. In addition, disruption of mei-P26 compromises BMP signaling, resulting in the inappropriate expression of bam in germ cells immediately adjacent to the cap cell niche. Loss of mei-P26 results in premature translation of the BMP antagonist Brat in germline stem cells. These data suggest that Mei-P26 has distinct functions in the ovary and participates in regulating the fates of both GSCs and their differentiating daughters.

Drosophila ataxin 2-binding protein 1 marks an intermediate step in the molecular differentiation of female germline cysts.

In the Drosophila ovary, extrinsic signaling from the niche and intrinsic translational control machinery regulate the balance between germline stem cell maintenance and the differentiation of their daughters. However, the molecules that promote the continued stepwise development of ovarian germ cells after their exit from the niche remain largely unknown. Here, we report that the early development of germline cysts depends on the Drosophila homolog of the human ataxin 2-binding protein 1 (A2BP1) gene. Drosophila A2BP1 protein expression is first observed in the cytoplasm of 4-, 8- and 16-cell cysts, bridging the expression of the early differentiation factor Bam with late markers such as Orb, Rbp9 and Bruno encoded by arrest. The expression of A2BP1 is lost in bam, sans-fille (snf) and mei-P26 mutants, but is still present in other mutants such as rbp9 and arrest. A2BP1 alleles of varying strength produce mutant phenotypes that include germline counting defects and cystic tumors. Phenotypic analysis reveals that strong A2BP1 alleles disrupt the transition from mitosis to meiosis. These mutant cells continue to express high levels of mitotic cyclins and fail to express markers of terminal differentiation. Biochemical analysis reveals that A2BP1 isoforms bind to each other and associate with Bruno, a known translational repressor protein. These data show that A2BP1 promotes the molecular differentiation of ovarian germline cysts.

Accumulation of a differentiation regulator specifies transit amplifying division number in an adult stem cell lineage.

A key feature of many adult stem cell lineages is that stem cell daughters destined for differentiation undergo several transit amplifying (TA) divisions before initiating terminal differentiation, allowing few and infrequently dividing stem cells to produce many differentiated progeny. Although the number of progenitor divisions profoundly affects tissue (re)generation, and failure to control these divisions may contribute to cancer, the mechanisms that limit TA proliferation are not well understood. Here, we use a model stem cell lineage, the Drosophila male germ line, to investigate the mechanism that counts the number of TA divisions. The Drosophila Bag of Marbles (Bam) protein is required for male germ cells to cease spermatogonial TA divisions and initiate spermatocyte differentiation [McKearin DM, et al. (1990) Genes Dev 4:2242-2251]. Contrary to models involving dilution of a differentiation repressor, our results suggest that the switch from proliferation to terminal differentiation is triggered by accumulation of Bam protein to a critical threshold in TA cells and that the number of TA divisions is set by the timing of Bam accumulation with respect to the rate of cell cycle progression.

Bam and Bgcn antagonize Nanos-dependent germ-line stem cell maintenance.

The balance between germ-line stem cell (GSC) self-renewal and differentiation in Drosophila ovaries is mediated by the antagonistic relationship between the Nanos (Nos)-Pumilio translational repressor complex, which promotes GSC self-renewal, and expression of Bam, a key differentiation factor. Here, we find that Bam and Nos proteins are expressed in reciprocal patterns in young germ cells. Repression of Nos in Bam-expressing cells depends on sequences in the nos 3'-UTR, suggesting that Nos is regulated by translational repression. Ectopic Bam causes differentiation of GSCs, and this activity depends on the endogenous nos 3'-UTR sequence. Previous evidence showed that Bgcn is an obligate factor for the ability of Bam to drive differentiation, and we now report that Bam forms a complex with Bgcn, a protein related to the RNA-interacting DExH-box polypeptides. Together, these observations suggest that Bam-Bgcn act together to antagonize Nos expression; thus, derepressing cystoblast-promoting factors. These findings emphasize the importance of translational repression in balancing stem cell self-renewal and differentiation.

Stonewalling Drosophila stem cell differentiation by epigenetic controls.

During Drosophila oogenesis, germline stem cell (GSC) identity is maintained largely by preventing the expression of factors that promote differentiation. This is accomplished via the activity of several genes acting either in the GSC or in its niche. The translational repressors Nanos and Pumilio act in GSCs to prevent differentiation, probably by inhibiting the translation of early differentiation factors, whereas niche signals prevent differentiation by silencing transcription of the differentiation factor Bam. We have found that the DNA-associated protein Stonewall (Stwl) is also required for GSC maintenance. stwl is required cell-autonomously; clones of stwl(-) germ cells were lost by differentiation, and ectopic Stwl caused an expansion of GSCs. stwl mutants acted as Suppressors of variegation, indicating that stwl normally acts in chromatin-dependent gene repression. In contrast to several previously described GSC maintenance factors, Stwl probably functions epigenetically to prevent GSC differentiation. Stwl-dependent transcriptional repression does not target bam, but rather Stwl represses the expression of many genes, including those that may be targeted by Nanos and Pumilio translational inhibition.

The miRNA pathway intrinsically controls self-renewal of Drosophila germline stem cells.

Stem cells uniquely self-renew and maintain tissue homoeostasis by differentiating into different cell types to replace aged or damaged cells [1]. During oogenesis of Drosophila melanogaster, self-renewal of germline stem cells (GSCs) requires both intrinsic signaling mechanisms and extrinsic signals from neighboring niche cells [2]. Emerging evidence suggests that microRNA (miRNA)-mediated translational regulation may also control Drosophila GSC self-renewal [3, 4]. It is unclear, however, whether the miRNA pathway functions within stem cells or niche cells to maintain GSCs. In Drosophila, Dicer-1 (Dcr-1) and the double-stranded RNA binding protein Loquacious (Loqs) catalyze miRNA biogenesis [3-5]. Here, we generate loqs knockout (loqs(KO)) flies by ends-out homologous recombination and show that loqs is essential for embryonic viability and ovarian GSC maintenance. Both developmental and miRNA processing defects are rescued by transgenic expression of Loqs-PB, but not Loqs-PA. Furthermore, mosaic germline analysis indicates that Loqs is required intrinsically for GSC maintenance. Consistently, GSCs are restored in loqs mutant ovaries by germline expression, but not somatic expression, of Loqs-PB. Together, these results demonstrate that Loqs-PB, but not Loqs-PA, is necessary and sufficient for Drosophila development and the miRNA pathway. Our study strongly suggests that miRNAs play an intrinsic, but not extrinsic, role in Drosophila female GSC self-renewal.

A discrete transcriptional silencer in the bam gene determines asymmetric division of the Drosophila germline stem cell.

The Drosophila germline lineage depends on a complex microenvironment of extrinsic and intrinsic factors that regulate the self-renewing and asymmetric divisions of dedicated stem cells. Germline stem cells (GSCs) must express components of the Dpp cassette and the translational repressors Nanos and Pumilio, whereas cystoblasts require the bam and bgcn genes. Bam is especially attractive as a target of GSC differentiation factors because current evidence indicates that bam is both necessary and sufficient for cystoblast differentiation. In this paper, we have sought to distinguish between mutually exclusive transcriptional or post-transcriptional mechanisms as the primary regulators of bam expression in GSCs and cystoblasts. We find that bam transcription is active in young germ cells but is repressed specifically in GSCs. Activation depends on a 50 bp fragment that carries at least one germ cell-specific enhancer element. A non-overlapping 18 bp sequence carries a transcriptional silencer that prevents bam expression in the GSC. Promoters lacking this silencer cause bam expression in the GSC and concomitant GSC loss. Thus, asymmetry of the GSC division can be reduced to identifying the mechanism that selectively activates the silencer element in GSCs.