DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we ...present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function. Mapping recombination in individual human malesSperm and eggs form from diploid cells that contain two copies of our genomic DNA. The haploid germ cells must undergo a special cell division, meiosis, which halves their DNA content. Meiosis involves a DNA recombination step between parental chromosomes. Recombination is initiated by a DNA double-strand break, which can exchange DNA between the chromosomes, a process that drives human genetic variation. Pratto et al. mapped meiotic recombination sites in individual human males (see the Perspective by de Massy). Recombination hotspots were influenced by variants of the histone-lysine N-methyltransferase protein, PRDM9, as well as by other factors. The recombination sites also influence genome evolution and the incidence of genetic disease.Science, this issue 10.1126/science.1256442; see also p. 808
Sex chromosomes in males of most eutherian mammals share only a small homologous segment, the pseudoautosomal region (PAR), in which the formation of double-strand breaks (DSBs), pairing and crossing ...over must occur for correct meiotic segregation
. How cells ensure that recombination occurs in the PAR is unknown. Here we present a dynamic ultrastructure of the PAR and identify controlling cis- and trans-acting factors that make the PAR the hottest segment for DSB formation in the male mouse genome. Before break formation, multiple DSB-promoting factors hyperaccumulate in the PAR, its chromosome axes elongate and the sister chromatids separate. These processes are linked to heterochromatic mo-2 minisatellite arrays, and require MEI4 and ANKRD31 proteins but not the axis components REC8 or HORMAD1. We propose that the repetitive DNA sequence of the PAR confers unique chromatin and higher-order structures that are crucial for recombination. Chromosome synapsis triggers collapse of the elongated PAR structure and, notably, oocytes can be reprogrammed to exhibit spermatocyte-like levels of DSBs in the PAR simply by delaying or preventing synapsis. Thus, the sexually dimorphic behaviour of the PAR is in part a result of kinetic differences between the sexes in a race between the maturation of the PAR structure, formation of DSBs and completion of pairing and synapsis. Our findings establish a mechanistic paradigm for the recombination of sex chromosomes during meiosis.
Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here, we unveil mouse ANKRD31 as a lynchpin governing multiple ...aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. They also have delayed and/or fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures—stochastic on autosomes, nearly absolute on X and Y—cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines a pleckstrin homology (PH) domain in REC114 and its direct intermolecular contacts with ANKRD31. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model in which ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, thereby regulating DSB formation.
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•REC114 directly interacts with ANKRD31, a novel factor required for normal fertility•ANKRD31 influences the distribution of double-strand breaks genome-wide•ANKRD31 is essential for the recombination between X and Y chromosomes•A crystal structure reveals a PH domain in REC114 and its contacts with ANKRD31
Boekhout et al. discover ANKRD31 as a REC114 interactor and key player in meiotic recombination. ANKRD31 acts as a molecular scaffold to regulate double-strand break formation and promote X-Y recombination. An atomic resolution structure illuminates the conserved features of REC114-ANKRD31 interaction, including an unexpected pleckstrin homology domain in REC114.
Meiosis is the specialized cell division during which parental genomes recombine to create genotypically unique gametes. Despite its importance, mammalian meiosis cannot be studied in vitro, greatly ...limiting mechanistic studies. In vivo, meiocytes progress asynchronously through meiosis and therefore the study of specific stages of meiosis is a challenge. Here, we describe a method for isolating pure sub-populations of nuclei that allows for detailed study of meiotic substages. Interrogating the H3K4me3 landscape revealed dynamic chromatin transitions between substages of meiotic prophase I, both at sites of genetic recombination and at gene promoters. We also leveraged this method to perform the first comprehensive, genome-wide survey of histone marks in meiotic prophase, revealing a heretofore unappreciated complexity of the epigenetic landscape at meiotic recombination hotspots. Ultimately, this study presents a straightforward, scalable framework for interrogating the complexities of mammalian meiosis.
How homologous chromosomes (homologs) find their partner, pair, and recombine during meiosis constitutes the central phenomenon in eukaryotic genetics. It is widely believed that, in most organisms, ...SPO11-mediated DNA double-strand breaks (DSBs) introduced during prophase I precede and are required for efficient homolog pairing. We now show that, in the mouse, a significant level of homolog pairing precedes programmed DNA cleavage. Strikingly, this early chromosome pairing still requires SPO11 but is not dependent on its ability to make DSBs or homologous recombination proteins. Intriguingly, SUN1, a protein required for telomere attachment to the nuclear envelope and for post-DSB synapsis, is also required for early pre-DSB homolog pairing. Furthermore, pre-DSB pairing at telomeres persists upon entry into prophase I and is most likely important for initiation of synapsis. Our findings suggest that the DSB-triggered homology search may mainly serve to proofread and stabilize the pre-DSB pairing of homologous chromosomes.
► Homolog pairing precedes SPO11-mediated programmed DSBs during meiosis in the mouse ► A DSB-independent activity of SPO11 is required for this preleptotene pairing ► SUN1 anchors telomeres to the nuclear periphery to facilitate the pre-DSB pairing ► Pre-DSB subtelomeric pairing is more stable and most likely aids synapsis initiation
DSB-independent meiotic pairing occurs in flies and worms, but its existence in yeast is controversial and has not been documented in mammals. Boateng et al. report pre-DSB meiotic pairing of homologous chromosomes in preleptotene mouse spermatocytes and show that these events require a DSB-independent function of SPO11 and SUN1.
The repair of programmed DNA double-strand breaks (DSBs) physically tethers homologous chromosomes in meiosis to allow for accurate segregation through meiotic cell divisions. This process, known as ...recombination, also results in the exchange of alleles between parental chromosomes and contributes to genetic diversity. In mammals, meiotic DSBs occur predominantly in a small fraction of the genome, at sites known as hotspots. Studies of the formation and repair of meiotic DSBs in mammals are challenging, because few cells undergo meiotic DSB formation at a given time. To better understand the initiation and control of meiotic recombination in mammals, we have devised a highly sensitive method to map the sites of meiotic DSBs genome wide. Our method first isolates DNA bound to DSB repair proteins and then specifically sequences the associated single-stranded DNA. This protocol has generated the first meiotic DSB maps in several mammals and the only map of meiotic DSBs in humans.
Vertebrate meiotic recombination events are concentrated in regions (hotspots) that display open chromatin marks, such as trimethylation of lysines 4 and 36 of histone 3 (H3K4me3 and H3K36me3). Mouse ...and human PRDM9 proteins catalyze H3K4me3 and H3K36me3 and determine hotspot positions, whereas other vertebrates lacking PRDM9 recombine in regions with chromatin already opened for another function, such as gene promoters. While these other vertebrate species lacking PRDM9 remain fertile, inactivation of the mouse Prdm9 gene, which shifts the hotspots to the functional regions (including promoters), typically causes gross fertility reduction; and the reasons for these species differences are not clear.
We introduced Prdm9 deletions into the Rattus norvegicus genome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-type Prdm9 allele shared 88% hotspots but strains with different Prdm9 alleles only 3%. After Prdm9 deletion, rat hotspots relocated to functional regions, about 40% to positions corresponding to Prdm9-independent mouse hotspots, including promoters. Despite the hotspot relocation and decreased fertility, Prdm9-deficient rats of the SHR/OlaIpcv strain produced healthy offspring. The percentage of normal pachytene spermatocytes in SHR-Prdm9 mutants was almost double than in the PWD male mouse oligospermic sterile mutants. We previously found a correlation between the crossover rate and sperm presence in mouse Prdm9 mutants. The crossover rate of SHR is more similar to sperm-carrying mutant mice, but it did not fully explain the fertility of the SHR mutants. Besides mild meiotic arrests at rat tubular stages IV (mid-pachytene) and XIV (metaphase), we also detected postmeiotic apoptosis of round spermatids. We found delayed meiosis and age-dependent fertility in both sexes of the SHR mutants.
We hypothesize that the relative increased fertility of rat versus mouse Prdm9 mutants could be ascribed to extended duration of meiotic prophase I. While rat PRDM9 shapes meiotic recombination landscapes, it is unnecessary for recombination. We suggest that PRDM9 has additional roles in spermatogenesis and speciation-spermatid development and reproductive age-that may help to explain male-specific hybrid sterility.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The PRDM9 protein determines sites of meiotic recombination in humans by directing meiotic DNA double-strand breaks to specific loci. Targeting specificity is encoded by a long array of C
2
H
2
zinc ...fingers that bind to DNA. This zinc finger array is hypervariable, and the resulting alleles each have a potentially different DNA binding preference. The assessment of
PRDM9
diversity is important for understanding the complexity of human population genetics, inheritance linkage patterns, and predisposition to genetic disease. Due to the repetitive nature of the
PRDM9
zinc finger array, the large-scale sequencing of human
PRDM9
is challenging. We, therefore, developed a long-read sequencing strategy to infer the diploid
PRDM9
zinc finger array genotype in a high-throughput manner. From an unbiased study of
PRDM9
allelic diversity in 720 individuals from seven human populations, we detected 69
PRDM9
alleles. Several alleles differ in frequency among human populations, and 32 alleles had not been identified by previous studies, which were heavily biased to European populations.
PRDM9
alleles are distinguished by their DNA binding site preferences and fall into two major categories related to the most common
PRDM9-A
and
PRDM9-C
alleles. We also found that it is likely that inter-conversion between allele types is rare. By mapping meiotic double-strand breaks (DSBs) in the testis, we found that small variations in
PRDM9
can substantially alter the meiotic recombination landscape, demonstrating that minor
PRDM9
variants may play an under-appreciated role in shaping patterns of human recombination. In summary, our data greatly expands knowledge of
PRDM9
diversity in humans.
Meiotic recombination and chromosome synapsis between homologous chromosomes are essential for proper chromosome segregation at the first meiotic division. While recombination and synapsis, as well ...as checkpoints that monitor these two events, take place in the context of a prophase I-specific axial chromosome structure, it remains unclear how chromosome axis components contribute to these processes. We show here that many protein components of the meiotic chromosome axis, including SYCP2, SYCP3, HORMAD1, HORMAD2, SMC3, STAG3, and REC8, become post-translationally modified by phosphorylation during the prophase I stage. We found that HORMAD1 and SMC3 are phosphorylated at a consensus site for the ATM/ATR checkpoint kinase and that the phosphorylated forms of HORMAD1 and SMC3 localize preferentially to unsynapsed chromosomal regions where synapsis has not yet occurred, but not to synapsed or desynapsed regions. We investigated the genetic requirements for the phosphorylation events and revealed that the phosphorylation levels of HORMAD1, HORMAD2, and SMC3 are dramatically reduced in the absence of initiation of meiotic recombination, whereas BRCA1 and SYCP3 are required for normal levels of phosphorylation of HORMAD1 and HORMAD2, but not of SMC3. Interestingly, reduced HORMAD1 and HORMAD2 phosphorylation is associated with impaired targeting of the MSUC (meiotic silencing of unsynapsed chromatin) machinery to unsynapsed chromosomes, suggesting that these post-translational events contribute to the regulation of the synapsis surveillance system. We propose that modifications of chromosome axis components serve as signals that facilitate chromosomal events including recombination, checkpoint control, transcription, and synapsis regulation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Vancomycin or erythromycin resistance and the stability determinants, δω and ωεζ, of Enterococci and Streptococci plasmids are genetically linked. To unravel the mechanisms that promoted the stable ...persistence of resistance determinants, the early stages of Streptococcus pyogenes pSM19035 partitioning were biochemically dissected. First, the homodimeric centromere-binding protein, ω2, bound parS DNA to form a short-lived partition complex 1 (PC1). The interaction of PC1 with homodimeric δ δ2 even in the apo form (Apo-δ2), significantly stimulated the formation of a long-lived ω2·parS complex (PC2) without spreading into neighbouring DNA sequences. In the ATP·Mg2+ bound form, δ2 bound DNA, without sequence specificity, to form a transient dynamic complex (DC). Second, parS bound ω2 interacted with and promoted δ2 redistribution to co-localize with the PC2, leading to transient segrosome complex (SC, parS·ω2·δ2) formation. Third, δ2, in the SC, interacted with a second SC and promoted formation of a bridging complex (BC). Finally, increasing ω2 concentrations stimulated the ATPase activity of δ2 and the BC was disassembled. We propose that PC, DC, SC and BC formation were dynamic processes and that the molar ω2:δ2 ratio and parS DNA control their temporal and spatial assembly during partition of pSM19035 before cell division.
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