Chromosoma

During meiosis, accurate chromosome segregation requires significant condensation and compaction. These processes are mediated by condensins, cohesins, and histone tail modifications. We identified that MET-2, a histone methyltransferase that catalyzes the dimethylation of histone H3 lysine 9 (H3K9me2), differentially impacts chromosome size in the male vs. female C. elegans germline. In met-2 null worms, autosomes during spermatogenesis are significantly larger than wild type, while chromosome size during oogenesis is unaffected. X-univalent size in males is also unaffected by loss of MET-2, indicating MET-2 differentially regulates autosomal and X-chromosome compaction in male spermatogenesis. Autosome size is not changed when males harbor a catalytically deficient MET-2 (met-2CD) or have mutations preventing germline histone H3K9 methylation (H3K9R). In addition, met-2 males, in contrast to met-2CD or H3K9R males, have more active RNA pol II in later stages of meiosis. These data suggest MET-2 plays a noncatalytic role in mediating chromosome structure and transcription. In met-2 male germ lines, genes on the X chromosome, which is typically enriched in H3K9me2, are significantly more likely to be upregulated than genes on autosomes, even though X-univalent size is unchanged. These results suggest that MET-2 plays a sex-specific role that is not limited to its enzymatic activity.

Human nucleoli are multivalent, involving contributions from up to ten NOR-bearing acrocentric chromosome p-arms. Precision mega-base scale chromosome engineering defines the requirements for this major genome organisational event. NOR deletions reveal that p-arm nucleolar association is rDNA independent. Deletion of all NOR-distal or proximal sequences individually have only a marginal effect on nucleolar association. Finally, deletion of an entire p-arm, while leaving centromere function intact, destroys the nucleolar association potential of that acrocentric. We propose that formation of multivalent nucleoli is not a nucleolar fusion event per se; rather it is driven by the surrounding chromosomal context of NORs.

Restricting the localization of centromere-specific histone H3 variant Cse4 (CENP-A in humans) to centromeric chromatin is essential for chromosome segregation. Mislocalization of overexpressed Cse4/CENP-A to non-centromeric regions contributes to chromosomal instability (CIN) in model organisms and human cells. CIN is an important hallmark of many cancers and hence defining mechanisms that prevent mislocalization of Cse4 is clinically significant. Here we report a role for YRA2 (Yeast RNA Annealing Protein 2) in ubiquitin mediated proteolysis of Cse4 to prevent its mislocalization for chromosomal stability. YRA2 was identified in a genome-wide screen for gene deletions that exhibit synthetic dosage lethality (SDL) upon overexpression of CSE4 (GALCSE4). We determined that yra2{Delta} strains exhibit increased Cse4 stability, enriched Cse4 chromatin association, reduced Cse4 ubiquitination, Cse4 mislocalization, and CIN. Defects in interaction of E3 ubiquitin ligase Psh1 with Cse4 contributes to stability of Cse4 in yra2{Delta} strains. Consistent with these results, overexpression of PSH1 suppresses GALCSE4 SDL in yra2{Delta} strain. We determined that Yra2 mediated proteolysis of Cse4 is independent of its RNA related functions as strain deleted for the C-terminal ChTOP domain of Yra2 with an intact N-terminal RNA binding domain exhibits GALCSE4 SDL and defects in Cse4 proteolysis. Furthermore, poly(A)+ RNA export mutants in YRA1 (yra1-2) and MEX67 (mex67-5), that interact with Yra2, do not exhibit GALCSE4 SDL and defects in RNA export are not observed in yra2{Delta} cells. In summary, we have defined a key role for Yra2 in preventing mislocalization of Cse4 by facilitating its proteolysis to preserve chromosomal stability. Article summaryAccurate segregation of chromosomes during cell division is essential because segregation errors are linked to cancer and developmental disorders. We investigated how cells prevent mislocalization of centromere-specific histone H3 variant Cse4, which is essential for faithful chromosome segregation. We found that the yeast RNA annealing protein Yra2 prevents Cse4 mislocalization by promoting Psh1 mediated ubiquitination and degradation of Cse4. Cells lacking Yra2 showed increased stability of Cse4, enhanced chromatin enrichment with mislocalization to non-centromeric regions and CIN. These defects were suppressed by induction of Psh1. Our findings reveal a novel role for Yra2 in regulating Cse4 levels for chromosomal stability.

Bipolar spindle assembly is essential for accurate chromosome segregation. KIFC1, a conserved Ran- regulated minus-end-directed kinesin-14 motor, accumulates in the nucleus during interphase and promotes chromatin-mediated spindle assembly during mitosis and meiosis. In human oocytes, reduced KIFC1 levels destabilize meiotic spindles, a defect that can be rescued by increasing KIFC1 expression. However, how KIFC1 expression levels affect mitotic spindle stability during cleavage divisions in vertebrates remains unclear. Here, we show that whereas an approximately 50% reduction in KIFC1 causes no detectable defects in spindle assembly, approximately 10-fold overexpression of KIFC1 induces monopolar spindle formation, leading to chromosome mis-segregation and embryonic lethality in medaka early embryos. KIFC1 overexpression results in ectopic centrosomal localization during interphase, impairing the separation of duplicated centrosomes before mitotic entry. Analyses of KIFC1 mutants demonstrated that these centrosome separation defects require KIFC1s microtubule-binding and motor activities and are further enhanced by deletion of KIFC1s nuclear localization sequences. Together, our findings demonstrate that tight regulation of KIFC1 expression and its nuclear sequestration is essential for the proper separation and positioning of duplicated centrosomes before mitotic entry, thereby ensuring efficient bipolar spindle assembly during the rapid cleavage divisions of vertebrate embryos. HighlightsO_LIKIFC1 accumulates in the nucleus and at the embryonic spindle midplane via the Ran pathway. C_LIO_LIPartial KIFC1 depletion does not impair spindle assembly in medaka early embryos. C_LIO_LIKIFC1 overexpression induces monopolar spindles by preventing centrosome separation. C_LIO_LICentrosome separation defects require KIFC1 microtubule-binding and motor activity. C_LI

HP1 proteins are highly evolutionarily conserved chromatin-associated factors known to play essential roles in genome stability and nuclear organization. In mammals, three HP1 isoforms, HP1, HP1{beta} and HP1{gamma}, have been described, but their individual functions remain incompletely characterized. Here, we inactivated HP1 or HP1{beta} in different cell lines and quantified chromosomal breaks on metaphase spreads in the presence or absence of aphidicolin-induced replication stress. Loss of HP1, but not of HP1{beta}, led to a significant increase of chromosomal breaks on chromosome arms and within pericentromeric heterochromatin under these conditions. Mechanistically, loss of HP1 was associated with a reduction in replication fork velocity, suggesting that HP1 deficiency induces a replication stress that sensitizes specific genomic loci to replication perturbation. Consistent with this, HP1 loss was associated with a moderate but consistent increase in {gamma}H2AX and 53BP1 foci, an increased occurrence of DNA synthesis during mitosis, and enhanced recruitment of FANCD2, all recognized as hallmarks of common fragile site (CFS) expression. In addition, rescue experiments using a chromodomain mutant HP1 (V22M) unable to bind H3K9me3 indicated that HP1 protective function over these specific foci did not require its interaction with this histone mark. Altogether, these data indicate that, independently of its binding to H3K9me3, HP1 stabilizes specific genomic regions that behave as HP1-dependent fragile sites, at least in part by regulating replication fork progression, limiting mitotic DNA synthesis possibly by competing with FANCD2 for chromatin access at these regions.

The germline-restricted chromosome (GRC) of the zebra finch Taeniopygia guttata represents a well-established model of programmed DNA elimination in vertebrates. Although the DNA composition of the GRC, as well as elimination processes during spermatogenesis and early embryogenesis, have been characterised previously, little is known about the cytogenetic features underlying its unusual behaviour, including its stable transmission through the maternal germline. Here, we provide a detailed characterisation of the zebra finch GRC at the diplotene stage of female meiosis, when chromosomes are actively transcribed and acquire the form of giant lampbrushes. We identified a transcriptionally repressed region on the GRC, which we term the belt. Microdissection and sequencing of the belt revealed that it is predominantly composed of a tandem repeat derived from the dph6 gene, robo1 gene fragments, and ERVs. Notably, the terminally located functional centromere of the GRC lacks typical zebra finch centromeric satellites and, conversely, consists of the newly identified GRC-specific tandem repeats Tgut16-201 and Tgut17-167. The canonical centromeric repeat Tgut716 was observed in the GRC belts. Moreover, belts, like the terminal GRC centromere, were associated with coilin-containing nuclear bodies, which serve as markers of centromeric regions on zebra finch lampbrush chromosomes. Together, our findings provide evidence for the presence of one functional and one putative centromeric region on the zebra finch GRC, suggesting their role in non-Mendelian inheritance of the GRC. Author summaryGermline-restricted chromosomes (GRCs) are unusual chromosomes that are retained in germ cells but eliminated from somatic cells during early development. They have evolved independently in several groups of organisms, but are particularly notable in passerine birds, a large monophyletic vertebrate clade ([~]6,700 species) in which GRCs have persisted for at least 44 million years. Passerine GRCs are normally transmitted to the next generation through the maternal germ cell, however, the mechanisms ensuring their inheritance remain unknown. To address this question, we examined the structure of the zebra finch GRC during female meiosis. We found that the GRC differs from all other chromosomes in possessing two distinct centromeric regions: a functional terminal centromere and an extended heterochromatic region exhibiting centromeric properties. These unusual features suggest a mechanism by which the GRC may achieve its preferential transmission through the female germline. Our findings substantially advance the understanding of the zebra finch GRC and the general biology of passerine GRCs. By revealing chromosome features that may underlie their non-Mendelian inheritance, this work provides new insights into the evolution and behaviour of GRCs and other selfish chromosomes that bias their own transmission.

The genus Rhynchospora Vahl (beak-sedges) comprises approximately 381 accepted species with a worldwide distribution, all of which possess holocentric chromosomes, where centromeric activity is distributed almost along the entire chromosome. Despite the recent advances, the mechanisms governing the dynamics of meiotic recombination in holocentric plants remain poorly understood. Here, we developed haplotype-specific oligo-FISH probes for chromosomes 1, 2, and 3 based on a haplotype-phased genome assembly of Rhynchospora breviuscula (n = 5), enabling homolog-specific chromosome painting. Each probe set was labelled with a distinct fluorophore and hybridised in situ to metaphase chromosomes of the reference plant and seven F1 individuals derived from self-crossed reference plants. This approach allowed the unambiguous discrimination of homologous haplotypes and the indirect visualisation of crossover (CO) events in recombined chromosomes. We observed that recombination events were predominantly located in terminal chromosomal regions, consistent across individuals. These results corroborate previous findings from single-cell recombination mapping and provide independent cytological validation of the recombination landscape in this species. Our study establishes haplotype-specific chromosome painting as a robust tool for high-resolution mapping of meiotic recombination in holocentric plants across generations. Furthermore, these probes provided a foundation for future investigations into inverted meiosis, a mechanism characterized by an alternative pattern of chromosome segregation in holocentric species.

BackgroundLamins are major regulators of the spatial and functional organization of chromatin. Lamins at the nuclear periphery form the lamina that anchors heterochromatin to the nuclear envelope. A subpool of A-type lamins localizes in the nuclear interior, where they also bind to euchromatic genomic regions. A-type lamin properties and chromatin association are regulated by lamin-associated polypeptide 2alpha (LAP2). Here we systematically analyze, how LAP2 depletion affects chromatin organization, accessibility and gene expression on a genome-wide level. ResultsLAP2 depletion in mouse dermal fibroblasts positively and negatively affects chromatin accessibility and gene expression throughout the genome, which correlates with changes in chromatin association of A-type lamins and the nucleosomal remodeler proteins BRG1 and CHD4. In particular, A-type lamins bind to open chromatin regions close to BRG1 and CHD4 binding sites and deregulated genes, but do not directly accumulate on genes and BRG1 and CHD4-enriched sites. Unsupervised clustering of the datasets on LAP2-bound genomic regions confirms spreading of A-type lamins to active chromatin regions containing deregulated genes and an enrichment of chromatin remodelers on a subset of these genomic regions. ConclusionsLAP2 depletion in fibroblasts leads to a gross rearrangement of chromatin. Genome-wide chromatin reorganization is linked to spreading of A-type lamins to active chromatin regions and accompanied by a restriction of chromatin remodelers to a subset of active genomic regions. These changes correlate with changes in chromatin accessibility and gene expression throughout the genome, particularly in regions where lamin binding is gained in LAP2 knockout versus wildtype cells.

The protein complex cohesin plays critical roles in genomic stability by tethering together sister chromatids at their pericentric regions and along their arms from S phase until anaphase. Cohesin-mediated pericentric cohesion prevents aneuploidy by ensuring bipolar attachment of sister kinetochores. Arm cohesion prevents loss of heterozygosity by biasing DNA repair via recombination between sister chromatids rather than between homologs. Here, we investigate in yeast whether cohesin also enhances genomic stability by suppressing unequal sister chromatid exchange (USCE) between repetitive sequences. In wild-type cells, the USCE rate between repeats 4kb apart (proximal) was 15X higher than repeats 68kb apart (distal). The USCE between distal repeats but not proximal repeats increased 4 to 7-fold in mutants with altered cohesin subunits or auxiliary factors. The level of increased distal USCE corresponded with reduced arm cohesion, reduced density of cohesion arm sites, and higher sister loci mobility. Our results suggest that high density of arm cohesion sites confines repair of DNA damage to local sequences. When the density of cohesion sites decreases, sister chromatid sequences are less confined, thereby enhancing distal repeat interactions and USCE. Another set of mutations disrupted both DNA replication and cohesin loading at the replication fork during S phase. Remarkably, distal USCE in these mutants increased approximately 100-fold and was 6-fold more likely than proximal USCE. This preferential hyperdistal USCE can be explained by an aberrant sister-chromatid structure that is normally prevented by proper coupling of cohesin function and replication.

The Gene Version Iteration Hypothesis (GVIH) proposes that mutant genes may originate from the Y chromosome, traverse through the X chromosome to autosomes, undergo interchromosomal transfer, and potentially return to the Y chromosome via the X chromosome. This hypothetical closed transmission loop may facilitate the storage, screening, and elimination of different versions of mutant genes. The hypothesis comprises five core propositions: (1) Mutation reservoir: The Y chromosome may serve as a specialized carrier for generating mutant genes, characterized by elevated mutation rates, reduced gene density, and accelerated evolutionary dynamics; (2) Closed-loop transmission: Mutant genes may follow a unidirectional pathway Y[->]X[->]autosomes[->]X[->]Y, forming a complete transmission circuit; (3) Coexistence of multiple versions: A single functional gene may exist in multiple versions across different chromosomes, constituting a dynamic gene version library; (4) Reproductive screening: Environmentally adaptive gene versions may persist across generations and potentially migrate to upstream chromosomes, while maladaptive versions may be eliminated; (5) Terminal elimination: Gene versions reaching the Y chromosome may undergo elimination processes, potentially preventing version monopolization and maintaining evolutionary dynamics. This hypothesis provides a novel framework for understanding adaptive evolution at the genetic level. If empirically validated, it may offer new insights into the molecular mechanisms underlying certain genetic phenomena and evolutionary processes.

Doublesex and mab-3 related (DMRT) genes encode a family of transcription factors central to sexual development across metazoa. DMRT genes are characterised by a highly conserved DNA binding domain (DM) while flanking regions may vary between species. Gene duplication and loss has shaped the diversity of the DMRT genes with several unresolved questions about their evolution. The most well characterised and conserved DMRT gene, DMRT1, functions as a sexual regulator universally in metazoans. In chicken, DMRT1 is located on the Z chromosome and acts as a dosage dependent primary sex determination gene. In therian mammals DMRT1 is autosomal, however, two copies are required for male development. Interestingly in the basal lineage of egg-laying mammals (monotremes), DMRT1 is localised on the X specific part of one of the X chromosomes. This provided the first evidence of a sex chromosome system with homology to the avian Z chromosome and raises questions about the function and evolution of DMRT1 in egg-laying mammals. To gain insight into the evolution of mammalian DMRT genes we performed sequence and expression analysis of monotreme DMRT genes and comparative analysis with other vertebrates. In monotremes, we identified DMRT genes 1-7, and show that DMRT8 is absent, suggesting that DMRT8 evolved in therian mammals after the divergence of monotremes. Sequence and expression analysis revealed multiple monotreme specific DMRT1 isoforms with additional protein-coding exons. The independent evolution of monotreme specific changes in DMRT1 may be the first indication of functional or regulatory differences in monotreme DMRT1. Article SummaryGenes in the Doublesex and mab-3 related (DMRT) family play important roles in sexual development across animals, but a comprehensive analysis of these transcription factors is lacking in the most basal mammalian lineage of monotremes. This comparative analysis of DMRT genes in monotremes and other vertebrates shows the conservation of DMRT genes 1- 7 but found no evidence of DMRT8 in monotremes or marsupial species, suggesting that this gene evolved in eutherians after the divergence of marsupials. The discovery of several monotreme specific isoforms and novel exons of the X linked DMRT1 reveals unique evolutionary changes in monotreme DMRT1.

Oocytes arrested at the dictyate stage of meiosis I must maintain genomic integrity for prolonged periods to preserve female fertility. During this extended arrest, DNA lesions arising from endogenous and exogenous sources threaten oocyte survival, yet the molecular mechanisms coordinating DNA repair in dormant oocytes remain poorly understood. Here, we identify cyclin-dependent kinase 1 (CDK1) as a critical regulator of the oocyte DNA damage response and homologous recombination (HR) repair under genotoxic stress. Using cisplatin-induced DNA damage models in fetal goat ovaries and neonatal mouse ovaries, we investigated repair mechanisms operating within the ovarian reserve. Label-free proteomic profiling revealed significant enrichment of DNA damage response pathways following cisplatin exposure, with CDK1 emerging as one of the most prominently upregulated kinases. Pharmacological inhibition of CDK1 had little effect on follicle survival under physiological conditions but aggravated oocyte and follicle loss following DNA damage, indicating a stress-dependent role for CDK1 in preserving ovarian follicle pool integrity. Mechanistically, DNA damage activated a Chk2-dependent signaling pathway that promoted p63 phosphorylation and altered the WEE1-CDK1 regulatory axis, resulting in reduced inhibitory CDK1 phosphorylation (Thr14/Tyr15) and increased activating phosphorylation (Thr161). Activated CDK1 was associated with enhanced RAD51 phosphorylation and accumulation at DNA damage foci, supporting homologous recombination (HR)-mediated repair in dictyate-arrested oocytes. In contrast, CDK1 inhibition reduced phospho-RAD51 levels, impaired RAD51 localization, increased persistent {gamma}H2AX accumulation, and elevated oocyte apoptosis. Notably, suppression of CDK1 was accompanied by increased expression of the non-homologous end joining (NHEJ) marker Ku80 and the nucleotide excision repair (NER) factor XPA, suggesting increased engagement of alternative DNA repair pathways. Furthermore, inhibition of Chk2 abolished the DNA damage-associated CDK1 activation signature and restored WEE1 expression, supporting a model in which CDK1 functions downstream of Chk2 signaling during the oocyte DNA damage response. Collectively, our findings identify a previously unrecognized Chk2-CDK1-RAD51 signaling axis that coordinates homologous recombination repair in dormant oocytes and safeguards ovarian follicular pool integrity under genotoxic stress. These findings provide new mechanistic insight into how dictyate-arrested oocytes maintain genome stability during prolonged meiotic arrest.

MLLT1 (also named ENL) is a chromatin reader protein whose encoding gene was originally identified as a chromosomal translocation partner with MLL(KMT2A) in acute leukemia. However, its role in normal hematopoiesis has not been investigated. This study uncovers a critical role of Mllt1 in normal B cell lymphopoiesis. We found Mllt1 to be essential for early B lymphocyte development using a conditional Mllt1 knockout mouse model that we developed. A significant decrease of bone marrow B-lineage progenitors, splenic transitional B cells and peripheral blood B cells were observed in Mllt1del mice compared to control Mllt1fl/fl mice. Similarly, Mllt1 deletion in in vitro cultured B-enriched progenitor cells from Mllt1fl/fl; Rosa26CreERT2/+ mice resulted in reduced B cells, demonstrating the cell-intrinsic role of Mllt1 in this process. Direct MLLT1 target genes including Il7r and critical B-lineage transcription factors, Ebf1 and Pax5, were decreased following Mllt1 deletion. Gene set enrichment, gene ontology, and functional analyses of Mllt1-deficient cells showed significant alterations related to B cell development, critical relevant signaling pathways, DNA replication, and mitochondrial function. In vitro complementation with MLLT1 rescued the B cell phenotype observed with endogenous Mllt1 deletion; however, specific MLLT1 YEATS domain mutants lacking chromatin reader and RNA-binding functions were unable to rescue the phenotype. Taken together, our research demonstrates a previously unappreciated role for MLLT1 as critical for maintenance of B cell lymphopoiesis.

A fraction of patients treated with radiotherapy are known to be more sensitive to ionizing radiations. Skin fibroblasts from such radiosensitive individuals exhibit a higher cellular toxicity after irradiation and a delay in DNA repair. Deciphering the molecular mechanisms underlying these cellular defects is thus of major importance. We previously observed that the transcription factor NFATc2 is expressed at a reduced level in fibroblasts from radiosensitive patients. The present work aimed to elucidate the role of NFATc2 in the regulation of DNA repair, particularly the repair of radiation-induced double-strand breaks. We demonstrate an interaction of NFATc2 with the NHEJ repair protein Ku80 and observe that the NFATc2 RHD domain is necessary and sufficient for this interaction. Moreover, we show that NFATc2-Ku80 complexes are not colocalized to DNA double-strand breaks sites suggesting an involvement upstream of the DNA repair pathway. The silencing of NFATc2 impairs the NHEJ repair activities by delaying Ku70-Ku80 interaction in the early steps of this pathway. Finally, stable over-expression of NFATc2 in patients fibroblasts partially rescues their defective DNA repair phenotype, especially in the most radiosensitive cells. Altogether, our data reveal that NFATc2 is a regulator of DNA repair in skin fibroblasts and therefore a potential modulator of cellular radiosensitivity.

Cisplatin and its analogues are valuable anti-cancer drugs that target the genome, block DNA replication, and induce apoptosis. As a counteractive response, cancer cells activate several mechanisms to maintain uninterrupted DNA replication, and those are yet to be fully elucidated. This study using head and neck squamous carcinoma cells (HNSCC) demonstrated the involvement of DNA polymerase Kappa (Pol{kappa}), a trans-lesion DNA synthesis (TLS) polymerase that primarily functions as a mismatch extender, in cisplatin resistance. Interestingly, the catalytic activity of Pol{kappa} plays a minimal role in adduct bypass; rather, tripartite interactions involving it, rewire and stabilize the stalled replication fork. While the Pol{kappa}-PCNA-Pol{delta} axis facilitates efficient proliferation of cisplatin-resistant cells, the Pol{kappa}-PCNA-USP18 axis stabilizes critical proteins of ATM-ATR, and HR and NHEJ pathways to protect replication fork, repair damage, and restart DNA synthesis under cisplatin-induced stress. In resistant cells, the efficiency of ubiquitin-mediated proteasomal degradation is low, which is further diminished by Pol{kappa}-recruited USP18 deubiquitinase, maintaining a cellular homeostasis. In conclusion, for the first time, we uncovered two critical Pol{kappa} axes crucial for regulating cisplatin toxicity in cells and provided foundation for future drug discovery against advance HNSCC by targeting this non-essential DNA polymerase.

The QxxR motif is evolutionarily conserved within DEAD-box RNA helicases, including Drosophila Me31B and human DDX6, which post-transcriptionally regulate gene expression during animal development. A pathogenic H372R substitution (QxHR to QxRR) in the QxxR motif of human DDX6 has been associated with various developmental defects, but how this motif contributes to DDX6-family protein function remains unclear. Here, we used Drosophila Me31B as an in vivo model to investigate the QxxR motifs developmental role. We generated a Drosophila strain carrying the corresponding H333R missense mutation in Me31B and characterized its effects on female fertility, embryonic viability, germline development, and Me31B-associated molecular pathways. The me31BH333R mutation reduced female fertility in a gene dose-dependent manner, with homozygous mutant females being sterile. Embryos from the mutant females also exhibited primordial germ cell defects. Despite these developmental phenotypes, the me31BH333R mutation did not significantly alter Me31B protein abundance, global ovarian transcriptome or proteome profiles, or representative germ plasm mRNA and protein localization. In contrast, bait-normalized IP-MS analysis revealed altered enrichment of selected Me31B-associated proteins, including increased association of known Me31B interactors Trailer hitch (Tral) and Ypsilon Schachtel (Yps). These findings establish Me31BH333R as an in vivo model for investigating the conserved QxxR motif and suggest that disruption of this motif compromises development not through broad changes in gene expression, but potentially through altered composition or regulation of Me31B-containing ribonucleoprotein complexes.

Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is a globally invasive pest of soft-skinned fruits that is currently controlled largely through the use of broad-spectrum insecticides. Increasing resistance to pesticides and regulatory pressures have motivated the development of genetic control strategies. We previously developed a CRISPR/Cas9-based homing gene drive targeting the coding sequence of the female-specific exon of the sex-determination gene doublesex, achieving highly efficient inheritance (94-99%) in both male and female germlines. A major limitation of homing gene drives is the formation of resistant alleles that evade cleavage yet retain gene function. Multiplexing guide RNAs (gRNAs) could reduce the formation of such functional resistance alleles. Here, we generated and tested homing constructs expressing one, two, or three gRNAs targeting different regions of the female-specific exon of doublesex, including a splice-junction target site. A single gRNA targeting the splice junction supported high inheritance in males but showed reduced efficiency in females. Combining this gRNA with a coding sequence-targeting guide further reduced drive efficiency, particularly in the female germline. Constructs expressing two gRNAs performed similarly whether guides were linked by transfer RNA (tRNA) sequences or expressed from independent promoters. Constructs expressing three gRNAs using tRNA processing showed consistently low drive inheritance in both sexes, likely a consequence of reduced cleavage efficiency due to inefficient gRNA production. Inheritance was significantly higher in male than female germlines for several constructs, indicating that germline context strongly influences drive performance. Our findings highlight that the approach used for multi-gRNA expression, target site choice and sex-specific germline environments can impact gene drive efficiency, and emphasize the need to optimize construct design within the biological context of the target species.

Adversity in early life has lasting effects on the physiology and behavior of exposed individuals and their descendants. In mice, early-life stress alters the RNA content of adult sperm, and this RNA is sufficient to transmit some of the effects to the offspring who were never exposed. However, sperm cells are not yet formed during the early postnatal window in which the exposure occurs. Spermatogonial cells (SPGs), which give rise to them, are present at that time, but whether they respond to the exposure and maintain a molecular signature of it into adulthood is unknown. Here we show that early-life stress alters both the transcriptome and the chromatin accessibility of mouse SPGs, and that a molecular signature of the exposure remains detectable in adulthood. One day after exposure ended, the transcriptional response was extensive, with proliferation and nucleosome-organization programs coordinately up-regulated. In adulthood, the transcriptional response was modest and dominated by coordinately down-regulated gene programs. Single-cell profiling of the whole testis localized the adult response to spermatogonial stem cells (SSCs) and to genes involved in spermatogenesis. At the chromatin level, accessibility shifted one day after exposure at binding motifs for signal-responsive transcription factor families, and in adulthood at a different set of families, in both cases at primed enhancers. These data demonstrate that SPGs respond to an early postnatal environmental exposure and identify them as a candidate origin of the molecular changes later found in adult sperm.

Centromeres ensure accurate chromosome segregation and are typically confined to a single, localised region in monocentric chromosomes. In contrast, holocentric chromosomes exhibit kinetochore activity distributed along the chromosome length. Although holocentricity is widespread in Cyperaceae, the composition and organisation of these centromeres, as well as their evolutionary diversification, remain poorly understood. Here, we investigated centromere organisation in representatives of the subfamilies Mapanioideae (Hypolytrum schraderianum Nees) and Cyperoideae (Cladium mariscus (L.) Pohl) by combining genome assemblies, repeatome characterisation (RepeatExplorer), fluorescence in situ hybridisation (FISH), and immunolocalisation. Comparative synteny analyses incorporating the genomes of Rhynchospora breviuscula (n = 5) and Carex littledalei (n = 29) identified conserved blocks, eventually expanding almost whole chromosomes of H. schraderianum (n = 30) and Cl. mariscus (n = 39), despite divergent chromosome numbers and deep evolutionary distances within Cyperaceae. Mobile elements showed very low abundances and were uniformly dispersed, with Ty1/Copia Angela being the most abundant in both species. In Cl. mariscus, holocentromeres showed an extended distribution of centromere- and kinetochore-associated proteins along the chromosomes, largely colocalised with two satellite DNA repeats that form dispersed clusters. In contrast, H. schraderianum also displayed kinetochore signals along chromatids, but the most abundant satellite DNA family was enriched in distal and interstitial chromosomal regions rather than interspersed along the chromatids. Together, these results reveal different genomic architectures underlying holocentric organisation in phylogenetically distinct Cyperaceae lineages, suggesting that holocentromeres in this family have diversified with variation in centromere organisation in regard to its association with repetitive DNA.

Meiosis is a fundamental process in producing both sperm and eggs, yet recombination landscapes often exhibit sexual differences, known as heterochiasmy. Since meiotic proteins are generally expressed in both sexes, the molecular mechanism driving heterochiasmy remains elusive. The -kleisin subunit gene of meiotic cohesin, Rec8, is expressed bisexually in mammals, while its putative teleost ortholog, rec8a, is expressed in a female-biased manner, presumably due to the presence of its paralog originating from the teleost-specific whole-genome duplication (TGD). Here, we elucidated the evolutionary history and expression dynamics of -kleisin genes across teleost lineages. Through comprehensive phylogenetic and synteny analyses, we revealed that major teleost lineages retain two copies of rec8 and rad21, with rec8 loci experiencing drastic chromosomal rearrangements immediately after the TGD. Using in situ hybridization and single-cell transcriptome data in medaka and zebrafish, we demonstrated a conserved sexually biased expression pattern: rec8a is predominantly female-biased, whereas rec8b exhibits male-biased expression during gametogenesis. Furthermore, comparative epigenetic analyses revealed that the conserved sexually biased expression is driven by lineage-specific cis-regulatory elements, rather than conserved ones. Motif analyses imply that regulatory rewiring by transcription factors, including foxl2l in particular, might have played a crucial role in the establishment and maintenance of this paralog divergence. Our findings highlight how whole-genome duplication and subsequent genomic and epigenetic rewiring subdivided the bisexual function of rec8, offering insights into sexually distinct meiotic regulation. HighlightsO_LITeleosts possess a unique -kleisin repertoire originating from the TGD. C_LIO_LITeleost rec8 paralogs exhibit conserved sex-biased expression during meiosis. C_LIO_LIDrastic genomic rearrangements after the duplication rewired the teleost rec8 loci. C_LIO_LIThe conserved expression pattern is governed by lineage-specific CREs. C_LIO_LIThose CREs harbor similar types of TFBSs such as Fox-family TFs. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/731870v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@c8c84dorg.highwire.dtl.DTLVardef@1d65668org.highwire.dtl.DTLVardef@c2d732org.highwire.dtl.DTLVardef@1be54a2_HPS_FORMAT_FIGEXP M_FIG C_FIG