Genes & Development

Faithful chromosome segregation during meiosis I requires tight control of interhomolog recombination. In budding yeast, the meiotic chromosome axis, built on Rec8-containing cohesin together with Red1 and Hop1, acts as a central platform regulating meiotic recombination from programmed DNA double-strand break (DSB) formation to checkpoint signaling and chromosome segregation, yet how cohesin recruits axis proteins remains unclear. Here, we identified a conserved cohesin-interacting motif (CIM) in Red1 that directly binds Rec8. AlphaFold3 modeling predicted that Red1-CIM forms a short -helix that docks into a conserved hydrophobic pocket within the Rec8 C-terminal winged-helix domain, which we confirmed biochemically. Disruption of the Red1-CIM preferentially impaired Red1 recruitment to Rec8-dependent chromosomal regions, while relative enrichment in Rec8-independent domains was preserved, leading to reduced DSB formation in Rec8-dependent domains. The Red1-CIM mutation also reduced crossover formation, increased chromosome missegregation, and reduced spore viability. Notably, this spore lethality exceeded that predicted by the reduction in DSB formation. Consistently, red1-CIM mutants failed to activate the meiotic checkpoint kinase Mek1. Finally, we provide evolutionary, structural, and biochemical evidence that this Red1-Rec8 interaction is conserved across fungi and plants. Together, these findings define a direct molecular bridge linking cohesin to chromosome-axis organization, spatial DSB regulation, and checkpoint signaling during meiosis.

Polycomb Repressive Complex 2 (PRC2) is a conserved epigenetic regulator that represses gene expression through methylation of histone H3 lysine 27 (H3K27me3). In animals, plants, and some fungi, PRC2-directed facultative heterochromatin plays essential roles in development and cellular differentiation. Here, we show that the replication-dependent histone chaperone Chromatin Assembly Factor 1 (CAF-1) is required for proper structure and function of facultative heterochromatin in the model fungus Neurospora crassa. Loss of CAF-1 causes widespread transcriptional misregulation, particularly within PRC2-repressed regions, and leads to redistribution of H3K27me3, reduced ASH1-dependent H3K36 methylation, and accumulation of chromatin marks associated with active transcription. CAF-1 was not required for repressive histone methylation within constitutive heterochromatin. A double mutant lacking both CAF-1 and PRC2 components displayed a synergistic silencing defect, suggesting these complexes make distinct contributions to facultative heterochromatin. Together, our findings indicate that CAF-1 works in concert with PRC2 to silence transcription within N. crassa facultative heterochromatin domains.

Heterochromatin and its core effectors from the Heterochromatin Protein 1 (HP1) family are essential for epigenetic silencing of repetitive regions and genome integrity from yeast to humans. However, how HP1s and associated factors regulate heterochromatin properties in vivo to sustain silencing across development, aging, and environmental factors, remains incompletely understood. Here, we identify Clump/CG30403 -- a previously uncharacterized MADF-BESS domain protein -- as a novel heterochromatin factor required for robust transposon silencing during Drosophila oogenesis and sustained fertility with age and temperature stress. Clump/CG30403 interacts with the main HP1-family silencing effector Su(var)205/HP1a through a noncanonical binding motif within its large intrinsically disordered region. Notably, in the absence of Clump/CG30403, HP1a mobility and silencing capacity are compromised despite largely unperturbed genomic distribution, showing that HP1a presence alone is insufficient for repression. We also show that Clump/CG30403 uniquely accumulates at its own promoter to self-repress and prevent ectopic aggregation, revealing a feedback mechanism to constrain protein dosage and phase behavior. Overall, we propose that Clump/CG30403 is a HP1 corepressor that acts as a tightly calibrated safeguard of the heterochromatin environment properties to ensure stable silencing, genome integrity, and persistent reproductive function.

Topoisomerase II (TOP2) resolves DNA topological constraints through a tightly regulated cycle of DNA double-strand cleavage and religation. Nearby DNA damage or chemotherapeutic agents such as etoposide block the DNA religation step, stabilizing TOP2-DNA cleavage complexes (TOP2ccs) at DNA double-strand breaks (DSBs). The SUMO E3 ligase ZATT (ZNF451) has recently emerged as a key effector of TOP2cc repair, but its mechanism of action remains poorly understood. Here, we show that ZATT is sufficient to resolve TOP2ccs independently of TDP2, TOP2 proteolysis, and canonical DSB repair pathways. Using Xenopus egg extracts and biochemical reconstitution, we find that ZATT salvages trapped TOP2 by promoting TOP2 release from its stalled cleavage complex. Structural modeling and targeted mutagenesis in Xenopus egg extracts and human cells identify a highly conserved hydrophobic pocket in the tower domain of TOP2 where the ZATT coiled-coil "hooks on" to promote TOP2cc resolution. Our findings reveal a new strategy to resolve TOP2ccs that bypasses the exposure of dangerous DNA breaks.

The PR-DUB complex is responsible for erasing the repressive histone modification, H2AK119ub1. ASXL1-3 proteins are mutually exclusive catalytic partners of BAP1 in the PR-DUB complex. Somatic heterozygous ASXL1-3 variants are associated with cancer, including myeloid malignancies, while de novo germline variants cause neurodevelopmental disorders such as Bohring-Opitz syndrome. These pathogenic variants are almost exclusively nonsense and frameshift and have been proposed to act as gain-of-function. However, the precise catalytic impact and mechanism of variant ASXL1-3 remains elusive. Using an isogenic embryonic stem cell model we have discovered that ASXL1 BOS variants drive reductions - but not global ablations - in H2AK119ub1, consistent with gain-of-function. This catalytic change occurs through the production of a truncated ASXL1 protein with enhanced stability. Hyper-stabilised ASXL1 drives a stoichiometric shift in PR-DUB assembly away from ASXL2 complexes. The drop in H2AK119ub1 levels ultimately reduces PRC2 binding and H3K27me3 deposition. Surprisingly, this phenotype is shared across PR-DUB loss-of-function models and indeed is emerging as a common phenotype across genetically and mechanistically distinct Polycomb-related chromatinopathies.

Processing of the precursor ribosomal RNAs (pre-rRNAs) is a key aspect of ribosomal subunit assembly that is closely coordinated with other maturation events. The ribonucleases that mediate pre-rRNA cleavages require regulation to ensure that their activities are exerted in a timely manner. Post-translational modifications can influence protein functions, and although many human ribosome assembly factors are reported to be post-translationally modified, most of these sites remain unconfirmed and functional insights are lacking. Here, we show that NOB1, the PIN domain endoribonuclease responsible for cleavage of the 3' end of the 18S rRNA, is phosphorylated within an evolutionarily conserved acidic tract that can be modified by casein kinase II in vitro. Association of NOB1 with pre-ribosomes is independent of these phosphorylations, and lack of NOB1 phosphorylation only mildly perturbs the efficiency of SSU maturation events upstream of 3' end cleavage of the 18S rRNA. Interestingly, our analyses of pre-rRNA levels in cells depleted of NOB1 or lacking its catalytic activity revealed not only accumulation of the 18SE precursor of the 18S rRNA, but also altered levels of pre-rRNAs containing 5' external transcribed spacer (ETS) sequences (43S, 26S and 30S). This suggests that lack of NOB1-mediated pre-rRNA cleavage impairs recycling of assembly factors required during early biogenesis steps, leading to altered kinetics of 5' ETS processing. Taken together these data provide new insights into the role of NOB1 during SSU biogenesis and the post-translational regulation of this ribonuclease.

G-quadruplexes (G4s) are non-canonical DNA secondary structures that can impede DNA replication and transcription and provoke genome instability, and DNA helicases of the PIF1 and RecQ families have long been regarded as the principal enzymes that resolve them. To directly test the relative contributions of these families, we measured the growth of Saccharomyces cerevisiae helicase mutants in the presence of the G4-stabilizing ligand pyridostatin (PDS). Unexpectedly, no single PIF1- or RecQ-family mutant was sensitized to PDS relative to wild type. Sensitivity emerged only in double mutants, and it did so for combinations both within a single family and across the two families. This pattern indicates that G4 tolerance is buffered by the combined, partially interchangeable, activity of multiple helicases rather than by any one family. To ask whether this redundancy extends beyond the canonical players, we tested two additional helicases whose human orthologs are implicated in G4 metabolism: Chl1 (DDX11/ChlR1) and Srs2 (RTEL1). Loss of Chl1 alone did not sensitize cells, and chl1{Delta} combined with PIF1- or RecQ-family mutations recapitulated the redundancy pattern - with one informative exception: chl1{Delta} hrq1{Delta} remained PDS-tolerant, placing Chl1 and Hrq1 in a shared genetic route. In contrast, srs2{Delta} was the sole single mutant sensitized to PDS, defining a non-redundant requirement that no other helicase compensates. We integrate these results into a two-layer model in which a redundant helicase pool resolves G4-associated genomic stress, while a non-redundant Srs2 function manages its recombinogenic consequences. Our findings reframe G4 maintenance from a family-specific activity into a distributed, buffered network. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/737069v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@18db695org.highwire.dtl.DTLVardef@ce0062org.highwire.dtl.DTLVardef@7b2a8corg.highwire.dtl.DTLVardef@267073_HPS_FORMAT_FIGEXP M_FIG C_FIG ARTICLE SUMMARYDNA helicases, enzymes that unwind DNA, are thought to dismantle G-quadruplexes (G4s), four-stranded DNA structures that can block DNA metabolism and destabilize genomes. In Saccharomyces cerevisiae, we used the chemical pyridostatin to stabilize G4s and measured the growth of helicase mutants. Losing any single helicase had no effect, but losing two together - even from different helicase families - impaired growth. The protein Chl1 works with the helicase Hrq1 in one shared pathway, while Srs2 is uniquely required on its own. G4 tolerance therefore depends on a redundant network of helicases. These findings interest researchers studying genome stability and related human cancer-predisposition disorders.

A central question in developmental biology is how a single signaling pathway can generate diverse developmental outcomes. One prominent example is Notch-dependent binary fate choice, which is reiteratively utilized during development to sequentially generate multiple distinct pairs of cell fates. To investigate the transcriptional mechanisms that diversify Notch signaling outputs, we profiled gene expression and chromatin accessibility simultaneously in single cells of the developing Drosophila medulla, identified and analyzed the cis-regulatory enhancer elements (CREs) for representative neuronal transcription factor (nTF) genes, whose expression depends on the Notch status. Canonical models predict that Notch target genes of the Hey/Hes family act primarily as transcriptional repressors and bind the CACGTG E-box motif to suppress Notch-off transcriptional programs in Notch-on cells. Contrary to the prediction, we found that Hey is not required for repression of Notch-off nTF genes. Instead, Hey is required for activation of a subset of Notch-on nTF genes. Using the Notch-on nTF gene bsh as an example, we identified a CRE that recapitulates the endogenous expression pattern and demonstrated that a single-base-pair mutation in a CACGTG type E-box present in the CRE abolishes its enhancer activity. We further identified additional relay factors that mediate Notch-dependent transcriptional outputs. The bHLH factors Sim/Tgo activate a Notch-on nTF through binding to the AACGTG variant E-boxes within its CRE, whereas the bHLH factor Tap activates a Notch-off nTF through a CRE containing different types of E-boxes. Together, these findings reveal that Notch signaling is not relayed through a single universal downstream effector. Instead, distinct bHLH factors decode Notch status through different classes of E-box motifs embedded within target enhancers. Finally, we show that enhancer architecture is modular, allowing temporal identity and Notch-status information to be integrated through the same or distinct CREs to generate precise patterns of nTF expression. We propose that diversification of Notch-dependent cell fates arises through a modular transcriptional relay and enhancer-decoding mechanism in which multiple bHLH factors act on distinct E-box motifs to convert a common signaling input into diverse developmental outcomes.

During organogenesis, epithelial tissues undergo extensive three-dimensional (3D) remodeling while simultaneously generating specialized cell types. Whether these transient architectural states actively instruct lineage allocation remains unclear. Here we identify a morphogenetic stage in which resolution of epithelial stratification is required for lineage allocation and establishment of endocrine cell mass. We show that loss of the Hippo pathway regulator Merlin disrupts lumen morphogenesis and prevents formation of the transient 3D epithelial architecture that characterizes normal pancreas development. Failure to establish this architectural state alters lineage allocation, impairing acinar differentiation, markedly reducing adult endocrine cell mass, and disrupting glucose homeostasis. Mosaic analyses reveal that these lineage defects arise non-cell autonomously, demonstrating that epithelial architecture itself instructs cell fate decisions. Mechanistically, Merlin coordinates PI3K-regulated polarized membrane trafficking required for apical membrane biogenesis and lumen formation. Together, these findings identify Merlin-dependent membrane trafficking as a mechanism coupling epithelial morphogenesis to lineage allocation and demonstrate that transient developmental architectures can determine the cellular composition of mature organs.

The Saccharomyces cerevisiae Pch2 protein is a conserved meiotic AAA+ ATPase whose activity must be tightly regulated to ensure proper chromosome dynamics during meiotic prophase I. Its function relies on remodeling the HORMA-domain protein Hop1, promoting conformational transitions that are essential for chromosome axis organization, checkpoint signaling, and recombination control. Here, we identify threonine 428 (T428), located within a conserved threonine-glutamine (TQ) putative phosphorylation motif, as a critical regulatory residue of Pch2. We found that, in zip1{Delta} cells, the meiotic recombination checkpoint response is partially or completely abolished in the pch2-T428A and pch2-T428D mutants, respectively. Both mutations alter Pch2 subcellular localization, leading to its increased nuclear accumulation; however, forced nuclear exclusion of Pch2-T428A, but not Pch2-T428D, restores the zip1{Delta} meiotic block, indicating an additional effect of the T428D substitution on checkpoint function beyond subcellular distribution. Analysis in synapsis-proficient strains reveals that this residue also plays a critical role in coordinating Hop1 chromosomal enrichment with Mek1 activation along the synaptonemal complex. In contrast to pch2{Delta} or the ATPase-defective pch2-E399Q mutant, introduction of a negative charge at the 428 position uncouples Hop1 accumulation from its phosphorylation, preventing Mek1 activation despite robust Hop1 association with meiotic chromosomes. These findings support emerging models in which Pch2 regulates Hop1 to control not only its chromosomal abundance, but also the maintenance of sufficient levels of Hop1 in a phosphorylation-competent conformation, thereby ensuring proper checkpoint signaling and faithful meiotic progression.

The rhombic lip (RL) gives rise to all cerebellar glutamatergic cell types, including unipolar brush cells (UBCs). Disruptions to UBC development can lead to the neurodevelopmental disorder Dandy-Walker Syndrome and the pediatric brain tumor medulloblastoma, but these diseases have not been adequately modeled in mice. To evaluate conservation of UBC development in mouse and human, we examined UBC localization, lineage decisions, and the underlying molecular mechanisms of UBC differentiation using multiplex immunofluorescence and single-cell RNA-seq of wild-type and conditional knockout animals of the primary UBC transcription factor Eomes. Similar to the human RL, the murine RL is molecularly compartmentalized, cycling EOMES+ UBC progenitors are highly abundant, and persist after birth. Eomes regulates the transcriptional networks important for UBC differentiation and migration, but not UBC fate. Overall, our findings suggest that murine UBC development recapitulates many features of human UBC development, with EOMES playing a central role in UBC maturation.

In female mammals, Xist, the master regulator of X-chromosome inactivation (XCI), is expressed monoallelically. This pattern is established during early embryonic development, when the active Xist allele is chosen at random in each cell. How this choice is made remains incompletely understood. Combining knock-down and overexpression strategies in differentiating mouse embryonic stem cells, which recapitulate the onset of random XCI, we identify a role for the repressive chromatin mark H3K9me3 in the XCI initiation. We show that H3K9me3 accumulates at the promoter-proximal region of the silent Xist allele in female cells as monoallelic expression is established. Unexpectedly, this accumulation requires prior transcription of Xist itself, likely during the initial phase of upregulation, when Xist is frequently transcribed in male cells and from both X chromosomes in females. A repressive function of Xist-dependent H3K9me3 accumulation is supported by our finding that premature, transient Xist overexpression primes an allele for future silencing and skews the choice of the inactive X. Xist-dependent H3K9me3 recruitment does not require its antisense transcript Tsix, which can nonetheless enhance subsequent maintenance of the mark. In addition, the X-linked Xist activator RNF12 counteracts H3K9me3 formation, independently of its known target REX1. Our results thus point to facultative heterochromatin formation as a key contributor to choice at the onset of XCI, where activating and repressing mechanisms are intertwined to establish monoallelic Xist expression.

Esophageal adenocarcinoma (EAC) is a genetically heterogeneous malignancy with few recurrent drivers, limiting effective targeted therapies. Although EAC arises from Barretts esophagus (BE), mechanisms driving progression from this premalignant state to invasive cancer remain unclear. We combined pooled CRISPR-Cas9 loss-of-function screening, in vivo tumorigenicity assays, and Perturb-seq profiling to define functional drivers of BE transformation. We identified 37 tumor suppressors whose loss promotes progression to EAC, defining a functional landscape of tumor initiation. Despite genetic diversity, these losses converged on four transcriptional programs involving metabolic reprogramming, cell cycle progression, RNA processing, and cellular motility. Furthermore, we identify loss of NIPBL, TGFBR2, and RPL22 as key mediators of resistance to platinum- and taxane-based chemotherapy. Collectively, these findings provide a unifying framework for genomic heterogeneity in EAC, uncover underappreciated tumor suppressor pathways, and establish a resource to guide mechanistic and translational studies aimed at improving treatment strategies in this aggressive cancer.

Premature termination of transcription (PTT), also known as attenuation, is a conserved gene regulatory mechanism that operates across all domains of life and in viruses. Attenuation enables rapid cellular responses to environmental and metabolic changes and fine-tunes expression of biosynthetic genes. In Saccharomyces cerevisiae, attenuation of RNA Polymerase II (Pol II) transcription was first linked to the Nrd1-Nab3-Sen1 (NNS) termination pathway for non-coding RNAs, and the mRNA 3-end processing factor Hrp1 has been implicated more recently. Substitutions in Hrp1 RNA Recognition Motifs (RRMs) cause attenuator readthrough and reduce RNA-binding affinity in vitro, but direct evidence for Hrp1 functioning at attenuators in vivo remains limited. Here, we characterized 5-end RNA terminator elements from several genes, including RAD3, SNG1, MNR2, and CPR8. Readthrough mutations clustered in AU-rich regions resembling polyadenylation site (pA) efficiency elements, consistent with Hrp1 binding targets. Amino acid substitutions of Hrp1 RRM residue F162 revealed a general requirement for aromaticity in RNA recognition that varied to some degree by gene context. To test Hrp1-RNA interactions independent of other yeast factors, we adapted a bacterial 3-hybrid (B3H) assay. Hrp1 interacted with RNA derived from the GAL7 3-end pA site and 5-end terminator regions of RAD3, MNR2, and CPR8. Mutations in AU-rich RNA regions that disrupted Pol II attenuation in yeast generally impaired B3H interactions. However, some Hrp1 mutants (M191T, I270T, D271G, M275V, T280I) retained binding to CPR8 terminator RNA, suggesting their defects require additional yeast components. These results demonstrate that Hrp1 is sufficient to bind multiple UA-rich attenuator RNAs in vivo, expanding Hrp1 function to include early transcription events.

Heterochromatin marked by histone H3 lysine 9 di- or trimethylation (H3K9me2/3) underpins transcriptional silencing and nuclear organization, yet its full complement of associated proteins remains incompletely defined. Here, we apply ChromID proximity labelling with the mouse HP1{beta} chromodomains to map the H3K9me3-proximal proteome in Caenorhabditis elegans, recovering known heterochromatin factors alongside previously uncharacterized candidates. We pursued one such candidate, the vaccinia-related kinase VRK-1, because of its established but poorly understood links to chromatin organization. Intriguingly, VRK-1 dynamically relocates from a broad nuclear distribution to the nuclear periphery upon azide or heat stress. Following these stresses, bulk chromatin exhibits similarly increased peripheral enrichment and apparent compaction, as assessed by radial fluorescence profiles. Although VRK-1 is not necessary for stress-induced chromatin reorganization, decompaction and repositioning of chromatin away from the nuclear envelope during recovery requires VRK-1. VRK-1 depletion leads to persistent perinuclear chromatin retention and compromises post-stress survival. Furthermore, loss of VRK-1 catalytic activity results in over-retention of chromatin at the nuclear periphery under normal growth conditions; this phenotype can be reversed by depletion of a key VRK-1 substrate at the nuclear envelope BAF-1. Our findings identify VRK-1 as a key regulator that controls the interaction of chromatin with the nuclear lamina through regulation of BAF-1.

The olfactory epithelium of adult mammals contains two populations of stem cells that support its remarkable ability to regenerate neuronal and non-neuronal cell types throughout life. How olfactory epithelial cell types are established during development, however, is not well understood. Here, we use genetic lineage tracing and single-cell RNA sequencing of the perinatal mouse olfactory epithelium to construct a developmental trajectory consisting of multiple lineages. We identify transitional states and lineage relationships between individual cells and establish Ascl1+ cells as the primary multipotent progenitors in the perinatal olfactory epithelium. Further, Ascl1+ cells become progressively restricted in their cell fate capacity over developmental time and appear to be transcriptionally primed toward specific lineages. We also predict signaling pathways that may contribute to lineage plasticity and niche permissiveness. Together, these results contribute to our understanding of how cell-intrinsic and-extrinsic signals contribute to the establishment of a stem cell niche.

During early embryogenesis, gene expression relies on maternally loaded mRNAs whose translation is controlled by RNA binding proteins. Here, we identify a critical role for the cyclin B3-CDK1 complex, known for its function in mitosis, in driving early embryonic gene expression in C. elegans. The cyclin B3-CDK1 complex works by marking the RNA binding OMA proteins (OMA-1 and OMA-2) for degradation, which ensures the de-repression and translation of their target mRNAs. OMA protein degradation relies on cyclin B3s conserved phosphate-binding pocket, which promotes multi-site OMA phosphorylation and the generation of phospho-degrons. Notably, the phosphate-binding pocket of cyclin B3 does not substantially contribute to its mitotic roles, indicating that the mitotic and translational functions of the cyclin B3-CDK1 complex are separable. These findings establish that embryonic activation of the cyclin B3-CDK1 complex drives both mitotic divisions and mRNA de-repression, which ensures that cell division is coupled to the early gene expression program in development.

The Origin Recognition Complex (ORC) is known for initiating DNA replication in eukaryotic cells, but increasing evidence suggests that ORC has multiple functions. Here we show that the organization and function of two major nuclear components, heterochromatin and the nucleolus, depend on multivalent interactions between Orc1 and Heterochromatin Protein 1a (HP1a) in Drosophila melanogaster. Specifically, binding requires two short motifs (R1 and R2) in the intrinsically disordered region (IDR) of Orc1, and two motifs (HGM and CTE) located in the N- and C-terminal regions of HP1a. Pairing of these four motifs promotes ORC-HP1 interactions, which also requires HP1 dimerization. Disrupting ORC-HP1a interactions by mutating the R1/R2 motifs causes defects in heterochromatin functions, specifically suppression of Position-Effect Variegation (PEV), ribosomal DNA (rDNA) decondensation, increased rRNA transcription, and nucleolar expansion, without global loss of H3K9me2 epigenetic modifications. Our findings indicate that ORC acts as a structural regulator that organizes heterochromatic regions and promotes proper genome regulation through multivalent interactions with HP1 and chromatin.

Germ granules are condensates in germ plasm, a specialized cytoplasm that segregates to the embryonic germline. In Drosophila, translation of nanos mRNA occurs at the surface of germ granules, suggesting that the granules promote translation. In C. elegans, however, germ (P) granules are not essential for Nanos expression. Using single-molecule imaging in C. elegans embryos, we map the distribution of translating and non-translating molecules of the Nanos homolog nos-2 and two other maternal mRNAs enriched in P granules. In early germline blastomeres, these mRNAs are not translated and distribute between the cytoplasm and P granules. At translation onset, mRNA molecules in the cytoplasm are translated, while most mRNA molecules in the P granules remain non-translating. nos-2 translation requires a rise in the concentration of the RNA-binding protein POS-1, which occurs independently of P granules. Consistent with low translation inside the granules, P granules are depleted of ribosomes and 43S pre-initiation complexes. Our observations suggest that germ granules promote Nanos protein expression by concentrating Nanos mRNA in germline precursors, but do not directly promote translation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/735846v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1875b06org.highwire.dtl.DTLVardef@16919f6org.highwire.dtl.DTLVardef@1278c27org.highwire.dtl.DTLVardef@1628645_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisGerm granules are condensates proposed to regulate the translation of mRNAs like Nanos that code for germ cell fate determinants. Using single-molecule imaging in C. elegans embryos, this study shows that P granule scaffolds concentrate mRNAs in germline precursors, but do not control the activity of translational regulators. - P granules concentrate mRNAs but are depleted of ribosomes and 43S pre-initiation complexes - Translation occurs mainly in the cytoplasm where ribosomes are most abundant - nanos translation onset is timed by a rise in POS-1, which counteracts the repressor SPN-4; both enrich in P granules but act independently.

Eukaryotic genomes often have fragile sites where chromosomes are particularly prone to break. In Drosophila, when dicentric ring chromosomes try to segregate, they break at nonrandom hotspots. Here, we precisely map breakage hotspots produced by dicentric ring chromosomes in Drosophila. Our study provides three key results about the nature of dicentric chromosome breakage. First, duplications produced by dicentric ring chromosome breakage are surprisingly complex and involve many structural rearrangements, indicating that healing of these breaks is not a simple process. Second, characterization of one particular hotspot showed that new termini all occurred within a single intron of a large testis-expressed gene, suggesting that replication-transcription conflict may be a key determinant of chromosome fragile sites. Third, the new ends are often located near preexisting transposons, suggesting that transposon insertions may contribute to fragility or participate in stabilization of broken ends.