BLM and BRCA1-BARD1 coordinate complementary mechanisms of joint DNA molecule resolution.

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.

Genetic dissection of crossover mutants defines discrete intermediates in mouse meiosis.

Crossovers (COs), the exchange of homolog arms, are required for accurate chromosome segregation during meiosis. Studies in yeast have described the single-end invasion (SEI) intermediate: a stabilized 3' end annealed with the homolog as the first detectible CO precursor. SEIs are thought to differentiate into double Holliday junctions (dHJs) that are resolved by MutLgamma (MLH1/MLH3) into COs. Currently, we lack knowledge of early steps of mammalian CO recombination or how intermediates are differentiated in any organism. Using comprehensive analysis of recombination in thirteen different genetic conditions with varying levels of compromised CO resolution, we infer CO precursors include asymmetric SEI-like intermediates and dHJs in mouse. In contrast to yeast, MLH3 is structurally required to differentiate CO precursors into dHJs. We verify conservation of aspects of meiotic recombination and show unique features in mouse, providing mechanistic insight into CO formation.

TDP1 phosphorylation by CDK1 in mitosis promotes MUS81-dependent repair of trapped Top1-DNA covalent complexes.

Topoisomerase 1 (Top1) controls DNA topology, relieves DNA supercoiling during replication and transcription, and is critical for mitotic progression to the G1 phase. Tyrosyl-DNA phosphodiesterase 1 (TDP1) mediates the removal of trapped Top1-DNA covalent complexes (Top1cc). Here, we identify CDK1-dependent phosphorylation of TDP1 at residue S61 during mitosis. A TDP1 variant defective for S61 phosphorylation (TDP1-S61A) is trapped on the mitotic chromosomes, triggering DNA damage and mitotic defects. Moreover, we show that Top1cc repair in mitosis occurs via a MUS81-dependent DNA repair mechanism. Replication stress induced by camptothecin or aphidicolin leads to TDP1-S61A enrichment at common fragile sites, which over-stimulates MUS81-dependent chromatid breaks, anaphase bridges, and micronuclei, ultimately culminating in the formation of 53BP1 nuclear bodies during G1 phase. Our findings provide new insights into the cell cycle-dependent regulation of TDP1 dynamics for the repair of trapped Top1-DNA covalent complexes during mitosis that prevents genomic instability following replication stress.

ATR Protects the Genome against R Loops through a MUS81-Triggered Feedback Loop.

R loops arising during transcription induce genomic instability, but how cells respond to the R loop-associated genomic stress is still poorly understood. Here, we show that cells harboring high levels of R loops rely on the ATR kinase for survival. In response to aberrant R loop accumulation, the ataxia telangiectasia and Rad3-related (ATR)-Chk1 pathway is activated by R loop-induced reversed replication forks. In contrast to the activation of ATR by replication inhibitors, R loop-induced ATR activation requires the MUS81 endonuclease. ATR protects the genome from R loops by suppressing transcription-replication collisions, promoting replication fork recovery, and enforcing a G2/M cell-cycle arrest. Furthermore, ATR prevents excessive cleavage of reversed forks by MUS81, revealing a MUS81-triggered and ATR-mediated feedback loop that fine-tunes MUS81 activity at replication forks. These results suggest that ATR is a key sensor and suppressor of R loop-induced genomic instability, uncovering a signaling circuitry that safeguards the genome against R loops.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

Network Rewiring of Homologous Recombination Enzymes during Mitotic Proliferation and Meiosis.

Homologous recombination (HR) is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the recombination machinery to avoid (mitosis) or enforce (meiosis) the formation of reciprocal exchanges-crossovers-between recombining chromosomes. To obtain molecular insight into how crossover control is achieved, we affinity purified 7 DNA-processing enzymes that channel HR intermediates into crossovers or noncrossovers from vegetative cells or cells undergoing meiosis. Using mass spectrometry, we provide a global characterization of their composition and reveal mitosis- and meiosis-specific modules in the interaction networks. Functional analyses of meiosis-specific interactors of MutLγ-Exo1 identified Rtk1, Caf120, and Chd1 as regulators of crossing-over. Chd1, which transiently associates with Exo1 at the prophase-to-metaphase I transition, enables the formation of MutLγ-dependent crossovers through its conserved ability to bind and displace nucleosomes. Thus, rewiring of the HR network, coupled to chromatin remodeling, promotes context-specific control of the recombination outcome.

Maintenance of neuronal identity in C. elegans and beyond: Lessons from transcription and chromatin factors.

Neurons are remarkably long-lived, non-dividing cells that must maintain their functional features (e.g., electrical properties, chemical signaling) for extended periods of time - decades in humans. How neurons accomplish this incredible feat is poorly understood. Here, we review recent advances, primarily in the nematode C. elegans, that have enhanced our understanding of the molecular mechanisms that enable post-mitotic neurons to maintain their functionality across different life stages. We begin with "terminal selectors" - transcription factors necessary for the establishment and maintenance of neuronal identity. We highlight new findings on five terminal selectors (CHE-1 [Glass], UNC-3 [Collier/Ebf1-4], LIN-39 [Scr/Dfd/Hox4-5], UNC-86 [Acj6/Brn3a-c], AST-1 [Etv1/ER81]) from different transcription factor families (ZNF, COE, HOX, POU, ETS). We compare the functions of these factors in specific neuron types of C. elegans with the actions of their orthologs in other invertebrate (D. melanogaster) and vertebrate (M. musculus) systems, highlighting remarkable functional conservation. Finally, we reflect on recent findings implicating chromatin-modifying proteins, such as histone methyltransferases and Polycomb proteins, in the control of neuronal terminal identity. Altogether, these new studies on transcription factors and chromatin modifiers not only shed light on the fundamental problem of neuronal identity maintenance, but also outline mechanistic principles of gene regulation that may operate in other long-lived, post-mitotic cell types.

Environmentally robust cis-regulatory changes underlie rapid climatic adaptation.

Changes in gene expression are thought to play a major role in adaptive evolution. While it is known that gene expression is highly sensitive to the environment, very few studies have determined the influence of genetic and environmental effects on adaptive gene expression differences in natural populations. Here, we utilize allele-specific expression to characterize cis and trans gene regulatory divergence in temperate and tropical house mice in two metabolic tissues under two thermal conditions. First, we show that gene expression divergence is pervasive between populations and across thermal conditions, with roughly 5 to 10% of genes exhibiting genotype-by-environment interactions. Second, we found that most expression divergence was due to cis-regulatory changes that were stable across temperatures. In contrast, patterns of expression plasticity were largely attributable to trans-effects, which showed greater sensitivity to temperature. Nonetheless, we found a small subset of temperature-dependent cis-regulatory changes, thereby identifying loci underlying expression plasticity. Finally, we performed scans for selection in wild house mice to identify genomic signatures of rapid adaptation. Genomic outliers were enriched in genes with evidence for cis-regulatory divergence. Notably, these genes were associated with phenotypes that affected body weight and metabolism, suggesting that cis-regulatory changes are a possible mechanism for adaptive body size evolution between populations. Our results show that gene expression plasticity, largely controlled in trans, may facilitate the colonization of new environments, but that evolved changes in gene expression are largely controlled in cis, illustrating the genetic and nongenetic mechanisms underlying the establishment of populations in new environments.

A Minimal Hybrid Sterility Genome Assembled by Chromosome Swapping Between Mouse Subspecies (Mus musculus).

Hybrid sterility is a reproductive isolation barrier between diverging taxa securing the early steps of speciation. Hybrid sterility is ubiquitous in the animal and plant kingdoms, but its genetic control is poorly understood. In our previous studies, we have uncovered the sterility of hybrids between musculus and domesticus subspecies of the house mouse, which is controlled by the Prdm9 gene, the X-linked Hstx2 locus, and subspecific heterozygosity for genetic background. To further investigate this form of genic-driven chromosomal sterility, we constructed a simplified hybrid sterility model within the genome of the domesticus subspecies by swapping domesticus autosomes with their homologous partners from the musculus subspecies. We show that the "sterility" allelic combination of Prdm9 and Hstx2 can be activated by a musculus/domesticus heterozygosity of as few as two autosomes, Chromosome 17 (Chr 17) and Chr 18 and is further enhanced when another heterosubspecific autosomal pair is present, whereas it has no effect on meiotic progression in the pure domesticus genome. In addition, we identify a new X-linked hybrid sterility locus, Hstx3, at the centromeric end of Chr X, which modulates the incompatibility between Prdm9 and Hstx2. These results further support our concept of chromosomal hybrid sterility based on evolutionarily accumulated divergence between homologous sequences. Based on these and previous results, we believe that future studies should include more information on the mutual recognition of homologous chromosomes at or before the first meiotic prophase in interspecific hybrids, as this may serve as a general reproductive isolation checkpoint in mice and other species.

Variation in the Spectrum of New Mutations among Inbred Strains of Mice.

The mouse serves as a mammalian model for understanding the nature of variation from new mutations, a question that has both evolutionary and medical significance. Previous studies suggest that the rate of single-nucleotide mutations (SNMs) in mice is ∼50% of that in humans. However, information largely comes from studies involving the C57BL/6 strain, and there is little information from other mouse strains. Here, we study the mutations that accumulated in 59 mouse lines derived from four inbred strains that are commonly used in genetics and clinical research (BALB/cAnNRj, C57BL/6JRj, C3H/HeNRj, and FVB/NRj), maintained for eight to nine generations by brother-sister mating. By analyzing Illumina whole-genome sequencing data, we estimate that the average rate of new SNMs in mice is ∼µ = 6.7 × 10-9. However, there is substantial variation in the spectrum of SNMs among strains, so the burden from new mutations also varies among strains. For example, the FVB strain has a spectrum that is markedly skewed toward C→A transversions and is likely to experience a higher deleterious load than other strains, due to an increased frequency of nonsense mutations in glutamic acid codons. Finally, we observe substantial variation in the rate of new SNMs among DNA sequence contexts, CpG sites, and their adjacent nucleotides playing an important role.

Multiple Genomic Landscapes of Recombination and Genomic Divergence in Wild Populations of House Mice-The Role of Chromosomal Fusions and Prdm9.

Chromosomal fusions represent one of the most common types of chromosomal rearrangements found in nature. Yet, their role in shaping the genomic landscape of recombination and hence genome evolution remains largely unexplored. Here, we take advantage of wild mice populations with chromosomal fusions to evaluate the effect of this type of structural variant on genomic landscapes of recombination and divergence. To this aim, we combined cytological analysis of meiotic crossovers in primary spermatocytes with inferred analysis of recombination rates based on linkage disequilibrium using single nucleotide polymorphisms. Our results suggest the presence of a combined effect of Robertsonian fusions and Prdm9 allelic background, a gene involved in the formation of meiotic double strand breaks and postzygotic reproductive isolation, in reshaping genomic landscapes of recombination. We detected a chromosomal redistribution of meiotic recombination toward telomeric regions in metacentric chromosomes in mice with Robertsonian fusions when compared to nonfused mice. This repatterning was accompanied by increased levels of crossover interference and reduced levels of estimated recombination rates between populations, together with high levels of genomic divergence. Interestingly, we detected that Prdm9 allelic background was a major determinant of recombination rates at the population level, whereas Robertsonian fusions showed limited effects, restricted to centromeric regions of fused chromosomes. Altogether, our results provide new insights into the effect of Robertsonian fusions and Prdm9 background on meiotic recombination.

Cdk8 and Hira mutations trigger X chromosome elimination in naive female hybrid mouse embryonic stem cells.

Mouse embryonic stem cells (ESCs) possess a pluripotent developmental potential and a stable karyotype. An exception is the frequent loss of one X chromosome in female ESCs derived from inbred mice. In contrast, female ESCs from crosses between different Mus musculus subspecies often maintain two X chromosomes and can model X chromosome inactivation. Here we report that combined mutations of Hira and Cdk8 induce rapid loss of one X chromosome in a Mus musculus castaneus hybrid female ESC line that originally maintains two X chromosomes. We show that MEK1 inhibition, which is used for culturing naive pluripotent ESCs is sufficient to induce X chromosome loss. In conventional ESC media, Hira and Cdk8 mutant ESCs maintain both X chromosomes. Induction of X chromosome loss by switching to naive culture media allows us to perform kinetic measurements for calculating the chromosome loss rate. Our analysis shows that X chromosome loss is not explained by selection of XO cells, but likely driven by a process of chromosome elimination. We show that elimination of the X chromosome occurs with a rate of 0.3% per cell per division, which exceeds reported autosomal loss rates by 3 orders of magnitude. We show that chromosomes 8 and 11 are stably maintained. Notably, Xist expression from one of the two X chromosomes rescues X chromosomal instability in ΔHiraΔCdk8 ESCs. Our study defines mutations of Hira and Cdk8 as molecular drivers for X chromosome elimination in naive female ESCs and describes a cell system for elucidating the underlying mechanism.

RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis.

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.

The alternative lengthening of telomeres mechanism jeopardizes telomere integrity if not properly restricted.

A substantial number of human cancers are telomerase-negative and elongate physiologically damaged telomeres through a break-induced replication (BIR)-based mechanism known as alternative lengthening of telomeres (ALT). We recently demonstrated that inhibiting the transcription of the telomeric long noncoding RNA TERRA suppresses telomere damage and ALT features, indicating that telomere transcription is a main trigger of ALT activity. Here we show that experimentally increased TERRA transcription not only increases ALT features, as expected, but also causes rapid loss of telomeric DNA through a pathway that requires the endonuclease Mus81. Our data indicate that the ALT mechanism can endanger telomere integrity if not properly controlled and point to TERRA transcription as a uniquely versatile target for therapy.

Developmental genetics with model organisms.

In Darwin's and Mendel's times, researchers investigated a wealth of organisms, chosen to solve particular problems for which they seemed especially well suited. Later, a focus on a few organisms, which are accessible to systematic genetic investigations, resulted in larger repertoires of methods and applications in these few species. Genetic animal model organisms with large research communities are the nematode Caenorhabditis elegans, the fly Drosophila melanogaster, the zebrafish Danio rerio, and the mouse Mus musculus. Due to their specific strengths, these model organisms have their strongest impacts in rather different areas of biology. C. elegans is unbeatable in the analysis of cell-to-cell contacts by saturation mutagenesis, as worms can be grown very fast in very high numbers. In Drosophila, a rich pattern is generated in the embryo as well as in adults that is used to unravel the underlying mechanisms of morphogenesis. The transparent larvae of zebrafish are uniquely suited to study organ development in a vertebrate, and the superb versatility of reverse genetics in the mouse made it the model organism to study human physiology and diseases. The combination of these models allows the in-depth genetic analysis of many fundamental biological processes using a plethora of different methods, finally providing many specific approaches to combat human diseases. The plant model Arabidopsis thaliana provides an understanding of many aspects of plant biology that might ultimately be useful for breeding crops.

Rapid Evolution of the Fine-scale Recombination Landscape in Wild House Mouse (Mus musculus) Populations.

Meiotic recombination is an important evolutionary force and an essential meiotic process. In many species, recombination events concentrate into hotspots defined by the site-specific binding of PRMD9. Rapid evolution of Prdm9's zinc finger DNA-binding array leads to remarkably abrupt shifts in the genomic distribution of hotspots between species, but the question of how Prdm9 allelic variation shapes the landscape of recombination between populations remains less well understood. Wild house mice (Mus musculus) harbor exceptional Prdm9 diversity, with >150 alleles identified to date, and pose a particularly powerful system for addressing this open question. We employed a coalescent-based approach to construct broad- and fine-scale sex-averaged recombination maps from contemporary patterns of linkage disequilibrium in nine geographically isolated wild house mouse populations, including multiple populations from each of three subspecies. Comparing maps between wild mouse populations and subspecies reveals several themes. First, we report weak fine- and broad-scale recombination map conservation across subspecies and populations, with genetic divergence offering no clear prediction for recombination map divergence. Second, most hotspots are unique to one population, an outcome consistent with minimal sharing of Prdm9 alleles between surveyed populations. Finally, by contrasting aggregate hotspot activity on the X versus autosomes, we uncover evidence for population-specific differences in the degree and direction of sex dimorphism for recombination. Overall, our findings illuminate the variability of both the broad- and fine-scale recombination landscape in M. musculus and underscore the functional impact of Prdm9 allelic variation in wild mouse populations.

How myeloid cells shape experimental autoimmune encephalomyelitis: At the crossroads of outside-in immunity.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of central nervous system (CNS) autoimmunity. It is most commonly used to mimic aspects of multiple sclerosis (MS), a demyelinating disorder of the human brain and spinal cord. The innate immune response displays one of the core pathophysiological features linked to both the acute and chronic stages of MS. Hence, understanding and targeting the innate immune response is essential. Microglia and other CNS resident MUs, as well as infiltrating myeloid cells, diverge substantially in terms of both their biology and their roles in EAE. Recent advances in the field show that antigen presentation, as well as disease-propagating and regulatory interactions with lymphocytes, can be attributed to specific myeloid cell types and cell states in EAE lesions, following a distinct temporal pattern during disease initiation, propagation and recovery. Furthermore, single-cell techniques enable the assessment of characteristic proinflammatory as well as beneficial cell states, and identification of potential treatment targets. Here, we discuss the principles of EAE induction and protocols for varying experimental paradigms, the composition of the myeloid compartment of the CNS during health and disease, and systematically review effects on myeloid cells for therapeutic approaches in EAE.

Genetically identical mice express alternative reproductive tactics depending on social conditions in the field.

In many species, establishing and maintaining a territory is critical to survival and reproduction, and an animal's ability to do so is strongly influenced by the presence and density of competitors. Here we manipulate social conditions to study the alternative reproductive tactics displayed by genetically identical, age-matched laboratory mice competing for territories under ecologically realistic social environmental conditions. We introduced adult males and females of the laboratory mouse strain C57BL/6J into a large, outdoor field enclosure containing defendable resource zones under one of two social conditions. We first created a low-density social environment, such that the number of available territories exceeded the number of males. After males established stable territories, we introduced a pulse of intruder males and observed the resulting defensive and invasive tactics employed. In response to this change in social environment, males with large territories invested more in patrolling but were less effective at excluding intruder males as compared with males with small territories. Intruding males failed to establish territories and displayed an alternative tactic featuring greater exploration as compared with genetically identical territorial males. Alternative tactics did not lead to equal reproductive success-males that acquired territories experienced greater survival and had greater access to females.

The effect of host admixture on wild house mouse gut microbiota is weak when accounting for spatial autocorrelation.

The question of how interactions between the gut microbiome and vertebrate hosts contribute to host adaptation and speciation is one of the major problems in current evolutionary research. Using bacteriome and mycobiome metabarcoding, we examined how these two components of the gut microbiota vary with the degree of host admixture in secondary contact between two house mouse subspecies (Mus musculus musculus and M. m. domesticus). We used a large data set collected at two replicates of the hybrid zone and model-based statistical analyses to ensure the robustness of our results. Assuming that the microbiota of wild hosts suffers from spatial autocorrelation, we directly compared the results of statistical models that were spatially naive with those that accounted for spatial autocorrelation. We showed that neglecting spatial autocorrelation can strongly affect the results and lead to misleading conclusions. The spatial analyses showed little difference between subspecies, both in microbiome composition and in individual bacterial lineages. Similarly, the degree of admixture had minimal effects on the gut bacteriome and mycobiome and was caused by changes in a few microbial lineages that correspond to the common symbionts of free-living house mice. In contrast to previous studies, these data do not support the hypothesis that the microbiota plays an important role in host reproductive isolation in this particular model system.

Epigenetic age estimation of wild mice using faecal samples.

Age is a key parameter in population ecology, with a myriad of biological processes changing with age as organisms develop in early life then later senesce. As age is often hard to accurately measure with non-lethal methods, epigenetic methods of age estimation (epigenetic clocks) have become a popular tool in animal ecology and are often developed or calibrated using captive animals of known age. However, studies typically rely on invasive blood or tissue samples, which limit their application in more sensitive or elusive species. Moreover, few studies have directly assessed how methylation patterns and epigenetic age estimates compare across environmental contexts (e.g. captive or laboratory-based vs. wild animals). Here, we built a targeted epigenetic clock from laboratory house mice (strain C57BL/6, Mus musculus) using DNA from non-invasive faecal samples, and then used it to estimate age in a population of wild mice (Mus musculus domesticus) of unknown age. This laboratory mouse-derived epigenetic clock accurately predicted adult wild mice to be older than juveniles and showed that wild mice typically increased in epigenetic age over time, but with wide variation in epigenetic ageing rate among individuals. Our results also suggested that, for a given body mass, wild mice had higher methylation across targeted CpG sites than laboratory mice (and consistently higher epigenetic age estimates as a result), even among the smallest, juvenile mice. This suggests wild and laboratory mice may display different CpG methylation levels from very early in life and indicates caution is needed when developing epigenetic clocks on laboratory animals and applying them in the wild.