Gene name - bag of marbles
Cytological map position - 96C1--96C9
Function - translational regulator
Symbol - bam
FlyBase ID: FBgn0000158
Genetic map position - 3-
Classification - novel protein
Cellular location - cytoplasmic
|Recent literature||Tokusumi, T., Tokusumi, Y., Hopkins, D. W. and Schulz, R. A. (2015). Bag of Marbles controls the size and organization of the Drosophila hematopoietic niche through interactions with the Insulin-like growth factor pathway and Retinoblastoma-family protein. Development [Epub ahead of print]. PubMed ID: 26041767
During Drosophila hematopoiesis, Bag of Marbles (Bam) is known to function as a positive regulator of hematopoietic progenitor maintenance in the lymph gland blood cell-forming organ. This study demonstrates a key function for Bam in cells of the lymph gland posterior signaling center (PSC), a cellular domain proven to function as a hematopoietic niche. Bam is expressed in PSC cells and gene loss-of-function results in PSC overgrowth and disorganization, indicating Bam plays a crucial role in controlling the proper development of the niche. It was previously shown that Insulin receptor (InR) pathway signaling was essential for proper PSC cell proliferation. This study analyzed PSC cell number in lymph glands that were double mutant for bam and InR pathway genes, and observed bam genetically interacts with pathway members in the formation of a normal PSC. The elF4A protein is a translation factor downstream of InR pathway signaling and functional knockdown of this critical regulator rescued the bam PSC overgrowth phenotype, further supporting the cooperative function of Bam with InR pathway members. Additionally, the Retinoblastoma-family protein (Rbf), a proven regulator of cell proliferation, is present in cells of the PSC, with this expression dependent on bam function. In contrast, perturbation of Decapentaplegic or Wingless signaling failed to affect Rbf niche cell expression. Together, these findings indicate InR pathway-Bam-Rbf functional interactions represent a newly identified means to regulate the correct size and organization of the PSC hematopoietic niche.
|Flores, H. A., Bubnell, J. E., Aquadro, C. F. and Barbash, D. A. (2015). The Drosophila bag of marbles gene interacts genetically with Wolbachia and shows female-specific effects of divergence. PLoS Genet 11: e1005453. PubMed ID: 26291077
Many reproductive proteins from diverse taxa evolve rapidly and adaptively. These proteins are typically involved in late stages of reproduction such as sperm development and fertilization, and are more often functional in males than females. Surprisingly, many germline stem cell (GSC) regulatory genes, which are essential for the earliest stages of reproduction, also evolve adaptively in Drosophila. One example is the bag of marbles (bam) gene, which is required for GSC differentiation and germline cyst development in females and for regulating mitotic divisions and entry to spermatocyte differentiation in males. This study shows that the extensive divergence of bam between Drosophila melanogaster and D. simulans affects bam function in females but has no apparent effect in males. It was further found that infection with Wolbachia pipientis, an endosymbiotic bacterium that can affect host reproduction through various mechanisms, partially suppresses female sterility caused by bam mutations in D. melanogaster and interacts differentially with bam orthologs from D. melanogaster and D. simulans. It is proposed that the adaptive evolution of bam has been driven at least in part by the long-term interactions between Drosophila species and Wolbachia. More generally, it is suggested that microbial infections of the germline may explain the unexpected pattern of evolution of several GSC regulatory genes.
Mottier-Pavie, V.I., Palacios, V., Eliazer, S., Scoggin, S. and Buszczak, M. (2016). The Wnt pathway limits BMP signaling outside of the germline stem cell niche in Drosophila ovaries. Dev Biol [Epub ahead of print]. PubMed ID: 27364467
|Ji, S., Li, C., Hu, L., Liu, K., Mei, J., Luo, Y., Tao, Y., Xia, Z., Sun, Q. and Chen, D. (2017). Bam-dependent deubiquitinase complex can disrupt germ-line stem cell maintenance by targeting cyclin A. Proc Natl Acad Sci U S A. PubMed ID: 28484036
Drosophila germ-line stem cells (GSCs) provide an excellent model to study the regulatory mechanisms of stem cells in vivo. Bag of marbles (bam) has been demonstrated to be necessary and sufficient to promote GSC and cystoblast differentiation. Despite extensive investigation of its regulation and genetic functions, the biochemical nature of the Bam protein has been unknown. This study reports that Bam is an ubiquitin-associated protein and controls the turnover of cyclin A (CycA). Mechanistically, it was found that Bam associated with Otu to form a deubiquitinase complex that stabilized CycA by deubiquitination, thus providing a mechanism to explain how ectopic expression of Bam in GSCs promotes differentiation. Collectively, these findings not only identify a biochemical function of Bam, which contributes to GSC fate determination, but also emphasizes the critical role of proper expression of cyclin proteins mediated by both ubiquitination and deubiquitination pathways in balancing stem cell self-renewal and differentiation.
|Keskeny, C., Raisch, T., Sgromo, A., Igreja, C., Bhandari, D., Weichenrieder, O. and Izaurralde, E. (2019). A conserved CAF40-binding motif in metazoan NOT4 mediates association with the CCR4-NOT complex. Genes Dev 33(3-4): 236-252. PubMed ID: 30692204
The multisubunit CCR4-NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. The NOT4 E3 ubiquitin ligase is a stable component of the CCR4-NOT complex in yeast but does not copurify with the human or Drosophila melanogaster complex. This study shows that the C-terminal regions of human and D. melanogaster NOT4 contain a conserved sequence motif that directly binds the CAF40 subunit of the CCR4-NOT complex (CAF40-binding motif [CBM]). In addition, nonconserved sequences flanking the CBM also contact other subunits of the complex. Crystal structures of the CBM-CAF40 complex reveal a mutually exclusive binding surface for NOT4 and Roquin or Bag of marbles mRNA regulatory proteins. Furthermore, CAF40 depletion or structure-guided mutagenesis to disrupt the NOT4-CAF40 interaction impairs the ability of NOT4 to elicit decay of tethered reporter mRNAs in cells. Together with additional sequence analyses, these results reveal the molecular basis for the association of metazoan NOT4 with the CCR4-NOT complex and show that it deviates substantially from yeast. They mark the NOT4 ubiquitin ligase as an ancient but nonconstitutive cofactor of the CCR4-NOT deadenylase with potential recruitment and/or effector functions (Keskeny, 2019).
|Sgromo, A., Raisch, T., Backhaus, C., Keskeny, C., Alva, V., Weichenrieder, O. and Izaurralde, E. (2017). Drosophila Bag-of-marbles directly interacts with the CAF40 subunit of the CCR4-NOT complex to elicit repression of mRNA targets. RNA [Epub ahead of print]. PubMed ID: 29255063
Drosophila melanogaster Bag-of-marbles (Bam) promotes germline stem cell (GSC) differentiation by repressing the expression of mRNAs encoding stem cell maintenance factors. Bam interacts with Benign gonial cell neoplasm (Bgcn) and the CCR4 deadenylase, a catalytic subunit of the CCR4-NOT complex. Bam has been proposed to bind CCR4 and displace it from the CCR4-NOT complex. This study investigated the interaction of Bam with the CCR4-NOT complex by using purified recombinant proteins. Unexpectedly, it was found that Bam does not interact with CCR4 directly but instead binds to the CAF40 subunit of the complex in a manner mediated by a conserved N-terminal CAF40-binding motif (CBM). The crystal structure of the Bam CBM bound to CAF40 reveals that the CBM peptide adopts an alpha-helical conformation after binding to the concave surface of the crescent-shaped CAF40 protein. It was further shown that Bam-mediated mRNA decay and translational repression depend entirely on Bam's interaction with CAF40. Thus, Bam regulates the expression of its mRNA targets by recruiting the CCR4-NOT complex through interaction with CAF40.
|Carbonell, A., Perez-Montero, S., Climent-Canto, P., Reina, O. and Azorin, F. (2017). The germline linker histone dBigH1 and the translational regulator Bam form a repressor loop essential for male germ stem cell differentiation. Cell Rep 21(11): 3178-3189. PubMed ID: 29241545
Drosophila spermatogenesis constitutes a paradigmatic system to study maintenance, proliferation, and differentiation of adult stem cell lineages. Each Drosophila testis contains 6-12 germ stem cells (GSCs) that divide asymmetrically to produce gonialblast cells that undergo four transit-amplifying (TA) spermatogonial divisions before entering spermatocyte differentiation. Mechanisms governing these crucial transitions are not fully understood. This study reports the essential role of the germline linker histone dBigH1 during early spermatogenesis. These results suggest that dBigH1 is a general silencing factor that represses Bam, a key regulator of spermatogonia proliferation that is silenced in spermatocytes. Reciprocally, Bam represses dBigH1 during TA divisions. This double-repressor mechanism switches dBigH1/Bam expression from off/on in spermatogonia to on/off in spermatocytes, regulating progression into spermatocyte differentiation. dBigH1 is also required for GSC maintenance and differentiation. These results show the critical importance of germline H1s for male GSC lineage differentiation, unveiling a regulatory interaction that couples transcriptional and translational repression.
|Tokusumi, T., Tokusumi, Y. and Schulz, R. A. (2018). The mir-7 and bag of marble genes regulate Hedgehog pathway signaling in blood cell progenitors in Drosophila larval lymph glands. Genesis e23210. PubMed ID: 29663653
Hedgehog (Hh) pathway signaling is crucial for the maintenance of blood cell progenitors in the lymph gland hematopoietic organ present in Drosophila third instar larvae. Previous studies have likewise shown the importance of the mir-7 and bag of marbles (bam) genes in maintaining the progenitor state. Thus attempts were made to investigate a possible interaction between the Hh pathway and mir-7/bam in the prohemocyte population within this hematopoietic tissue. Gain of function mir-7 was able to rescue a blood cell progenitor depletion phenotype caused by Patched (Ptc) inhibition of Hh pathway signaling in these cells. Similarly, expression of a dominant/negative version of Ptc was able to rescue the severe reduction of prohemocytes due to bam loss of function. Furthermore, it was demonstrated that Suppressor of fused [Su(fu)], another known inhibitor of Hh signaling, likely serves as a translational repression target of the mir-7 miRNA. These results suggest the mir-7/bam combination regulates the Hh signaling network through repression of Su(fu) to maintain hemocyte progenitors in the larval lymph gland.
|Zhao, S., Fortier, T. M. and Baehrecke, E. H. (2018). Autophagy promotes tumor-like stem cell niche occupancy. Curr Biol 28(19): 3056-3064.e3053. PubMed ID: 30270184
Adult stem cells usually reside in specialized niche microenvironments. Accumulating evidence indicates that competitive niche occupancy favors stem cells with oncogenic mutations, also known as tumor-like stem cells. However, the mechanisms that regulate tumor-like stem cell niche occupancy are largely unknown. This study used Drosophila ovarian germline stem cells as a model and use bam mutant cells as tumor-like stem cells. Interestingly, it was found that autophagy is low in wild-type stem cells but elevated in bam mutant stem cells. Significantly, autophagy is required for niche occupancy by bam mutant stem cells. Although loss of either atg6 or Fip200 alone in stem cells does not impact their competitiveness, loss of these conserved regulators of autophagy decreases bam mutant stem cell niche occupancy. In addition, starvation enhances the competition of bam mutant stem cells for niche occupancy in an autophagy-dependent manner. Of note, loss of autophagy slows the cell cycle of bam mutant stem cells and does not influence stem cell death. In contrast to canonical epithelial cell competition, loss of regulators of tissue growth, either the insulin receptor or cyclin-dependent kinase 2 function, influences the competition of bam mutant stem cells for niche occupancy. Additionally, autophagy promotes the tumor-like growth of bam mutant ovaries. Autophagy is known to be induced in a wide variety of tumors. Therefore, these results suggest that specifically targeting autophagy in tumor-like stem cells has potential as a therapeutic strategy.
|Herrera, S. C. and Bach, E. A. (2018). JNK signaling triggers spermatogonial dedifferentiation during chronic stress to maintain the germline stem cell pool in the Drosophila testis. Elife 7. PubMed ID: 29985130
Exhaustion of stem cells is a hallmark of aging. In the Drosophila testis, dedifferentiated germline stem cells (GSCs) derived from spermatogonia increases during lifespan, leading to the model that dedifferentiation counteracts the decline of GSCs in aged males. To test this, dedifferentiation was blocked by mis-expressing the differentiation factor bag of marbles (bam) in spermatogonia while lineage-labeling these cells. Strikingly, blocking bam-lineage dedifferentiation under normal conditions in virgin males has no impact on the GSC pool. However, in mated males or challenging conditions, inhibiting bam-lineage dedifferentiation markedly reduced the number of GSCs and their ability to proliferate and differentiate. bam-lineage derived GSCs have significantly higher proliferation rates than sibling GSCs in the same testis. Jun N-terminal kinase (JNK) activity is autonomously required for bam-lineage dedifferentiation. Overall, this study shows that dedifferentiation provides a mechanism to maintain the germline and ensure fertility under chronically stressful conditions.
|Ji, S., Luo, Y., Cai, Q., Cao, Z., Zhao, Y., Mei, J., Li, C., Xia, P., Xie, Z., Xia, Z., Zhang, J., Sun, Q. and Chen, D. (2019). LC domain-mediated coalescence is essential for Otu enzymatic activity to extend Drosophila lifespan. Mol Cell. PubMed ID: 30879902
In eukaryotic cells, RNA-binding proteins (RBPs) interact with RNAs to form ribonucleoprotein complexes (RNA granules) that have long been thought to regulate RNA fate or activity. Emerging evidence suggests that some RBPs not only bind RNA but also possess enzymatic activity related to ubiquitin regulation, raising important questions of whether these RBP-formed RNA granules regulate ubiquitin signaling and related biological functions. This study shows that Drosophila Otu binds RNAs and coalesces to membrane-less biomolecular condensates via its intrinsically disordered low-complexity domain, and coalescence represents a functional state for Otu exerting deubiquitinase activity. Notably, coalescence-mediated enzymatic activity of Otu is positively regulated by its bound RNAs and co-partner Bam. Further genetic analysis reveals that the Otu/Bam deubiquitinase complex and dTraf6 constitute a feedback loop to maintain intestinal immune homeostasis during aging, thereby controlling longevity. Thus, regulated biomolecular condensates may represent a mechanism that controls dynamic enzymatic activities and related biological processes.
The early processes of Drosophila gametogenesis in both ovaries and testes are remarkably similar to one another: germ-line stem cells are sequestered at the anterior end of either organ. When a germ-line cell divides, one of the daughters remains attached and continues to function as a stem cell, whereas the other daughter becomes the 'founder' of a 16-cell syncytial cyst. Four successive divisions of this cystoblast or spermatoblast (within the ovary or testis, respectively) produce a clone of 16 sister germ line cells that remain interconnected due to an unusual process of incomplete cytokinesis, which generates interconnecting ring canals. In the ovary, a complement of somatic cells encloses each 16-cell cyst sometime after it is formed, as it passes through a specific region of the germarium. By comparison, in the testis during spermatogenesis the founding spermatoblast is enclosed by just two somatic cells prior to the four successive spermatoblast divisions. These differences aside, both processes generate cysts of 16 germ-line-derived cells surrounded by a layer of somatic cells. Shortly after this developmental juncture has been reached, the pathways diverge: female cysts go on to produce a single egg, whereas male cysts produce 64 spermatids. (See betaTub85D for a more detailed account of spermatogenesis; see also the oocyte and alpha Spectrin sites for information about oogenesis).
Of particular interest is the fate transition of descendents of the self-renewing stem cell population to become more specialized daughter cells (termed the 'cystoblast' in oogenesis, and the 'primary spermatogonial cell' in spermatogenesis). Several genes have been characterized in Drosophila that carry a common defect in oogenesis. These include ovarian tumor (otu), ovo, benign gonial cell neoplasm, sans fille, Sex lethal and the topic of this site, bag of marbles (bam). Mutations at these loci result in the absence of mature germ cells, and in the overproliferation of small cells with morphological characteristics of undifferentiated germ cells. The tumorous cyst phenotype points to the importance of gene action in the transition from stem cell to daughter cells of distinct fate. Only bag of marbles and benign gonial cell neoplasm (bgcn) act both in oogeneis and spermatogenesis, suggesting that these two genes regulate a pathway shared in the two processes (Gonczy, 1997).
In bam mutant ovaries, the overproliferating germ cells appear to behave as stem cells, according to two criteria. (1) Germ cells overproliferating in bam mutant ovaries are either not connected cytoplasmically to a neighbouring cell, or are connected to a single neighbouring cell, with a fusome passing between the two cells (see alpha Spectrin for information about the fusome). This pattern of connections is identical to that of wild-type germline stem cells, but different from that of wild-type amplifying germ cells (the differentiating progeny of stem cells), which are connected to several neighbouring germ cells as a result of incomplete cytokineses. Similarly, branched fusomes are rarely observed in bgcn mutant ovaries. (2) Germ cells overproliferating in bam mutant ovaries undergo S phase asynchronously. Again, this behavior is like that observed among germline stem cells, but not among their dividing differentiating progeny, which progress synchronously through the cell cycle. These observations led to the conclusion that bam mutant germ cells behave as stem cells and to the postulate that bam function is normally required in females to assign the fate of the cystoblast (McKearin, 1995).
Does bag of marbles play a similar role in spermatogenesis, that is, does bam mutation result in the overproliferation of germ-line stem cells in the male? Examination of bam testes suggests that gametogenesis is also disrupted at an early stage in males. These testes contain abnormal cysts populated with an excessive number of small cells the size of primary spermatocytes. Normally, the 16 primary spermatocytes, derived from the primary spermatogonial cell by four cell divisions, increase 25-fold in size prior to the onset of meiosis. However, bam spermatocytes remain about the size of early spermatocytes. They never undergo subsequent morphological changes characteristic of meiosis and spermatogenesis. Consistent with an early developmental arrest, expression of betaTub85D (also known as beta2-tubulin) is not detected in mutant testes. Mutant cysts progress into a highly refractile state (McKearin, 1990).
Evidence points to different consequences for bam mutation, depending on whether it occurs in spermatogenesis or oogenesis. In either case, mutations in bam and its kindred gene bgcn result in the overproliferation of undifferentiated germ cells. In males, bam and bgcn appear to be required to restrict the proliferation of amplifying germ cells (the progeny of primary spermatogonial cells) or to promote their entry into the meiotic cell cycle, in the case of spermatogenesis. Germ cells accumulating in bam and bgcn mutant testes have several characteristics of amplifying germ cells: (1) cytoplasmic Bam protein is expressed in bgcn mutant germ cells; (2) bgcn and bam mutant germ cells undergo incomplete cytokinesis, and (3) they proliferate in synchrony within a cyst. All three aspects are characteristic of amplifying germ cells, but not of germline stem cells. It is concluded that bam and bgcn most likely do not regulate the decision to adopt a primary spermatogonial cell fate; rather, they act to restrict the proliferation of amplifying germ cells or promote their entry into the meiotic cell cycle (Gonczy, 1997).
In females, overexpression of bam has a deleterious effect on oogenic germline stem cells, one that is not evident in spermatogenic germline stem cells. Female germ cells are progressively depleted with bam overexpression, suggesting that ectopic bam expression blocks stem cell function. It is though that ectopic bam causes stem cells to divide as committed cystoblasts: as contractile ring formation and closure takes place, fusomes grow and cell-cycle coordination is evident. It is suggested that a presumptive cystoblast daughter (pre-cystoblast) of the stem cell division undergoes a maturation process during which bam+ activation initiates cystoblast/cystocyte mitoses by modifying the germ-cell division cycle. Surprisingly, ectopic bam+ has no deleterious consequences for male germline cells, suggesting again that Bam may regulate somewhat different steps of germ-cell development in oogenesis as compared to spermatogenesis (Ohlstein, 1997).
What is the basis of the difference between functions of Bam in males and females? The function of bam and bgcn may have been adapted to play an essential role in the cystoblast due to a unique requirement of the female germline stem cell lineage. In females, only one of the sixteen amplifying germ cells adopts the oocyte fate, while its fifteen sisters become nurse cells. Oocyte determination relies on polarized microtubule-dependent intercellular transport from the nurse cells into the oocyte through ring canals. The basis for this polarity appears to be established as early as in the cystoblast. Since the fusome is associated with microtubules, it has been suggested that the fusome helps establish and maintain polarity during cystoblast divisions and the amplifying of germ cell divisions. Since a form of Bam protein is associated with the fusome (McKearin, 1995), it is possible that Bam itself plays a role in establishing polarity in the cystoblast. In bam mutant ovaries, polarity cannot be established, and the cystoblast cell fate cannot be assigned, resulting in an overproliferation of cells with stem cell characteristics. By contrast, in males, all sixteen amplifying germ cells are equivalent; there is no evidence of polarity analogous to that observed in females. This might explain why bam and bgcn are not strictly necessary for assigning primary spermatogonial cell fate (Gonczy, 1997).
What is the biochemical function of Bag of marbles? Little can be gleaned from the protein sequence since Bam is a novel protein. Some information is provided by the phenotype especially with regard to fusome appearance. In the female, each round of cystocyte mitosis is accompanied by the growth of a germ cell-specific organelle, the fusome. The fusome contains four membrane cytoskeletal proteins: alpha-Spectrin, beta-Spectrin, the adducin-like Hu-li tai shao and Ankyrin. Stem cells and cystocytes contain a large sphere of fusomal material, termed the spectrosome. During the four cystocyte mitoses, one pole of each spindle associates with the fusome, and following each mitosis, as the spindles disaggregate, additional fusomal material accumulates. Thus, by the fourth division, the fusome forms one large branched structure that extends though the ring canals into all the cells in a cyst. alpha-Spectrin deficient cells were generated in fly ovaries and the effects on cyst formation and oocyte differentiation were observed. This work shows that the fusome acts as a pole for each mitotic spindle by capturing a centriole and, in this capacity, serves to orient the planes of cell division at each cystocyte mitosis (reviewed in McKearin, 1997).
Bam is a component of the fusome. It has been noted the bam mutant fusomes are deficient in the membranous tubular reticulum that normally fills the fusome core. This observation prompts the suggestion that Bam protein might be required to recruit vesicular material into the reticulum. A role for the fusome reticulum in directing a switch from stem cell to cystoblast-like divisions could explain both the bam loss-of-function and ectopic expression phenotypes. It is proposed that fusome biogenesis is an obligate step for cystoblast cell fate and that Bam is the limiting factor for fusome maturation in female germ cells (Ohlstein, 1997).
Epigenetic silencing is critical for maintaining germline stem cells in Drosophila ovaries. However, it remains unclear how the differentiation factor, Bag-of-marbles (Bam), counteracts transcriptional silencing. This study found that the trimethylation of lysine 36 on histone H3 (H3K36me3), a modification that is associated with gene activation, is enhanced in Bam-expressing cells. H3K36me3 levels were reduced in flies deficient in Bam. Inactivation of the Set2 methyltransferase, which confers the H3K36me3 modification, in germline cells markedly reduced H3K36me3 and impaired differentiation. Genetic analyses revealed that Set2 acts downstream of Bam. Furthermore, orb expression, which is required for germ cell differentiation, was activated by Set2, probably through direct H3K36me3 modification of the orb locus. These data indicate that H3K36me3-mediated epigenetic regulation is activated by bam, and that this modification facilitates germ cell differentiation, probably through transcriptional activation. This work provides a novel link between Bam and epigenetic transcriptional control (Mukai, 2015).
To examine histone modifications in differentiating germ cells, wild-type ovaries were stained using monoclonal antibodies specific for histone modifications. The H3K36me3 histone modification, associated with active genes, accumulated in differentiating cystoblasts. H3K36me3 signals were increased in the differentiating cystoblasts that expressed the bam reporter gene (bam-GFP). By contrast, the H3K27me3 modification associated with gene repression accumulated in early germ cells, and its signals decreased as the cells differentiated. These results suggest that the H3K36me3 levels were upregulated in differentiating cystoblasts. Next, H3K36me3 levels were examined in the ovaries of the third instar larvae and bam86 mutant adult females, both of which contain undifferentiated germ cells. Although H3K27me3 signals were detected in these undifferentiated germ cells, strong H3K36me3 signals were not detected. Taken together, these data supported the idea that H3K36me3-mediated epigenetic regulation may be involved in germ cell differentiation. (Mukai, 2015).
Set2 methyltransferase is responsible for the H3K36me3 modification. Immunostaining revealed that, in the germarium region, Set2 was expressed in most of the germline cells, and that nuclear Set2 levels increased in differentiating cystoblasts. To determine whether Set2 participates in H3K36me3 accumulation and differentiation, Set2 expression was inhibited by using an UAS-Set2.IR line. Set2 levels in germ cells were reduced by the expression of Set2 RNAi. Specifically, while Set2 signals in differentiating cystoblasts were detected in 100% of control (nanos-Gal4/+) germaria, the Set2 signals in the cystoblasts were significantly reduced in 57% of the germaria, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver. Next, H3K36me3 levels were investigated in the ovaries expressing Set2 RNAi. As expected, H3K36me3 levels were reduced as a consequence of Set2 RNAi treatment. In control ovaries, H3K36me3 signals in differentiating cystoblasts were detected in 97% of germaria. By contrast, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver, H3K36me3 signals in cystoblasts were severely reduced in 41% of the germaria. Moreover, germ cell differentiation was impaired because of the expression of Set2 RNAi. In 96% of the control germaria, cysts with branched fusomes were observed. However, fragmented fusomes were detected in 34% of the germaria expressing Set2 RNAi. These results indicate that Set2 was required for both H3K36me3 accumulation and cyst formation. Mosaic analysis was performed by using a Set2 null allele Set21. Strong H3K36me3 signals were observed in 80% of the control germline clones. By contrast, H3K36me3 levels were considerably reduced in 74% of the Set2- cystoblasts. Furthermore, a differentiation defect was observed that was similar to that induced by Set2 RNAi treatment in 84% of Set2- mutant cysts. These results suggest that Set2 is intrinsically required both for H3K36me3 accumulation in cystoblasts and for differentiation (Mukai, 2015).
To investigate the potential regulatory link between Set2 and Bam, their genetic interaction was analyzed. Reduction in Set2 activity by introduction of a single copy of Set21 dominantly increased the number of germaria with the differentiation defect in bam86/+ flies. Fragmented fusomes were observed in 26% of germaria from the Set21/+; bam86/+ females , as compared to 5% in bam86/+ and 3% in Set21/+ females. These results indicated that Set2 cooperates with bam to promote cyst formation. To determine whether bam expression requires Set2 activity, Bam expression in Set2- germline clones by immunostaining. Indeed, Set2 activity in germ cells was dispensable for bam expression. Conversely, nuclear Set2 expression in the germ cells was significantly reduced by bam mutation, suggesting that bam is involved in the regulation of Set2 in these cells. This result is consistent with the observation that H3K36me3 levels were reduced by bam mutation. Moreover, reducing of bam activity by introducing of a single copy of bam86 dominantly increased the number of germaria with weaker H3K36me3 signals in Set21/+ flies. Decreased H3K36me3 signals in the cystoblasts were observed in 29% of germaria from the Set21/+; bam86/+ females, as compared to 3% in Set21/+ and 2% in bam86/+ females. These data prompted an exploration of the mechanism of regulation of Set2 activity by bam (Mukai, 2015).
To address whether bambam is sufficient for H3K36me3 accumulation, H3K36me3 levels were examined in the ovaries carrying the hs-bam transgene, which is used to ectopically express bam+ by heat shock treatment (Ohlstein and McKearin, 1997). No GSCs with a strong H3K36me3 signal were observed in germaria from wild-type females 1 hour post-heat shock (PHS; n = 42). However, H3K36me3 levels in the GSCs were significantly increased in 51% of the germaria from hs-bam females 1 hour PHS (n = 65), indicating that ectopic bam expression is sufficient for H3K36me3 accumulation. Because Set2 is responsible for H3K36me3, it is speculated that bam may regulate Set2 activity to control H3K36me3 accumulation and GSC differentiation. To determine whether Set2 activity is required for these bam-mediated processes, the effect was studied of a reduction in Set2 activity on the GSC differentiation induced by bam. When bam+ was ectopically expressed by heat shock, GSC differentiation was induced as previously reported. In 71% of ovaries from hs-bam flies dissected 24 hours PHS, it was found that differentiating cysts, instead of GSCs, occupied the tip of germaria. By contrast, when both bam and Set2 RNAi were ectopically expressed, GSC loss was significantly suppressed. These data suggest that Set2 activity is regulated by Bam, and that Set2 acts downstream of bam and promotes differentiation (Mukai, 2015).
Nuclear Set2 levels were increased in differentiating cystoblasts. Furthermore, nuclear Set2 levels in germ cells were reduced by bam mutation. It is speculated that bam may regulate Set2 nuclear localization. Therefore, whether bam expression is sufficient for Set2 nuclear accumulation was examined. The subcellular localization of Set2 was examined in hs-bam flies cultured at 30°C. First, H3K36me3 levels were examined in the GSCs. H3K36me3 levels in GSCs were increased in 36% of the germaria from the hs-bam females, as compared to 6% in wild-type females. This result suggests that the ectopic expression of bam is sufficient for H3K36me3 accumulation. Next, Set2 subcellular localization was examined in GSCs of hs-bam females cultured at 30°C. Nuclear Set2 levels in GSCs were increased in 54% of the germaria from the hs-bam females, as compared to 12% in wild-type females. These results suggest that bam promotes the nuclear accumulation of Set2 (Mukai, 2015).
To understand the mechanism by which Set2 regulates germ cell differentiation, the genetic interaction between Set2 and the differentiation genes A2BP1 and orb, both of which are required for cyst differentiation, were examined. Reduction of Set2 activity by introduction of a single dose of Set21 dominantly increased the number of germaria exhibiting a differentiation defect in orbdec/+ flies. In 24% of germaria from the Set21/+; orbdec/+ females, fragmented fusomes were observed, as compared with 4% in orbdec/+ and 7% in Set21/+ females. By contrast, the reduction of Set2 activity did not significantly affect cyst formation in A2BP1KG06463/+ ovaries). These results implied that Set2 function is required to specifically regulate orb expression and promote cyst formation. To confirm this, orb expression was examined in Set2- cyst clones. Deletion of Set2 led to the delayed activation of orb. Although 74% of the control cyst clones located at the boundary of germarium regions 1 and 2a initiated orb expression, only 31% of Set2- cyst clones expressed orb. Most (61%) of the Set2- cyst clones in germarium region 2b recovered orb expression. These observations suggest that Set2 was required for the proper activation of orb in differentiating cysts. Next, the H3K36me3 state of the orb locus was investigated in the ovaries. ChIP assays demonstrated that the H3K36me3 enrichment in the 3'-UTR region of orb was significantly higher than in the 5'-UTR region. It has been reported that the H3K36me3 modification exhibits a 3'-bias, such that H3K36me3 is preferentially enriched at the 3' regions of actively transcribed genes. These results support the idea that orb expression in differentiating cysts is controlled in part by H3K36me3-mediated epigenetic regulation (Mukai, 2015).
Next, the H3K36me3 status was investigated in the orb gene in bam86 mutant ovaries. ChIP assays showed that bam mutation reduced the amount of H3K36me3 in the 3'-UTR region of the orb gene. The H3K36me3 modification is linked to transcriptional elongation. Therefore, the results suggested that bam activates orb expression through the epigenetic control. Additionally, H3K4me3 and RNA polymerase II levels in the 5'-UTR region of the orb gene were also reduced by bam mutation, implying a role for bam in transcriptional initiation. To investigate this possibility, further investigation will be needed in order to identify the enzymes responsible for H3K4me3 and exploring the interactions between bam and those enzymes (Mukai, 2015).
These results have shown that H3K36me3 levels are regulated by bam. As a cytoplasmic protein, Bam may indirectly regulate Set2 nuclear localization. Set2 exerts its functions through the interactions with cofactors. Understanding the mechanism by which Bam regulates Set2 will require the identification of the cofactors that mediate the nuclear transport of Set2. These data suggest a link between Bam and epigenetic transcriptional control. Bam may counteract epigenetic silencing in GSCs through H3K36me3-mediated epigenetic regulation. This study showed that orb expression is activated by epigenetic regulation. Because orb encodes a cytoplasmic polyadenylation element-binding protein, Orb may control translation in differentiating cysts in a polyadenylation-associated manner. Bam antagonizes the Nanos/Pumilio complex, which suppresses the translation of target mRNAs that encode differentiation factors . However, the ientity of the target mRNAs and the mechanisms for transcriptional activation have not yet been elucidated. Because Set2 is required for bam-induced GSC differentiation, studies focused on identifying the genes marked by H3K36me3 and on their epigenetic regulation will aid in the identification of the differentiation genes. Because Set2 is linked to transcriptional elongation, differentiation genes in GSCs might be poised for expression, but may be kept awaiting bam expression for full activation. It is anticipated that these results will facilitate a better understanding of the epigenetic mechanisms that regulate gametogenesis (Mukai, 2015).
The available experimental data support the hypothesis that the cap cells (CpCs) at the anterior tip of the germarium form an environmental niche for germline stem cells (GSCs) of the Drosophila ovary. Each GSC undergoes an asymmetric self-renewal division that gives rise to both a GSC, which remains associated with the CpCs, and a more posterior located cystoblast (CB). The CB upregulates expression of the novel gene, bag of marbles (bam), which is necessary for germline differentiation. Decapentaplegic (Dpp), a BMP2/4 homolog, has been postulated to act as a highly localized niche signal that maintains a GSC fate solely by repressing bam transcription. The role of Dpp in GSC maintenance has been examined in more detail. In contrast to the above model, it is found that an enhancer trap inserted near the Dpp target gene, Daughters against Dpp (Dad), is expressed in additional somatic cells within the germarium, suggesting that Dpp protein may be distributed throughout the anterior germarium. However, Dad-lacZ expression within the germline is present only in GSCs and to a lower level in CBs, suggesting there are mechanisms that actively restrict Dpp signaling in germ cells. One function of Bam is to block Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes. Moreover, in females doubly mutant for bam and the ubiquitin protein ligase Smurf, the number of germ cells responsive to Dpp is greatly increased relative to the number observed in either single mutant. These data indicate that there are multiple, genetically redundant mechanisms that act within the germline to downregulate Dpp signaling in the Cb and its descendants, and raise the possibility that a Cb and its descendants must become refractory to Dpp signaling in order for germline differentiation to occur (Casanueva, 2004).
The prevalent model for Dpp action within the ovary is that it is a local niche signal whose activity is permissive for GSC maintenance. In this model, only GSCs within the niche are exposed to Dpp protein and removal of the CB from the niche lessens or eliminates exposure to the ligand. Moreover, the only postulated function of Dpp is to repress the transcription of bam within the GSCs. The data presented in this paper reveal additional aspects of Dpp function in GSC maintenance. The results strongly suggest that Dpp ligand is not restricted to the niche but rather is present throughout the anterior germarium. Data is presented that the observed specificity of Dpp signaling to the GSCs and CBs is due to functionally redundant mechanisms that operate in the germline to actively downregulate Dpp signaling during GSC differentiation. One of these mechanisms is Bam itself, thus establishing a negative feedback loop between the actions of the two genes. These findings indicate GSC differentiation is correlated with downregulation of Dpp signaling, raising the possibility that Dpp signaling plays an active role in GSC maintenance, and that GSC differentiation requires both the presence of Bam and the absence of Dpp signaling (Casanueva, 2004).
If GSCs and CBs are exposed to equivalent amounts of Dpp protein, as is suggested by both the transcription pattern of the Dpp gene and the expression of Dad-lacZ in the CpCs of the niche and the ISCs posterior to the niche, then it is likely that the observed reduction in Dad-lacZ expression between the GSC and the CB results from intracellular modulation of the strength of the Dpp signal. One hallmark of the GSC is its invariant plane of division. It is proposed that the differential Dpp signaling between the GSC and CB sign results from an intracellular modulation of Dpp signal strength between the two daughter cells, either by the asymmetric segregation of one or more cellular components that modulate Dpp signaling, or by loss of a contact-based niche signal that elevates Dpp signaling preferentially within the GSCs. Removal of the CB cell from the niche thus results in partial downregulation of Dpp signaling. A lower level of Dpp signaling in the CB cell results in the transcription of Bam, which plays multiple roles in CB differentiation, one of which is to cause the daughters of the CB cell to become refractory to further Dpp signaling. Thus, sequential regulatory mechanisms cooperate to ensure an irreversible change in the fate of the GSC cell within two generations (Casanueva, 2004).
Loss-of-function mutations in Smurf and gain-of-function mutations in sax increase the number of GSCs, suggesting these genes may perturb the proposed intracellular modulation of Dpp signaling that occurs between the GSC and CB. However, these data are not sufficient to determine whether this proposed modulatory pathway acts through direct regulation of the functions of one or both of these gene products, or whether the proposed pathway acts in parallel to these genes. In the embryo, loss of Smurf activity results in a ligand-dependent elevation of Dpp signaling that has greater, but not indefinite, perdurance (Podos, 2001), suggesting that Dpp signaling in Smurf mutants, and by inference sax mutants, is still responsive both to the amount of ligand and to the presence of other negative regulatory mechanisms. In the ovary, the Dad-lacZ-expressing germ cells in the Smurf and sax mutants fill the region of the anterior germarium that roughly corresponds to the spatial extent of Dad-lacZ expression in the somatic cells of region 1 and 2A of a wild-type germarium, suggesting that potentially all germ cells in region 1 and 2A of the Smurf and sax germaria are equally and fully responsive to the Dpp ligand. It is proposed that GSCs in the Smurf and sax germaria ultimately undergo normal differentiation because in the more posterior regions of the germaria the amount of Dpp ligand may be reduced to a level that allows bam transcription, which further reduces Dpp signaling and causes cyst differentiation (Casanueva, 2004).
The reduction in Dpp signaling between the GSC and the CB releases Bam from Dpp-dependent transcriptional repression, and one, but not the only, function of Bam is to downregulate Dpp signaling downstream of receptor activation prior to overt GSC differentiation. This is the first molecular action ascribed to Bam, and these data could provide an entry point to elucidate the biochemical basis of the function of Bam in CB differentiation. Further work will be necessary to determine whether the action of Bam on the Dpp pathway is direct or indirect, whether Bam action results in the reduction or complete elimination of Dpp signaling in the developing cysts, and which step in the intracellular Dpp signal transduction pathway or expression of Dpp target genes is affected by Bam action. However, it is possible that initial insights into Bam function can be made by comparing the thresholds for Dpp signaling readouts in the developing wing disc of the larva to the data obtained in the germarium. In the wing disc, Dpp diffuses from a limited source to form a gradient throughout the disc that displays different thresholds for multiple signaling readouts. Specifically, Dad-lacZ is transcribed in response to high and intermediate levels of Dpp, but does not respond to the lowest levels of ligand. An antibody exists that recognizes the active phosphorylated form of Mad, pMad. In the wing disc, high level staining with the pMad antibody is present in only a subset of cells that express high levels of Dad-lacZ, suggesting that in this tissue the pMad antibody is less sensitive to Dpp signaling than is Dad-lacZ expression. Intriguingly, in the ovariole pMad staining is visible in the GSCs, CBs and the developing cysts. Because Dad-lacZ expression was never observed in the developing cysts, these results could suggest that the relative sensitivities of these two reagents are reversed within the germline. Alternatively, if the reagents have the same relative sensitivities in the two tissues, the data suggest that Bam could act, probably at a post-transcriptional level, to downregulate Dpp signaling downstream of Mad activation (Casanueva, 2004).
The pattern of Dad-lacZ expression observed in the Smurf; bam and sax; bam double mutant ovarioles is qualitatively different from that observed in any of the single mutant ovarioles. Although Dad-lacZ expression is observed only at the anterior tip of the germarium of each single mutant, many, but not all, of the double mutant ovarioles contain germ cells throughout the ovariole that express high levels of Dad-lacZ. From these data, it is concluded that two redundant pathways downregulate Dpp signaling in the germline, and that in the single mutants, the action of the remaining active pathway is sufficient to constrain Dpp responsiveness to the anterior tip of the germarium. However, not all doubly mutant ovarioles display a spatial expansion of Dpp signaling, and this variability can even be observed in ovarioles from a single female. It is proposed that the observed variability results because the Smurf and sax mutations have modulatory effects on Dpp signaling that are both dependent on the presence of ligand and are sensitive to additional mechanisms that downregulate Dpp signaling. In both the Smurf; bam and sax; bam ovarioles, the germ cells that express Dad-lacZ are observed throughout the ovariole, but are more likely to be near somatic cells. It is possible that the variability in Dad-lacZ expression occurs because of a non-uniform distribution of the Dpp ligand. Nevertheless, there is not a consistent correlation between the domains of Dad-lacZ expression in the somatic and germ cells, suggesting that there may be additional germline intrinsic factors that affect Dpp signaling (Casanueva, 2004).
Stem cell self-renewal is controlled by concerted actions of extrinsic niche signals and intrinsic factors in a variety of systems. Drosophila ovarian germline stem cells (GSCs) have been one of the most productive systems for identifying the factors controlling self-renewal. The differentiation factor BAM is necessary and sufficient for GSC differentiation, but it still remains expressed in GSCs at low levels. However, it is unclear how its function is repressed in GSCs to maintain self-renewal. This study reports the identification of the translation initiation factor eIF4A for its essential role in self-renewal by directly inactivating BAM function. eIF4A can physically interact with BAM in Drosophila S2 cells and yeast cells. eIF4A exhibits dosage-specific interactions with bam in the regulation of GSC differentiation. It is required intrinsically for controlling GSC self-renewal and proliferation but not survival. In addition, it is required for maintaining E-cadherin expression but not BMP signaling activity. Furthermore, BAM and BGCN together repress translation of E-cadherin through its 3' UTR in S2 cells. Therefore, it is proposed that BAM functions as a translation repressor by interfering with translation initiation and eIF4A maintains self-renewal by inhibiting BAM function and promoting E-cadherin expression (Shen, 2009).
This study has revealed the biochemical function of the BAM/BGCN complex as a translational repressor. eIF4A in the regulation of GSC self-renewal to be a direct antagonist of BAM function in the Drosophila ovary. A model is proposed explaining how GSC self-renewal is controlled by concerted actions of intrinsic factors and the extrinsic BMP signal. BMP signaling directly represses bam expression, yet leaves low levels of BAM protein expression in the GSC. eIF4A and other unidentified germline factors in the GSC can effectively dismantle BAM/BGCN's repression of GSC maintenance factors, including E-cadherin, through physical interactions, leading to high expression of maintenance factors in the GSC. In the cystoblast (CB), high levels of BAM along with BGCN can keep eIF4A proteins out of the active pool and thus effectively repress GSC maintenance factors, promoting CB differentiation. Therefore, this study has significantly advanced current understanding of how GSC self-renewal and differentiation are regulated by translation factors (Shen, 2009).
bam and bgcn genetically require each other's function to control CB differentiation. Although they are expressed at low levels in GSCs, they have an important role in regulating GSC competition. However, their biochemical functions remained unclear until this study. This study showed that BAM specifically interacts with BGCN, but not other RNA-binding proteins VASA, Rm62, and Me31B, to form a protein complex. In addition, BAM and BGCN are shown to act together; BAM or BGCN alone are not capable of suppressing the expression of the reporter containing the shg 3' UTR. Furthermore, BAM and BGCN do not affect the stability of the reporter mRNA, further supporting that they regulate mRNA translation but not stability. To reveal the role of BGCN in the function of the BAM/BCGN complex, this study showed that direct tethering of BAM to the 3' UTR of the target mRNA can bypass the requirement of BGCN and sufficiently suppress the expression of the reporter. Based on the fact that BGCN contains a putative DEXH RNA binding domain, it is proposed that BGCN helps bring BAM to its target mRNAs to repress their translation. Therefore, this study has revealed the biochemical functions of BAM and BGCN (Shen, 2009).
Previous genetic study showed that BAM and BGCN negatively regulate E-cadherin expression in GSCs to control GSC competition, but the underlying molecular mechanism remains defined. This study showed that in Drosophila S2 cells BAM and BGCN could repress E-cadherin expression through its 3' UTR at the translational level. Along with previous observation that BAM and BGCN negatively regulate E-cadherin expression in GSCs in vivo, it is proposed that BAM and BGCN likely repress E-cadherin expression in GSCs at the translational level. In the future, it will be important to show if BAM and BGCN directly bind to the shg 3' UTR to repress E-cadherin expression in the GSC (Shen, 2009).
eIF4A, an RNA helicase, is one component of the translation initiation complex eIF4F, which is required for loading the small 40S ribosome subunit onto the target mRNA to initiate its translation. The helicase activity of eIF4A itself is weak but is enhanced upon binding to eIF4G, another component of eIF4F. Such helicase activity is important to remove the secondary structure of the 5' UTR, facilitating the ribosome scanning along mRNA to find the initiation codon ATG. To reveal how BAM and BGCN confer translation repression, the yeast 2-hybrid screen was used to identify eIF4A as a BAM interacting protein. Then, two pieces of genetic of evidence were provided supporting the idea that eIF4A and bam function together to control the balance between GSC self-renewal and differentiation. First, one copy of the mutations in eIF4A can dramatically promote germ cell differentiation in the hypomorphic bamZ/bamδ86 transheterozygous ovaries. However, a mutation in eIF4A cannot suppress the tumorous phenotype of the bam?86 homozygous ovaries (no bam function at all), suggesting that the reduction of eIF4A dosage helps enhance the remaining BAM function. Second, overexpression of eIF4A can enhance the differentiation defect in the bam?86 heterozygote. These genetic results support the antagonizing relationship between bam and eIF4A (Shen, 2009).
The antagonizing genetic relationship between bam and eIF4A suggests that eIF4A favors GSC maintenance over differentiation. The genetic analysis of the marked eIF4A mutant GSC clones shows that eIF4A is indeed required in GSCs for their self-renewal and division. To uncover the genetic mechanism underlying the function of eIF4A in maintaining GSCs, it was also shown that the marked eIF4A mutant GSC has normal BMP signaling activities in comparison with its neighboring wild-type GSC based on expression results from 2 BMP responses genes, bam and Dad, but has significantly reduced E-cadherin expression in comparison with its neighboring wild-type GSC. These genetic and cell biological results demonstrate that eIF4A controls GSC maintenance at least partly by maintaining E-cadherin expression. In mammalian cells, overexpression of translation initiation factors, such as eIF4A, 4G, and 4E, is implicated in different kinds of cancer due to their ability to increase cell proliferation. In the Drosophila imaginal disc, the block in cell proliferation caused by mutations in eIF4A can be bypassed by E2F overexpression, indicating that eIF4A regulates cell cycle progression and consequently cell proliferation. In this study, it was shown that eIF4A is also required for controlling GSC division. Therefore, it is proposed that eIF4A controls GSC proliferation by regulating cell cycle progression like in Drosophila imaginal tissues (Shen, 2009).
Bases in 5' UTR - 112
Exons - 3
Bases in 3' UTR - 479
Bam has some sequence homology to the ovarian tumor gene of Drosophila. Weak similarity is detected within three regions spanning about 150 amino acids in the central region of the Bam protein. About 20% of the residues are identical and 15% correspond to conserved replacements within these regions (McKearin, 1990).
date revised: 2 December 2018
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