Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination.

Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.

The impact of replication stress on replication dynamics and DNA damage in vertebrate cells.

The interplay between replication stress and the S phase checkpoint is a key determinant of genome maintenance, and has a major impact on human diseases, notably, tumour initiation and progression. Recent studies have yielded insights into sequence-dependent and sequence-independent sources of endogenous replication stress. These stresses result in nuclease-induced DNA damage, checkpoint activation and genome-wide replication fork slowing. Several hypotheses have been proposed to account for the mechanisms involved in this complex response. Recent results have shown that the slowing of the replication forks most commonly results from DNA precursor starvation. By concomitantly increasing the density of replication initiation, the cell elicits an efficient compensatory strategy to avoid mitotic anomalies and the inheritance of damage over cell generations.

Trajectory and uniqueness of mutational signatures in yeast mutators.

The acquisition of mutations plays critical roles in adaptation, evolution, senescence, and tumorigenesis. Massive genome sequencing has allowed extraction of specific features of many mutational landscapes but it remains difficult to retrospectively determine the mechanistic origin(s), selective forces, and trajectories of transient or persistent mutations and genome rearrangements. Here, we conducted a prospective reciprocal approach to inactivate 13 single or multiple evolutionary conserved genes involved in distinct genome maintenance processes and characterize de novo mutations in 274 diploid Saccharomyces cerevisiae mutation accumulation lines. This approach revealed the diversity, complexity, and ultimate uniqueness of mutational landscapes, differently composed of base substitutions, small insertions/deletions (InDels), structural variants, and/or ploidy variations. Several landscapes parallel the repertoire of mutational signatures in human cancers while others are either novel or composites of subsignatures resulting from distinct DNA damage lesions. Notably, the increase of base substitutions in the homologous recombination-deficient Rad51 mutant, specifically dependent on the Polζ translesion polymerase, yields COSMIC signature 3 observed in BRCA1/BRCA2-mutant breast cancer tumors. Furthermore, "mutome" analyses in highly polymorphic diploids and single-cell bottleneck lineages revealed a diverse spectrum of loss-of-heterozygosity (LOH) signatures characterized by interstitial and terminal chromosomal events resulting from interhomolog mitotic cross-overs. Following the appearance of heterozygous mutations, the strong stimulation of LOHs in the rad27/FEN1 and tsa1/PRDX1 backgrounds leads to fixation of homozygous mutations or their loss along the lineage. Overall, these mutomes and their trajectories provide a mechanistic framework to understand the origin and dynamics of genome variations that accumulate during clonal evolution.

Bread wheat TaSPO11-1 exhibits evolutionarily conserved function in meiotic recombination across distant plant species.

The manipulation of meiotic recombination in crops is essential to develop new plant varieties rapidly, helping to produce more cultivars in a sustainable manner. One option is to control the formation and repair of the meiosis-specific DNA double-strand breaks (DSBs) that initiate recombination between the homologous chromosomes and ultimately lead to crossovers. These DSBs are introduced by the evolutionarily conserved topoisomerase-like protein SPO11 and associated proteins. Here, we characterized the homoeologous copies of the SPO11-1 protein in hexaploid bread wheat (Triticum aestivum). The genome contains three SPO11-1 gene copies that exhibit 93-95% identity at the nucleotide level, and clearly the A and D copies originated from the diploid ancestors Triticum urartu and Aegilops tauschii, respectively. Furthermore, phylogenetic analysis of 105 plant genomes revealed a clear partitioning between monocots and dicots, with the seven main motifs being almost fully conserved, even between clades. The functional similarity of the proteins among monocots was confirmed through complementation analysis of the Oryza sativa (rice) spo11-1 mutant by the wheat TaSPO11-1-5D coding sequence. Also, remarkably, although the wheat and Arabidopsis SPO11-1 proteins share only 55% identity and the partner proteins also differ, the TaSPO11-1-5D cDNA significantly restored the fertility of the Arabidopsis spo11-1 mutant, indicating a robust functional conservation of the SPO11-1 protein activity across distant plants. These successful heterologous complementation assays, using both Arabidopsis and rice hosts, are good surrogates to validate the functionality of candidate genes and cDNA, as well as variant constructs, when the transformation and mutant production in wheat is much longer and more tedious.

Human papilloma virus (HPV) integration signature in Cervical Cancer: identification of MACROD2 gene as HPV hot spot integration site.

BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality with infection by human papilloma virus (HPV) being the most important risk factor. We analysed the association between different viral integration signatures, clinical parameters and outcome in pre-treated CCs. METHODS: Different integration signatures were identified using HPV double capture followed by next-generation sequencing (NGS) in 272 CC patients from the BioRAIDs study [NCT02428842]. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed. RESULTS: Episomal HPV was much less frequent in CC as compared to anal carcinoma (p < 0.0001). We identified >300 different HPV-chromosomal junctions (inter- or intra-genic). The most frequent integration site in CC was in MACROD2 gene followed by MIPOL1/TTC6 and TP63. HPV integration signatures were not associated with histological subtype, FIGO staging, treatment or PFS. HPVs were more frequently episomal in PIK3CA mutated tumours (p = 0.023). Viral integration type was dependent on HPV genotype (p < 0.0001); HPV18 and HPV45 being always integrated. High HPV copy number was associated with longer PFS (p = 0.011). CONCLUSIONS: This is to our knowledge the first study assessing the prognostic value of HPV integration in a prospectively annotated CC cohort, which detects a hotspot of HPV integration at MACROD2; involved in impaired PARP1 activity and chromosome instability.

Thermodynamically stable and genetically unstable G-quadruplexes are depleted in genomes across species.

G-quadruplexes play various roles in multiple biological processes, which can be positive when a G4 is involved in the regulation of gene expression or detrimental when the folding of a stable G4 impairs DNA replication promoting genome instability. This duality interrogates the significance of their presence within genomes. To address the potential biased evolution of G4 motifs, we analyzed their occurrence, features and polymorphisms in a large spectrum of species. We found extreme bias of the short-looped G4 motifs, which are the most thermodynamically stable in vitro and thus carry the highest folding potential in vivo. In the human genome, there is an over-representation of single-nucleotide-loop G4 motifs (G4-L1), which are highly conserved among humans and show a striking excess of the thermodynamically least stable G4-L1A (G3AG3AG3AG3) sequences. Functional assays in yeast showed that G4-L1A caused the lowest levels of both spontaneous and G4-ligand-induced instability. Analyses across 600 species revealed the depletion of the most stable G4-L1C/T quadruplexes in most genomes in favor of G4-L1A in vertebrates or G4-L1G in other eukaryotes. We discuss how these trends might be the result of species-specific mutagenic processes associated to a negative selection against the most stable motifs, thus neutralizing their detrimental effects on genome stability while preserving positive G4-associated biological roles.

Accurate Tracking of the Mutational Landscape of Diploid Hybrid Genomes.

Mutations, recombinations, and genome duplications may promote genetic diversity and trigger evolutionary processes. However, quantifying these events in diploid hybrid genomes is challenging. Here, we present an integrated experimental and computational workflow to accurately track the mutational landscape of yeast diploid hybrids (MuLoYDH) in terms of single-nucleotide variants, small insertions/deletions, copy-number variants, aneuploidies, and loss-of-heterozygosity. Pairs of haploid Saccharomyces parents were combined to generate ancestor hybrids with phased genomes and varying levels of heterozygosity. These diploids were evolved under different laboratory protocols, in particular mutation accumulation experiments. Variant simulations enabled the efficient integration of competitive and standard mapping of short reads, depending on local levels of heterozygosity. Experimental validations proved the high accuracy and resolution of our computational approach. Finally, applying MuLoYDH to four different diploids revealed striking genetic background effects. Homozygous Saccharomyces cerevisiae showed a ∼4-fold higher mutation rate compared with its closely related species S. paradoxus. Intraspecies hybrids unveiled that a substantial fraction of the genome (∼250 bp per generation) was shaped by loss-of-heterozygosity, a process strongly inhibited in interspecies hybrids by high levels of sequence divergence between homologous chromosomes. In contrast, interspecies hybrids exhibited higher single-nucleotide mutation rates compared with intraspecies hybrids. MuLoYDH provided an unprecedented quantitative insight into the evolutionary processes that mold diploid yeast genomes and can be generalized to other genetic systems.

Assessment of the roles of SPO11-2 and SPO11-4 in meiosis in rice using CRISPR/Cas9 mutagenesis.

In Arabidopsis, chromosomal double-strand breaks at meiosis are presumably catalyzed by two distinct SPO11 transesterases, AtSPO11-1 and AtSPO11-2, together with M-TOPVIB. To clarify the roles of the SPO11 paralogs in rice, we used CRISPR/Cas9 mutagenesis to produce null biallelic mutants in OsSPO11-1, OsSPO11-2, and OsSPO11-4. Similar to Osspo11-1, biallelic mutations in the first exon of OsSPO11-2 led to complete panicle sterility. Conversely, all Osspo11-4 biallelic mutants were fertile. To generate segregating Osspo11-2 mutant lines, we developed a strategy based on dual intron targeting. Similar to Osspo11-1, the pollen mother cells of Osspo11-2 progeny plants showed an absence of bivalent formation at metaphase I, aberrant segregation of homologous chromosomes, and formation of non-viable tetrads. In contrast, the chromosome behavior in Osspo11-4 male meiocytes was indistinguishable from that in the wild type. While similar numbers of OsDMC1 foci were revealed by immunostaining in wild-type and Osspo11-4 prophase pollen mother cells (114 and 101, respectively), a surprisingly high number (85) of foci was observed in the sterile Osspo11-2 mutant, indicative of a divergent function between OsSPO11-1 and OsSPO11-2. This study demonstrates that whereas OsSPO11-1 and OsSPO11-2 are the likely orthologs of AtSPO11-1 and AtSPO11-2, OsSPO11-4 has no major role in wild-type rice meiosis.

Hard to wake up? The cerebral correlates of sleep inertia assessed using combined behavioral, EEG and fMRI measures.

The first minutes following awakening from sleep are typically marked by reduced vigilance, increased sleepiness and impaired performance, a state referred to as sleep inertia. Although the behavioral aspects of sleep inertia are well documented, its cerebral correlates remain poorly understood. The present study aimed at filling this gap by measuring in 34 participants the changes in behavioral performance (descending subtraction task, DST), EEG spectral power, and resting-state fMRI functional connectivity across three time points: before an early-afternoon 45-min nap, 5 min after awakening from the nap and 25 min after awakening. Our results showed impaired performance at the DST at awakening and an intrusion of sleep-specific features (spectral power and functional connectivity) into wakefulness brain activity, the intensity of which was dependent on the prior sleep duration and depth for the functional connectivity (14 participants awakened from N2 sleep, 20 from N3 sleep). Awakening in N3 (deep) sleep induced the most robust changes and was characterized by a global loss of brain functional segregation between task-positive (dorsal attention, salience, sensorimotor) and task-negative (default mode) networks. Significant correlations were observed notably between the EEG delta power and the functional connectivity between the default and dorsal attention networks, as well as between the percentage of mistake at the DST and the default network functional connectivity. These results highlight (1) significant correlations between EEG and fMRI functional connectivity measures, (2) significant correlations between the behavioral aspect of sleep inertia and measures of the cerebral functioning at awakening (both EEG and fMRI), and (3) the important difference in the cerebral underpinnings of sleep inertia at awakening from N2 and N3 sleep.

Short loop length and high thermal stability determine genomic instability induced by G-quadruplex-forming minisatellites.

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids, with various biological roles. However, structural features dictating their formation and/or function in vivo are unknown. In S. cerevisiae, the pathological persistency of G4 within the CEB1 minisatellite induces its rearrangement during leading-strand replication. We now show that several other G4-forming sequences remain stable. Extensive mutagenesis of the CEB25 minisatellite motif reveals that only variants with very short (≤ 4 nt) G4 loops preferentially containing pyrimidine bases trigger genomic instability. Parallel biophysical analyses demonstrate that shortening loop length does not change the monomorphic G4 structure of CEB25 variants but drastically increases its thermal stability, in correlation with the in vivo instability. Finally, bioinformatics analyses reveal that the threat for genomic stability posed by G4 bearing short pyrimidine loops is conserved in C. elegans and humans. This work provides a framework explanation for the heterogeneous instability behavior of G4-forming sequences in vivo, highlights the importance of structure thermal stability, and questions the prevailing assumption that G4 structures with short or longer loops are as likely to form in vivo.

Engineering meiotic recombination pathways in rice.

In the last 15 years, outstanding progress has been made in understanding the function of meiotic genes in the model dicot and monocot plants Arabidopsis and rice (Oryza sativa L.), respectively. This knowledge allowed to modulate meiotic recombination in Arabidopsis and, more recently, in rice. For instance, the overall frequency of crossovers (COs) has been stimulated 2.3- and 3.2-fold through the inactivation of the rice FANCM and RECQ4 DNA helicases, respectively, two genes involved in the repair of DNA double-strand breaks (DSBs) as noncrossovers (NCOs) of the Class II crossover pathway. Differently, the programmed induction of DSBs and COs at desired sites is currently explored by guiding the SPO11-1 topoisomerase-like transesterase, initiating meiotic recombination in all eukaryotes, to specific target regions of the rice genome. Furthermore, the inactivation of 3 meiosis-specific genes, namely PAIR1, OsREC8 and OsOSD1, in the Mitosis instead of Meiosis (MiMe) mutant turned rice meiosis into mitosis, thereby abolishing recombination and achieving the first component of apomixis, apomeiosis. The successful translation of Arabidopsis results into a crop further allowed the implementation of two breakthrough strategies that triggered parthenogenesis from the MiMe unreduced clonal egg cell and completed the second component of diplosporous apomixis. Here, we review the most recent advances in and future prospects of the manipulation of meiotic recombination in rice and potentially other major crops, all essential for global food security.

Programming sites of meiotic crossovers using Spo11 fusion proteins.

Meiotic recombination shapes the genetic diversity transmitted upon sexual reproduction. However, its non-random distribution along the chromosomes constrains the landscape of potential genetic combinations. For a variety of purposes, it is desirable to expand the natural repertoire by changing the distribution of crossovers in a wide range of eukaryotes. Toward this end, we report the local stimulation of meiotic recombination at a number of chromosomal sites by tethering the natural Spo11 protein to various DNA-binding modules: full-length DNA binding proteins, zinc fingers (ZFs), transcription activator-like effector (TALE) modules, and the CRISPR-Cas9 system. In the yeast Saccharomyces cerevisiae, each strategy is able to stimulate crossover frequencies in naturally recombination-cold regions. The binding and cleavage efficiency of the targeting Spo11 fusions (TSF) are variable, being dependent on the chromosomal regions and potential competition with endogenous factors. TSF-mediated genome interrogation distinguishes naturally recombination-cold regions that are flexible and can be warmed-up (gene promoters and coding sequences), from those that remain refractory (gene terminators and centromeres). These results describe new generic experimental strategies to increase the genetic diversity of gametes, which should prove useful in plant breeding and other applications.

Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion.

In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction.

Functional Assessment of Genetic Variants with Outcomes Adapted to Clinical Decision-Making.

Understanding the medical effect of an ever-growing number of human variants detected is a long term challenge in genetic counseling. Functional assays, based on in vitro or in vivo evaluations of the variant effects, provide essential information, but they require robust statistical validation, as well as adapted outputs, to be implemented in the clinical decision-making process. Here, we assessed 25 pathogenic and 15 neutral missense variants of the BRCA1 breast/ovarian cancer susceptibility gene in four BRCA1 functional assays. Next, we developed a novel approach that refines the variant ranking in these functional assays. Lastly, we developed a computational system that provides a probabilistic classification of variants, adapted to clinical interpretation. Using this system, the best functional assay exhibits a variant classification accuracy estimated at 93%. Additional theoretical simulations highlight the benefit of this ready-to-use system in the classification of variants after functional assessment, which should facilitate the consideration of functional evidences in the decision-making process after genetic testing. Finally, we demonstrate the versatility of the system with the classification of siRNAs tested for human cell growth inhibition in high throughput screening.

Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.

Correction: Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

[This corrects the article DOI: 10.1371/journal.pgen.1006226.].

Smith-Magenis Syndrome: Molecular Basis of a Genetic-Driven Melatonin Circadian Secretion Disorder.

Smith-Magenis syndrome (SMS), linked to Retinoic Acid Induced (RAI1) haploinsufficiency, is a unique model of the inversion of circadian melatonin secretion. In this regard, this model is a formidable approach to better understand circadian melatonin secretion cycle disorders and the role of the RAI1 gene in this cycle. Sleep-wake cycle disorders in SMS include sleep maintenance disorders with a phase advance and intense sleepiness around noon. These disorders have been linked to a general disturbance of sleep-wake rhythm and coexist with inverted secretion of melatonin. The exact mechanism underlying the inversion of circadian melatonin secretion in SMS has rarely been discussed. We suggest three hypotheses that could account for the inversion of circadian melatonin secretion and discuss them. First, inversion of the circadian melatonin secretion rhythm could be linked to alterations in light signal transduction. Second, this inversion could imply global misalignment of the circadian system. Third, the inversion is not linked to a global circadian clock shift but rather to a specific impairment in the melatonin secretion pathway between the suprachiasmatic nuclei (SCN) and pinealocytes. The development of diurnal SMS animal models that produce melatonin appears to be an indispensable step to further understand the molecular basis of the circadian melatonin secretion rhythm.

Relationship Between G-Quadruplex Sequence Composition in Viruses and Their Hosts.

A subset of guanine-rich nucleic acid sequences has the potential to fold into G-quadruplex (G4) secondary structures, which are functionally important for several biological processes, including genome stability and regulation of gene expression. Putative quadruplex sequences (PQSs) G3+N1-7G3+N1-7G3+N1-7G3+ are widely found in eukaryotic and prokaryotic genomes, but the base composition of the N1-7 loops is biased across species. Since the viruses partially hijack their hosts' cellular machinery for proliferation, we examined the PQS motif size, loop length, and nucleotide compositions of 7370 viral genome assemblies and compared viral and host PQS motifs. We studied seven viral taxa infecting five distant eukaryotic hosts and created a resource providing a comprehensive view of the viral quadruplex motifs. Overall, short-looped PQSs are predominant and with a similar composition across viral taxonomic groups, albeit subtle trends emerge upon classification by hosts. Specifically, there is a higher frequency of pyrimidine loops in viruses infecting animals irrespective of the viruses' genome type. This observation is confirmed by an in-depth analysis of the Herpesviridae family of viruses, which showed a distinctive accumulation of thermally stable C-looped quadruplexes in viruses infecting high-order vertebrates. The occurrence of viral C-looped G4s, which carry binding sites for host transcription factors, as well as the high prevalence of viral TTA-looped G4s, which are identical to vertebrate telomeric motifs, provide concrete examples of how PQSs may help viruses impinge upon, and benefit from, host functions. More generally, these observations suggest a co-evolution of virus and host PQSs, thus underscoring the potential functional significance of G4s.

A role for the yeast CLIP170 ortholog, the plus-end-tracking protein Bik1, and the Rho1 GTPase in Snc1 trafficking.

The diversity of microtubule functions is dependent on the status of tubulin C-termini. To address the physiological role of the C-terminal aromatic residue of α-tubulin, a tub1-Glu yeast strain expressing an α-tubulin devoid of its C-terminal amino acid was used to perform a genome-wide-lethality screen. The identified synthetic lethal genes suggested links with endocytosis and related processes. In the tub1-Glu strain, the routing of the v-SNARE Snc1 was strongly impaired, with a loss of its polarized distribution in the bud, and Abp1, an actin patch or endocytic marker, developed comet-tail structures. Snc1 trafficking required dynamic microtubules but not dynein and kinesin motors. Interestingly, deletion of the microtubule plus-end-tracking protein Bik1 (a CLIP170 ortholog), which is preferentially recruited to the C-terminal residue of α-tubulin, similarly resulted in Snc1 trafficking defects. Finally, constitutively active Rho1 rescued both Bik1 localization at the microtubule plus-ends in tub1-Glu strain and a correct Snc1 trafficking in a Bik1-dependent manner. Our results provide the first evidence for a role of microtubule plus-ends in membrane cargo trafficking in yeast, through Rho1- and Bik1-dependent mechanisms, and highlight the importance of the C-terminal α-tubulin amino acid in this process.