Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis.

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ cell genes during differentiation, and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ cell genes into a silenced state and activating a group of oocyte genes and nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, cross-talk between genome architecture and NPCs is essential for successful cell fate transitions.

A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate.

The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ∼1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.

Macrophage mitochondrial bioenergetics and tissue invasion are boosted by an Atossa-Porthos axis in Drosophila.

Cellular metabolism must adapt to changing demands to enable homeostasis. During immune responses or cancer metastasis, cells leading migration into challenging environments require an energy boost, but what controls this capacity is unclear. Here, we study a previously uncharacterized nuclear protein, Atossa (encoded by CG9005), which supports macrophage invasion into the germband of Drosophila by controlling cellular metabolism. First, nuclear Atossa increases mRNA levels of Porthos, a DEAD-box protein, and of two metabolic enzymes, lysine-α-ketoglutarate reductase (LKR/SDH) and NADPH glyoxylate reductase (GR/HPR), thus enhancing mitochondrial bioenergetics. Then Porthos supports ribosome assembly and thereby raises the translational efficiency of a subset of mRNAs, including those affecting mitochondrial functions, the electron transport chain, and metabolism. Mitochondrial respiration measurements, metabolomics, and live imaging indicate that Atossa and Porthos power up OxPhos and energy production to promote the forging of a path into tissues by leading macrophages. Since many crucial physiological responses require increases in mitochondrial energy output, this previously undescribed genetic program may modulate a wide range of cellular behaviors.

Dynamic regulation of ribosome levels and translation during development.

The ability of ribosomes to translate mRNAs into proteins is the basis of all life. While ribosomes are essential for cell viability, reduction in levels of ribosomes can affect cell fate and developmental transitions in a tissue specific manner and can cause a plethora of related diseases called ribosomopathies. How dysregulated ribosomes homeostasis influences cell fate and developmental transitions is not fully understood. Model systems such as Drosophila and C. elegans oogenesis have been used to address these questions since defects in conserved steps in ribosome biogenesis result in stem cell differentiation and developmental defects. In this review, we first explore how ribosome levels affect stem cell differentiation. Second, we describe how ribosomal modifications and incorporation of ribosomal protein paralogs contribute to development. Third, we summarize how cells with perturbed ribosome biogenesis are sensed and eliminated during organismal growth.

Msl3 promotes germline stem cell differentiation in female Drosophila.

Gamete formation from germline stem cells (GSCs) is essential for sexual reproduction. However, the regulation of GSC differentiation is incompletely understood. Set2, which deposits H3K36me3 modifications, is required for GSC differentiation during Drosophila oogenesis. We discovered that the H3K36me3 reader Male-specific lethal 3 (Msl3) and histone acetyltransferase complex Ada2a-containing (ATAC) cooperate with Set2 to regulate GSC differentiation in female Drosophila. Msl3, acting independently of the rest of the male-specific lethal complex, promotes transcription of genes, including a germline-enriched ribosomal protein S19 paralog RpS19b. RpS19b upregulation is required for translation of RNA-binding Fox protein 1 (Rbfox1), a known meiotic cell cycle entry factor. Thus, Msl3 regulates GSC differentiation by modulating translation of a key factor that promotes transition to an oocyte fate.

The Drosophila ribosome protein S5 paralog RpS5b promotes germ cell and follicle cell differentiation during oogenesis.

Emerging evidence suggests that ribosome heterogeneity may have important functional consequences in the translation of specific mRNAs within different cell types and under various conditions. Ribosome heterogeneity comes in many forms, including post-translational modification of ribosome proteins (RPs), absence of specific RPs and inclusion of different RP paralogs. The Drosophila genome encodes two RpS5 paralogs: RpS5a and RpS5b. While RpS5a is ubiquitously expressed, RpS5b exhibits enriched expression in the reproductive system. Deletion of RpS5b results in female sterility marked by developmental arrest of egg chambers at stages 7-8, disruption of vitellogenesis and posterior follicle cell (PFC) hyperplasia. While transgenic rescue experiments suggest functional redundancy between RpS5a and RpS5b, molecular, biochemical and ribo-seq experiments indicate that RpS5b mutants display increased rRNA transcription and RP production, accompanied by increased protein synthesis. Loss of RpS5b results in microtubule-based defects and in mislocalization of Delta and Mindbomb1, leading to failure of Notch pathway activation in PFCs. Together, our results indicate that germ cell-specific expression of RpS5b promotes proper egg chamber development by ensuring the homeostasis of functional ribosomes.

Germ cells are forever.

Germ cells are the only cell type capable of generating an entirely new organism. In order to execute germline-specific functions and to retain the capacity for totipotency, germ cells repress somatic differentiation, interact with a specialized microenvironment, and use germline-specific networks of RNA regulation.

A switch in the mode of Wnt signaling orchestrates the formation of germline stem cell differentiation niche in Drosophila.

Germline stem cell (GSC) self-renewal and differentiation into gametes is regulated by both intrinsic factors in the germ line as well as extrinsic factors from the surrounding somatic niche. dWnt4, in the escort cells of the adult somatic niche promotes GSC differentiation using the canonical ß-catenin-dependent transcriptional pathway to regulate escort cell survival, adhesion to the germ line and downregulation of self-renewal signaling. Here, we show that in addition to the ß-catenin-dependent canonical pathway, dWnt4 also uses downstream components of the Wnt non-canonical pathway to promote escort cell function earlier in development. We find that the downstream non-canonical components, RhoA, Rac1 and cdc42, are expressed at high levels and are active in escort cell precursors of the female larval gonad compared to the adult somatic niche. Consistent with this expression pattern, we find that the non-canonical pathway components function in the larval stages but not in adults to regulate GSC differentiation. In the larval gonad, dWnt4, RhoA, Rac1 and cdc42 are required to promote intermingling of escort cell precursors, a function that then promotes proper escort cell function in the adults. We find that dWnt4 acts by modulating the activity of RhoA, Rac1 and cdc42, but not their protein levels. Together, our results indicate that at different points of development, dWnt4 switches from using the non-canonical pathway components to using a ß-catenin-dependent canonical pathway in the escort cells to facilitate the proper differentiation of GSCs.

Transient transcriptional silencing alters the cell cycle to promote germline stem cell differentiation in Drosophila.

Transcriptional silencing is a conserved process used by embryonic germ cells to repress somatic fate and maintain totipotency and immortality. In Drosophila, this transcriptional silencing is mediated by polar granule component (pgc). Here, we show that in the adult ovary, pgc is required for timely germline stem cell (GSC) differentiation. Pgc is expressed transiently in the immediate GSC daughter (pre-cystoblast), where it mediates a pulse of transcriptional silencing. This transcriptional silencing mediated by pgc indirectly promotes the accumulation of Cyclin B (CycB) and cell cycle progression into late-G2 phase, when the differentiation factor bag of marbles (bam) is expressed. Pgc mediated accumulation of CycB is also required for heterochromatin deposition, which protects the germ line genome against selfish DNA elements. Our results suggest that transient transcriptional silencing in the pre-cystoblast "re-programs" it away from self-renewal and toward the gamete differentiation program.

Transposon Dysregulation Modulates dWnt4 Signaling to Control Germline Stem Cell Differentiation in Drosophila.

Germline stem cell (GSC) self-renewal and differentiation are required for the sustained production of gametes. GSC differentiation in Drosophila oogenesis requires expression of the histone methyltransferase dSETDB1 by the somatic niche, however its function in this process is unknown. Here, we show that dSETDB1 is required for the expression of a Wnt ligand, Drosophila Wingless type mouse mammary virus integration site number 4 (dWnt4) in the somatic niche. dWnt4 signaling acts on the somatic niche cells to facilitate their encapsulation of the GSC daughter, which serves as a differentiation cue. dSETDB1 is known to repress transposable elements (TEs) to maintain genome integrity. Unexpectedly, we found that independent upregulation of TEs also downregulated dWnt4, leading to GSC differentiation defects. This suggests that dWnt4 expression is sensitive to the presence of TEs. Together our results reveal a chromatin-transposon-Wnt signaling axis that regulates stem cell fate.

From stem cell to egg cell.

Experiments on female fruit flies reveal more about the molecular mechanisms involved as germline stem cells transition to become egg cells.

Loss Of Chromodomain of Male-Specific Lethal 3 (MSL3) Does Not Affect Spermatogenesis In Rodents.

Msl3 is a member of the chromatin-associated male-specific lethal MSL complex which is responsible for the transcriptional upregulation of genes on the X chromosome in males Drosophila. Although the dosage complex operates differently in mammals, the Msl3 gene is conserved from flies to humans. Msl3 is required for meiotic entry during Drosophila oogenesis. Recent reports indicate that also in primates, Msl3 is expressed in undifferentiated germline cells before meiotic entry. However, if Msl3 plays a role in the meiotic entry of mammals has yet to be explored. To study this, we used mouse spermatogenesis as a study model. Analyses of single cells RNA-seq data revealed that, in mice, Msl3 is mostly expressed in meiotic cells. To test the role of Msl3 in meiosis, we used a male germline-specific Stra8-iCre driver and a newly generated Msl3flox conditional knock-out mouse line. Msl3 conditional loss-of-function in spermatogonia did not cause spermatogenesis defects or changes in the expression of genes related to meiosis. Our data suggest that, in mice, Msl3 exhibits delayed expression compared to Drosophila and primates, and loss-of-function mutations disrupting the chromodomain of Msl3 alone do not impede meiotic entry in rodents.

A feedback loop between heterochromatin and the nucleopore complex controls germ-cell-to-oocyte transition during Drosophila oogenesis.

Germ cells differentiate into oocytes that launch the next generation upon fertilization. How the highly specialized oocyte acquires this distinct cell fate is poorly understood. During Drosophila oogenesis, H3K9me3 histone methyltransferase SETDB1 translocates from the cytoplasm to the nucleus of germ cells concurrently with oocyte specification. Here, we discovered that nuclear SETDB1 is required for silencing a cohort of differentiation-promoting genes by mediating their heterochromatinization. Intriguingly, SETDB1 is also required for upregulating 18 of the ∼30 nucleoporins (Nups) that compose the nucleopore complex (NPC), promoting NPC formation. NPCs anchor SETDB1-dependent heterochromatin at the nuclear periphery to maintain H3K9me3 and gene silencing in the egg chambers. Aberrant gene expression due to the loss of SETDB1 or Nups results in the loss of oocyte identity, cell death, and sterility. Thus, a feedback loop between heterochromatin and NPCs promotes transcriptional reprogramming at the onset of oocyte specification, which is critical for establishing oocyte identity.

Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis.

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ-cell genes during differentiation and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we find that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ-cell genes into a silenced state and activating a group of oocyte genes and Nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, crosstalk between genome architecture and NPCs is essential for successful cell fate transitions.

A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate.

The association of genomic loci to the nuclear periphery is proposed to facilitate cell-type specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ~1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein, Stonewall (Stwl), as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.

H/ACA snRNP-dependent ribosome biogenesis regulates translation of polyglutamine proteins.

Stem cells in many systems, including Drosophila germline stem cells (GSCs), increase ribosome biogenesis and translation during terminal differentiation. Here, we show that the H/ACA small nuclear ribonucleoprotein (snRNP) complex that promotes pseudouridylation of ribosomal RNA (rRNA) and ribosome biogenesis is required for oocyte specification. Reducing ribosome levels during differentiation decreased the translation of a subset of messenger RNAs that are enriched for CAG trinucleotide repeats and encode polyglutamine-containing proteins, including differentiation factors such as RNA-binding Fox protein 1. Moreover, ribosomes were enriched at CAG repeats within transcripts during oogenesis. Increasing target of rapamycin (TOR) activity to elevate ribosome levels in H/ACA snRNP complex-depleted germlines suppressed the GSC differentiation defects, whereas germlines treated with the TOR inhibitor rapamycin had reduced levels of polyglutamine-containing proteins. Thus, ribosome biogenesis and ribosome levels can control stem cell differentiation via selective translation of CAG repeat-containing transcripts.

Oo-site: A dashboard to visualize gene expression during Drosophila oogenesis suggests meiotic entry is regulated post-transcriptionally.

Determining how stem cell differentiation is controlled has important implications for understanding the etiology of degenerative disease and designing regenerative therapies. In vivo analyses of stem cell model systems have revealed regulatory paradigms for stem cell self-renewal and differentiation. The germarium of the female Drosophila gonad, which houses both germline and somatic stem cells, is one such model system. Bulk mRNA sequencing (RNA-seq), single-cell RNA-seq (scRNA-seq), and bulk translation efficiency (polysome-seq) of mRNAs are available for stem cells and their differentiating progeny within the Drosophila germarium. However, visualizing those data is hampered by the lack of a tool to spatially map gene expression and translational data in the germarium. Here, we have developed Oo-site (https://www.ranganlab.com/Oo-site), a tool for visualizing bulk RNA-seq, scRNA-seq, and translational efficiency data during different stages of germline differentiation, which makes these data accessible to non-bioinformaticians. Using this tool, we recapitulated previously reported expression patterns of developmentally regulated genes and discovered that meiotic genes, such as those that regulate the synaptonemal complex, are regulated at the level of translation.

A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis.

Ribosomal defects perturb stem cell differentiation, and this is the cause of ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. Here, we discover that three DExD/H-box proteins govern ribosome biogenesis (RiBi) and Drosophila oogenesis. Loss of these DExD/H-box proteins, which we name Aramis, Athos, and Porthos, aberrantly stabilizes p53, arrests the cell cycle, and stalls germline stem cell (GSC) differentiation. Aramis controls cell-cycle progression by regulating translation of mRNAs that contain a terminal oligo pyrimidine (TOP) motif in their 5' UTRs. We find that TOP motifs confer sensitivity to ribosome levels that are mediated by La-related protein (Larp). One such TOP-containing mRNA codes for novel nucleolar protein 1 (Non1), a conserved p53 destabilizing protein. Upon a sufficient ribosome concentration, Non1 is expressed, and it promotes GSC cell-cycle progression via p53 degradation. Thus, a previously unappreciated TOP motif in Drosophila responds to reduced RiBi to co-regulate the translation of ribosomal proteins and a p53 repressor, coupling RiBi to GSC differentiation.

Sequence-selective purification of biological RNAs using DNA nanoswitches.

Nucleic acid purification is a critical aspect of biomedical research and a multibillion-dollar industry. Here we establish sequence-selective RNA capture, release, and isolation using conformationally responsive DNA nanoswitches. We validate purification of specific RNAs ranging in size from 22 to 401 nt with up to 75% recovery and 99.98% purity in a benchtop process with minimal expense and equipment. Our method compared favorably with bead-based extraction of an endogenous microRNA from cellular total RNA, and can be programmed for multiplexed purification of multiple individual RNA targets from one sample. Coupling our approach with downstream LC/MS, we analyzed RNA modifications in 5.8S ribosomal RNA, and found 2'-O-methylguanosine, 2'-O-methyluridine, and pseudouridine in a ratio of ~1:7:22. The simplicity, low cost, and low sample requirements of our method make it suitable for easy adoption, and the versatility of the approach provides opportunities to expand the strategy to other biomolecules.

RNA degradation is required for the germ-cell to maternal transition in Drosophila.

In sexually reproducing animals, the oocyte contributes a large supply of RNAs that are essential to launch development upon fertilization. The mechanisms that regulate the composition of the maternal RNA contribution during oogenesis are unclear. Here, we show that a subset of RNAs expressed during the early stages of oogenesis is subjected to regulated degradation during oocyte specification. Failure to remove these RNAs results in oocyte dysfunction and death. We identify the RNA-degrading Super Killer complex and No-Go Decay factor Pelota as key regulators of oogenesis via targeted degradation of specific RNAs expressed in undifferentiated germ cells. These regulators target RNAs enriched for cytidine sequences that are bound by the polypyrimidine tract binding protein Half pint. Thus, RNA degradation helps orchestrate a germ cell-to-maternal transition that gives rise to the maternal contribution to the zygote.