Genome-wide recombination is essential for genome stability, evolution, and speciation. Mouse Tex11, an X-linked meiosis-specific gene, promotes meiotic recombination and chromosomal synapsis. Here, we report that TEX11 is mutated in infertile men with non-obstructive azoospermia and that an analogous mutation in the mouse impairs meiosis. Genetic screening of a large cohort of idiopathic infertile men reveals that TEX11 mutations, including frameshift and splicing acceptor site mutations, cause infertility in 1% of azoospermic men. Functional evaluation of three analogous human TEX11 missense mutations in transgenic mouse models identified one mutation (V748A) as a potential infertility allele and found two mutations non-causative. In the mouse model, an intronless autosomal Tex11 transgene functionally substitutes for the X-linked Tex11 gene, providing genetic evidence for the X-to-autosomal retrotransposition evolution phenomenon. Furthermore, we find that TEX11 protein levels modulate genome-wide recombination rates in both sexes. These studies indicate that TEX11 alleles affecting expression level or substituting single amino acids may contribute to variations in recombination rates between sexes and among individuals in humans.

Although the mammalian X and Y chromosomes evolved from a single pair of autosomes, they are highly differentiated: the Y chromosome is dramatically smaller than the X and has lost most of its genes. The surviving genes are a specialized set with extraordinary evolutionary longevity. Most mammalian lineages have experienced delayed, or relatively recent, loss of at least one conserved Y-linked gene. An extreme example of this phenomenon is in the Japanese spiny rat, where the Y chromosome has disappeared altogether. In this species, many Y-linked genes were rescued by transposition to new genomic locations, but until our work presented here, this has been considered an isolated case.

Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative. Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.

Amplicons--large, nearly identical repeats in direct or inverted orientation--are abundant in the male-specific region of the human Y chromosome (MSY) and provide targets for intrachromosomal non-allelic homologous recombination (NAHR). Thus far, NAHR events resulting in deletions, duplications, inversions, or isodicentric chromosomes have been reported only for amplicon pairs located exclusively on the short arm (Yp) or the long arm (Yq). Here we report our finding of four men with Y chromosomes that evidently formed by intrachromosomal NAHR between inverted repeat pairs comprising one amplicon on Yp and one amplicon on Yq. In two men with spermatogenic failure, sister-chromatid crossing-over resulted in pseudoisoYp chromosome formation and loss of distal Yq. In two men with normal spermatogenesis, intrachromatid crossing-over generated pericentric inversions. These findings highlight the recombinogenic nature of the MSY, as intrachromosomal NAHR occurs for nearly all Y-chromosome amplicon pairs, even those located on opposing chromosome arms.

Zero-growth processes are a promising strategy for the production of reduced molecules and depict a steady transition from aerobic to anaerobic conditions. To investigate the adaptation of Corynebacterium glutamicum to altering oxygen availabilities, we conceived a triple-phase fermentation process that describes a gradual reduction of dissolved oxygen with a shift from aerobiosis via microaerobiosis to anaerobiosis. The distinct process phases were clearly bordered by the bacteria’s physiologic response such as reduced growth rate, biomass substrate yield and altered yield of fermentation products. During the process, sequential samples were drawn at six points and analyzed via RNA-sequencing, for metabolite concentrations and for enzyme activities. We found transcriptional alterations of almost 50% (1421 genes) of the entire protein coding genes and observed an upregulation of fermentative pathways, a rearrangement of respiration, and mitigation of the basic cellular mechanisms such as transcription, translation and replication as a transient response related to the installed oxygen dependent process phases. To investigate the regulatory regime, 18 transcriptionally altered (putative) transcriptional regulators were deleted, but none of the deletion strains showed noticeable growth kinetics under an oxygen restricted environment. However, the described transcriptional adaptation of C. glutamicum resolved to varying oxygen availabilities provides a useful basis for future process and strain engineering.

Wild-type C. glutamicum ATCC 13032 is known to possess two enzymes with anaplerotic (C4-directed) carboxylation activity, namely phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx). On the other hand, C3-directed decarboxylation can be catalyzed by the three enzymes phosphoenolpyruvate carboxykinase (PEPCk), oxaloacetate decarboxylase (ODx), and malic enzyme (ME). The resulting high metabolic flexibility at the anaplerotic node compromises the unambigous determination of its carbon and energy flux in C. glutamicum wild type. To circumvent this problem we performed a comprehensive analysis of selected single or double deletion mutants in the anaplerosis of wild-type C. glutamicum under defined d-glucose conditions. By applying well-controlled lab-scale bioreactor experiments in combination with untargeted proteomics, quantitative metabolomics and whole-genome sequencing hitherto unknown, and sometimes counter-intuitive, genotype-phenotype relationships in these mutants could be unraveled. In comparison to the wild type the four mutants C. glutamiucm Δpyc, C. glutamiucm Δpyc Δodx, C. glutamiucm Δppc Δpyc, and C. glutamiucm Δpck showed lowered specific growth rates and d-glucose uptake rates, underlining the importance of PCx and PEPCk activity for a balanced carbon and energy flux at the anaplerotic node. Most interestingly, the strain C. glutamiucm Δppc Δpyc could be evolved to grow on d-glucose as the only source of carbon and energy, whereas this combination was previously considered lethal. The prevented anaplerotic carboxylation activity of PEPCx and PCx was found in the evolved strain to be compensated by an up-regulation of the glyoxylate shunt, potentially in combination with the 2-methylcitrate cycle.

Little is known about how human Y-Chromosome gene expression directly contributes to differences between XX (female) and XY (male) individuals in nonreproductive tissues. Here, we analyzed quantitative profiles of Y-Chromosome gene expression across 36 human tissues from hundreds of individuals. Although it is often said that Y-Chromosome genes are lowly expressed outside the testis, we report many instances of elevated Y-Chromosome gene expression in a nonreproductive tissue. A notable example is EIF1AY, which encodes eukaryotic translation initiation factor 1A Y-linked, together with its X-linked homolog EIF1AX Evolutionary loss of a Y-linked microRNA target site enabled up-regulation of EIF1AY, but not of EIF1AX, in the heart. Consequently, this essential translation initiation factor is nearly twice as abundant in male as in female heart tissue at the protein level. Divergence between the X and Y Chromosomes in regulatory sequence can therefore lead to tissue-specific Y-Chromosome-driven sex biases in expression of critical, dosage-sensitive regulatory genes.

Precise execution of recombination during meiosis is essential for forming chromosomally-balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these remain poorly understood, particularly in mammals. Here we report that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated resected DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

It is widely believed that at least three nonoverlapping regions of the human Y chromosome-AZFa, AZFb, and AZFc ("azoospermia factors" a, b, and c)-are essential for normal spermatogenesis. These intervals are defined by interstitial Y-chromosome deletions that impair or extinguish spermatogenesis. Deletion breakpoints, mechanisms, and lengths, as well as inventories of affected genes, have been elucidated for deletions of AZFa and of AZFc but not for deletions of AZFb or of AZFb plus AZFc. We studied three deletions of AZFb and eight deletions of AZFb plus AZFc, as assayed by the STSs defining these intervals. Guided by Y-chromosome sequence, we localized breakpoints precisely and were able to sequence nine of the deletion junctions. Homologous recombination can explain seven of these deletions but not the remaining two. This fact and our discovery of breakpoint hotspots suggest that factors in addition to homology underlie these deletions. The deletions previously thought to define AZFb were found to extend from palindrome P5 to the proximal arm of palindrome P1, 1.5 Mb within AZFc. Thus, they do not define a genomic region separate from AZFc. We also found that the deletions of AZFb plus AZFc, as assayed by standard STSs heretofore available, in fact extend from P5 to the distal arm of P1 and spare distal AZFc. Both classes of deletions are massive: P5/proximal-P1 deletions encompass up to 6.2 Mb and remove 32 genes and transcripts; P5/distal-P1 deletions encompass up to 7.7 Mb and remove 42 genes and transcripts. To our knowledge, these are the largest of all human interstitial deletions for which deletion junctions and complete intervening sequence are available. The restriction of the associated phenotype to spermatogenic failure indicates the remarkable functional specialization of the affected regions of the Y chromosome.

The human Y chromosome began to evolve from an autosome hundreds of millions of years ago, acquiring a sex-determining function and undergoing a series of inversions that suppressed crossing over with the X chromosome. Little is known about the recent evolution of the Y chromosome because only the human Y chromosome has been fully sequenced. Prevailing theories hold that Y chromosomes evolve by gene loss, the pace of which slows over time, eventually leading to a paucity of genes, and stasis. These theories have been buttressed by partial sequence data from newly emergent plant and animal Y chromosomes, but they have not been tested in older, highly evolved Y chromosomes such as that of humans. Here we finished sequencing of the male-specific region of the Y chromosome (MSY) in our closest living relative, the chimpanzee, achieving levels of accuracy and completion previously reached for the human MSY. By comparing the MSYs of the two species we show that they differ radically in sequence structure and gene content, indicating rapid evolution during the past 6 million years. The chimpanzee MSY contains twice as many massive palindromes as the human MSY, yet it has lost large fractions of the MSY protein-coding genes and gene families present in the last common ancestor. We suggest that the extraordinary divergence of the chimpanzee and human MSYs was driven by four synergistic factors: the prominent role of the MSY in sperm production, 'genetic hitchhiking' effects in the absence of meiotic crossing over, frequent ectopic recombination within the MSY, and species differences in mating behaviour. Although genetic decay may be the principal dynamic in the evolution of newly emergent Y chromosomes, wholesale renovation is the paramount theme in the continuing evolution of chimpanzee, human and perhaps other older MSYs.

The Mre11 complex (Mre11, Rad50, and Nbs1) is a central component of the DNA damage response (DDR), governing both double-strand break repair and DDR signaling. Rad50 contains a highly conserved Zn(2+)-dependent homodimerization interface, the Rad50 hook domain. Mutations that inactivate the hook domain produce a null phenotype. In this study, we analyzed mutants with reduced hook domain function in an effort to stratify hook-dependent Mre11 complex functions. One of these alleles, Rad50(46), conferred reduced Zn(2+) affinity and dimerization efficiency. Homozygous Rad50(46/46) mutations were lethal in mice. However, in the presence of wild-type Rad50, Rad50(46) exerted a dominant gain-of-function phenotype associated with chronic DDR signaling. At the organismal level, Rad50(+/46) exhibited hydrocephalus, liver tumorigenesis, and defects in primitive hematopoietic and gametogenic cells. These outcomes were dependent on ATM, as all phenotypes were mitigated in Rad50(+/46) Atm(+/-) mice. These data reveal that the murine Rad50 hook domain strongly influences Mre11 complex-dependent DDR signaling, tissue homeostasis, and tumorigenesis.

A future bioeconomy relies on the efficient use of renewable resources for energy and material product supply. In this context, biorefineries have been developed and play a key role in converting lignocellulosic residues. Although a holistic use of the biomass feed is desired, side streams evoke in current biorefinery approaches. To ensure profitability, efficiency, and sustainability of the overall conversion process, a meaningful valorization of these materials is needed. Here, a so far unexploited side stream derived from fast pyrolysis of wheat straw-pyrolysis water-was used for production of 1,2-propanediol in microbial fermentation with engineered Corynebacterium glutamicum.

In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.

Transcriptional silencing by heritable cytosine-5 methylation is an ancient strategy to repress transposable elements. It was previously thought that mammals possess four DNA methyltransferase paralogs-Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l-that establish and maintain cytosine-5 methylation. Here we identify a fifth paralog, Dnmt3c, that is essential for retrotransposon methylation and repression in the mouse male germline. From a phenotype-based forward genetics screen, we isolated a mutant mouse called 'rahu', which displays severe defects in double-strand-break repair and homologous chromosome synapsis during male meiosis, resulting in sterility. rahu is an allele of a transcription unit (Gm14490, renamed Dnmt3c) that was previously mis-annotated as a Dnmt3-family pseudogene. Dnmt3c encodes a cytosine methyltransferase homolog, and Dnmt3crahu mutants harbor a non-synonymous mutation of a conserved residue within one of its cytosine methyltransferase motifs, similar to a mutation in human DNMT3B observed in patients with immunodeficiency, centromeric instability and facial anomalies syndrome. The rahu mutation lies at a potential dimerization interface and near the potential DNA binding interface, suggesting that it compromises protein-protein and/or protein-DNA interactions required for normal DNMT3C function. Dnmt3crahu mutant males fail to establish normal methylation within LINE and LTR retrotransposon sequences in the germline and accumulate higher levels of transposon-derived transcripts and proteins, particularly from distinct L1 and ERVK retrotransposon families. Phylogenetic analysis indicates that Dnmt3c arose during rodent evolution by tandem duplication of Dnmt3b, after the divergence of the Dipodoidea and Muroidea superfamilies. These findings provide insight into the evolutionary dynamics and functional specialization of the transposon suppression machinery critical for mammalian sexual reproduction and epigenetic regulation.

Amplicons-large, highly identical segmental duplications-are a prominent feature of mammalian Y chromosomes. Although they encode genes essential for fertility, these amplicons differ vastly between species, and little is known about the selective constraints acting on them. Here, we develop computational tools to detect amplicon copy number with unprecedented accuracy from high-throughput sequencing data. We find that one-sixth (16.9%) of 1,216 males from the 1000 Genomes Project have at least one deleted or duplicated amplicon. However, each amplicon's reference copy number is scrupulously maintained among divergent branches of the Y chromosome phylogeny, including the ancient branch A00, indicating that the reference copy number is ancestral to all modern human Y chromosomes. Using phylogenetic analyses and simulations, we demonstrate that this pattern of variation is incompatible with neutral evolution and instead displays hallmarks of mutation-selection balance. We also observe cases of amplicon rescue, in which deleted amplicons are restored through subsequent duplications. These results indicate that, contrary to the lack of constraint suggested by the differences between species, natural selection has suppressed amplicon copy number variation in diverse human lineages.

Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes. However, the meiotic functions of the PIKK ATR have remained elusive, because germline-specific depletion of this kinase is challenging. Here we uncover roles for ATR in male mouse prophase I progression. ATR deletion causes chromosome axis fragmentation and germ cell elimination at mid pachynema. This elimination cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germline. ATR is required for synapsis, in a manner genetically dissociable from DSB formation. ATR also regulates loading of recombinases RAD51 and DMC1 to DSBs and recombination focus dynamics on synapsed and asynapsed chromosomes. Our studies reveal ATR as a critical regulator of mouse meiosis.

Somatic cells of human males and females have 45 chromosomes in common, including the "active" X chromosome. In males the 46th chromosome is a Y; in females it is an "inactive" X (Xi). Through linear modeling of autosomal gene expression in cells from individuals with zero to three Xi and zero to four Y chromosomes, we found that Xi and Y impact autosomal expression broadly and with remarkably similar effects. Studying sex chromosome structural anomalies, promoters of Xi- and Y-responsive genes, and CRISPR inhibition, we traced part of this shared effect to homologous transcription factors-ZFX and ZFY-encoded by Chr X and Y. This demonstrates sex-shared mechanisms by which Xi and Y modulate autosomal expression. Combined with earlier analyses of sex-linked gene expression, our studies show that 21% of all genes expressed in lymphoblastoid cells or fibroblasts change expression significantly in response to Xi or Y chromosomes.

In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation. However, improperly repaired DSBs can cause meiotic arrest or mutation; thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse and SPO11 and its accessory factors remain abundant long after most DSB formation ceases, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency.

Gene conversion is GC-biased across a wide range of taxa. Large palindromes on mammalian sex chromosomes undergo frequent gene conversion that maintains arm-to-arm sequence identity greater than 99%, which may increase their susceptibility to the effects of GC-biased gene conversion. Here, we demonstrate a striking history of GC-biased gene conversion in 12 palindromes conserved on the X chromosomes of human, chimpanzee, and rhesus macaque. Primate X-chromosome palindrome arms have significantly higher GC content than flanking single-copy sequences. Nucleotide replacements that occurred in human and chimpanzee palindrome arms over the past 7 million years are one-and-a-half times as GC-rich as the ancestral bases they replaced. Using simulations, we show that our observed pattern of nucleotide replacements is consistent with GC-biased gene conversion with a magnitude of 70%, similar to previously reported values based on analyses of human meioses. However, GC-biased gene conversion since the divergence of human and rhesus macaque explains only a fraction of the observed difference in GC content between palindrome arms and flanking sequence, suggesting that palindromes are older than 29 million years and/or had elevated GC content at the time of their formation. This work supports a greater than 2:1 preference for GC bases over AT bases during gene conversion and demonstrates that the evolution and composition of mammalian sex chromosome palindromes is strongly influenced by GC-biased gene conversion.

The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1-Cullin-F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, γH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.