CXXC1 is not essential for normal DNA double-strand break formation and meiotic recombination in mouse.

In most mammals, including mice and humans, meiotic recombination is determined by the meiosis specific histone methytransferase PRDM9, which binds to specific DNA sequences and trimethylates histone 3 at lysine-4 and lysine-36 at the adjacent nucleosomes. These actions ensure successful DNA double strand break formation and repair that occur on the proteinaceous structure forming the chromosome axis. The process of hotspot association with the axis after their activation by PRDM9 is poorly understood. Previously, we and others have identified CXXC1, an ortholog of S. cerevisiae Spp1 in mammals, as a PRDM9 interactor. In yeast, Spp1 is a histone methyl reader that links H3K4me3 sites with the recombination machinery, promoting DSB formation. Here, we investigated whether CXXC1 has a similar function in mouse meiosis. We created two Cxxc1 conditional knockout mouse models to deplete CXXC1 generally in germ cells, and before the onset of meiosis. Surprisingly, male knockout mice were fertile, and the loss of CXXC1 in spermatocytes had no effect on PRDM9 hotspot trimethylation, double strand break formation or repair. Our results demonstrate that CXXC1 is not an essential link between PRDM9-activated recombination hotspot sites and DSB machinery and that the hotspot recognition pathway in mouse is independent of CXXC1

A quantitative assay for crossover and noncrossover molecular events at individual recombination hotspots in both male and female gametes

AbstractMeiotic recombination is a fundamental process in all eukaryotes. Among organisms in which recombination initiates prior to synapsis, recombination preferentially occurs in short 1-to 2-kb regions, known as recombination hotspots. Among mammals, genotyping sperm DNA has provided a means of monitoring recombination events at specific hotspots in male meiosis. To complement these current techniques, we developed an assay for amplifying all copies of a hotspot from the DNA of male and female germ cells, cloning the products into Escherichia coli, and SNP genotyping the resulting colonies using fluorescence technology. This approach examines the molecular details of crossover and noncrossover events of individual meioses directly at active hotspots while retaining the simplicity of using pooled DNA. Using this technique, we analyzed recombination events at the Hlx1 hotspot located on mouse chromosome 1, finding that the results agree well with a prior genetic characterization of 3026 male and 3002 female meioses

On the dependent recognition of some long zinc finger proteins

The human genome contains about 800 C2H2 zinc finger proteins (ZFPs), and most of them are composed of long arrays of zinc fingers. Standard ZFP recognition model asserts longer finger arrays should recognize longer DNA-binding sites. However, recent experimental efforts to identify in vivo ZFP binding sites contradict this assumption, with many exhibiting short motifs. Here we use ZFY, CTCF, ZIM3, and ZNF343 as examples to address three closely related questions: What are the reasons that impede current motif discovery methods? What are the functions of those seemingly unused fingers and how can we improve the motif discovery algorithms based on long ZFPs\u27 biophysical properties? Using ZFY, we employed a variety of methods and find evidence for \u27dependent recognition\u27 where downstream fingers can recognize some previously undiscovered motifs only in the presence of an intact core site. For CTCF, high-throughput measurements revealed its upstream specificity profile depends on the strength of its core. Moreover, the binding strength of the upstream site modulates CTCF\u27s sensitivity to different epigenetic modifications within the core, providing new insight into how the previously identified intellectual disability-causing and cancer-related mutant R567W disrupts upstream recognition and deregulates the epigenetic control by CTCF. Our results establish that, because of irregular motif structures, variable spacing and dependent recognition between sub-motifs, the specificities of long ZFPs are significantly underestimated, so we developed an algorithm, ModeMap, to infer the motifs and recognition models of ZIM3 and ZNF343, which facilitates high-confidence identification of specific binding sites, including repeats-derived elements. With revised concept, technique, and algorithm, we can discover the overlooked specificities and functions of those \u27extra\u27 fingers, and therefore decipher their broader roles in human biology and diseases

Parental origin of chromosomes influences crossover activity within the Kcnq1 transcriptionally imprinted domain of Mus musculus

BACKGROUND: Among the three functions of DNA, mammalian replication and transcription can be subject to epigenetic imprinting specified by the parental origin of chromosomes, and although there is suggestive indication that this is also true for meiotic recombination, no definitive evidence has yet been reported. RESULTS: We have now obtained such evidence on mouse chromosome 7 by assaying meiotic recombination as it occurs in reciprocal F1 mice. A 166 kb region near the Kcnq1 transcriptionally imprinted domain showed significantly higher recombination activity in the CAST x B6 parental direction (p \u3c 0.03). Characterizing hotspots within this domain revealed a cluster of three hotspots lying within a 100 kb span, among these hotspots, Slc22a18 showed a definitive parent of origin effect on recombination frequency (p \u3c 0.02). Comparing recombination activity in the mouse Kcnq1 and neighboring H19-Igf2 imprinted domains with their human counterparts, we found that elevated recombination activity in these domains is a consequence of their chromosomal position relative to the telomere and not an intrinsic characteristic of transcriptionally imprinted domains as has been previously suggested. CONCLUSION: Similar to replication and transcription, we demonstrate that meiotic recombination can be subjected to epigenetic imprinting and hotspot activity can be influenced by the parental origin of chromosomes. Furthermore, transcriptionally imprinted regions exhibiting elevated recombination activity are likely a consequence of their chromosomal location rather than their transcriptional characteristic

Substrate surface patterning by optical near field modulation around colloidal particles immersed in a liquid

Optical near field enhancements in the vicinity of particles illuminated by laser light are increasingly recognized as a powerful tool for nanopatterning applications, but achieving sub-wavelength details from the near-field distribution remains a challenge. Here we present a quantitative analysis of the spatial modulation of the near optical fields generated using single 8 ps, 355 nm (and 532 nm) laser pulses around individual colloidal particles and small close packed arrays of such particles on silicon substrates. The analysis is presented for particles in air and, for the first time, when immersed in a range of liquid media. Immersion in a liquid allows detailed exploration of the effects on the near field of changing not just the magnitude but also the sign of the refractive index difference between the particle and the host medium. The level of agreement between the results of ray tracing and Mie scattering simulations, and the experimentally observed patterns on solid surfaces, should encourage further modelling, predictions and demonstrations of the rich palette of sub-wavelength surface profiles that can be achieved using colloidal particles immersed in liquids

Evidence of a Large-Scale Functional Organization of Mammalian Chromosomes

Evidence from inbred strains of mice indicates that a quarter or more of the mammalian genome consists of chromosome regions containing clusters of functionally related genes. The intense selection pressures during inbreeding favor the coinheritance of optimal sets of alleles among these genetically linked, functionally related genes, resulting in extensive domains of linkage disequilibrium (LD) among a set of 60 genetically diverse inbred strains. Recombination that disrupts the preferred combinations of alleles reduces the ability of offspring to survive further inbreeding. LD is also seen between markers on separate chromosomes, forming networks with scale-free architecture. Combining LD data with pathway and genome annotation databases, we have been able to identify the biological functions underlying several domains and networks. Given the strong conservation of gene order among mammals, the domains and networks we find in mice probably characterize all mammals, including humans

The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots

: In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11β and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes

The Recombinational Anatomy of a Mouse Chromosome

Among mammals, genetic recombination occurs at highly delimited sites known as recombination hotspots. They are typically 1–2 kb long and vary as much as a 1,000-fold or more in recombination activity. Although much is known about the molecular details of the recombination process itself, the factors determining the location and relative activity of hotspots are poorly understood. To further our understanding, we have collected and mapped the locations of 5,472 crossover events along mouse Chromosome 1 arising in 6,028 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. Crossovers were mapped to a minimum resolution of 225 kb, and those in the telomere-proximal 24.7 Mb were further mapped to resolve individual hotspots. Recombination rates were evolutionarily conserved on a regional scale, but not at the local level. There was a clear negative-exponential relationship between the relative activity and abundance of hotspot activity classes, such that a small number of the most active hotspots account for the majority of recombination. Females had 1.2× higher overall recombination than males did, although the sex ratio showed considerable regional variation. Locally, entirely sex-specific hotspots were rare. The initiation of recombination at the most active hotspot was regulated independently on the two parental chromatids, and analysis of reciprocal crosses indicated that parental imprinting has subtle effects on recombination rates. It appears that the regulation of mammalian recombination is a complex, dynamic process involving multiple factors reflecting species, sex, individual variation within species, and the properties of individual hotspots

Sexual dimorphism in the meiotic requirement for PRDM9: A mammalian evolutionary safeguard.

In many mammals, genomic sites for recombination are determined by the histone methyltransferase PRMD9. Some mouse strains lacking PRDM9 are infertile, but instances of fertility or semifertility in the absence of PRDM9 have been reported in mice, canines, and a human female. Such findings raise the question of how the loss of PRDM9 is circumvented to maintain fertility. We show that genetic background and sex-specific modifiers can obviate the requirement for PRDM9 in mice. Specifically, the meiotic DNA damage checkpoint protein CHK2 acts as a modifier allowing female-specific fertility in the absence of PRDM9. We also report that, in the absence of PRDM9, a PRDM9-independent recombination system is compatible with female meiosis and fertility, suggesting sex-specific regulation of meiotic recombination, a finding with implications for speciation