Pavel P. Khil

The mouse X chromosome is enriched for sex-biased genes not subject to selection by meiotic sex chromosome inactivation

Sex chromosomes are subject to sex-specific selective evolutionary forces. One model predicts that genes with sex-biased expression should be enriched on the X chromosome. In agreement with Rices hypothesis, spermatogonial genes are over-represented on the X chromosome of mice and sex- and reproduction-related genes are over-represented on the human X chromosome. Male-biased genes are under-represented on the X chromosome in worms and flies, however. Here we show that mouse spermatogenesis genes are relatively under-represented on the X chromosome and female-biased genes are enriched on it. We used Spo11−/− mice blocked in spermatogenesis early in meiosis to evaluate the temporal pattern of gene expression in sperm development. Genes expressed before the Spo11 block are enriched on the X chromosome, whereas those expressed later in spermatogenesis are depleted. Inactivation of the X chromosome in male meiosis may be a universal driving force for X-chromosome demasculinization.

Genome-wide analysis reveals novel molecular features of mouse recombination hotspots.

Meiotic recombination predominantly occurs at discrete genomic loci called recombination hotspots, but the features defining these areas are still largely unknown (reviewed in refs 1–5). To allow a comprehensive analysis of hotspot-associated DNA and chromatin characteristics, we developed a direct molecular approach for mapping meiotic DNA double-strand breaks that initiate recombination. Here we present the genome-wide distribution of recombination initiation sites in the mouse genome. Hotspot centres are mapped with approximately 200-nucleotide precision, which allows analysis of the fine structural details of the preferred recombination sites. We determine that hotspots share a centrally distributed consensus motif, possess a nucleotide skew that changes polarity at the centres of hotspots and have an intrinsic preference to be occupied by a nucleosome. Furthermore, we find that the vast majority of recombination initiation sites in mouse males are associated with testis-specific trimethylation of lysine 4 on histone H3 that is distinct from histone H3 lysine 4 trimethylation marks associated with transcription. The recombination map presented here has been derived from a homogeneous mouse population with a defined genetic background and therefore lends itself to extensive future experimental exploration. We note that the mapping technique developed here does not depend on the availability of genetic markers and hence can be easily adapted to other species with complex genomes. Our findings uncover several fundamental features of mammalian recombination hotspots and underline the power of the new recombination map for future studies of genetic recombination, genome stability and evolution.

Genetic recombination is directed away from functional genomic elements in mice

Genetic recombination occurs during meiosis, the key developmental programme of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyltransferase, PR domain containing 9 (PRDM9), the product of the only known speciation-associated gene in mammals. PRDM9 is thought to determine the preferred recombination sites—recombination hotspots—through sequence-specific binding of its highly polymorphic multi-Zn-finger domain. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination. Here we map and analyse the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F1 hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the exception of the pseudo-autosomal region (PAR)—the only area of the genome that undergoes recombination in 100% of cells. Surprisingly, hotspots are still observed in Prdm9 knockout mice, and as in wild type, these hotspots are found at H3 lysine 4 (H3K4) trimethylation marks. However, in the absence of PRDM9, most recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation. Such sites are rarely targeted in wild-type mice, indicating an unexpected role of the PRDM9 protein in sequestering the recombination machinery away from gene-promoter regions and other functional genomic elements.

Over 1000 genes are involved in the DNA damage response of Escherichia coli

Changes in gene expression after treatment of Escherichia coli cultures with mitomycin C were assessed using gene array technology. Unexpectedly, a large number of genes (nearly 30% of all genes) displayed significant changes in their expression level. Analysis and classification of expression profiles of the corresponding genes allowed us to assign this large number of genes into one or two dozen small clusters of genes with similar expression profiles. This assignment allowed us to describe systematically the changes in the level of gene expression in response to DNA damage. Among the damage‐induced genes, more than 100 are novel. From those genes involved in DNA metabolism that have not previously been shown to be induced by DNA damage, the mutS gene involved in mismatch repair is especially noteworthy. In addition to the SOS response, we observed the induction of other stress response pathways, such as those of oxidative stress and osmotic protection. Among the genes that are downregulated in response to DNA damage are numerous protein biosynthesis genes. Analysis of the gene expression data highlighted the essential involvement of σs‐regulated genes and the general stress response network in the response to DNA damage.

Recombination initiation maps of individual human genomes

Introduction The dramatic events of meiotic recombination culminate in the exchange of genetic information between parental chromosomes and ensure the production of genetically distinct gametes. Recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs), and most DSBs occur at discrete hotspots defined by the DNA binding specificity of the PRDM9 protein. The tandem array of PRDM9 zinc fingers that binds DNA is highly polymorphic, and different variants have different DNA binding preferences. Subsequent to binding, PRDM9 is thought to modify the local chromatin environment and to recruit SPO11 for DSB formation. Meiotic DSBs are predominantly repaired through homologous recombination, giving rise to either genetic crossovers, where a reciprocal genetic exchange occurs between homologous chromosomes, or noncrossovers. Meiotic DSB maps in human male individuals. In meiotic cells, the formation of programmed meiotic DSBs facilitates the subsequent exchange of genetic material between parental homologous chromosomes. All current methods to study the sites of meiotic recombination rely on detection of these genetic exchanges. The DMC1 protein binds to DNA around meiotic DSBs, and in this work, we used testis biopsies from individual males to pull down the DNA bound by the DMC1 protein. We thus identified the sites of meiotic DSBs in five individual males. Analysis and comparison of the resultant PRDM9-specific, personal genome-wide maps offers insights into the mechanisms that initiate meiosis, genome evolution, crossover formation, human population structure, and predisposition to genomic disorders. Rationale Despite recent progress in our understanding of recombination hotspot formation, the initiation of recombination remains poorly understood. Current approaches to study the early steps of meiotic recombination in humans primarily detect genetic crossovers, only one of the possible outcomes of DSB repair. Furthermore, these methods are limited by resolution, by sex and population averaging, or by an inability to extend the analysis genome-wide. To overcome these limitations, we built and analyzed high-resolution, individual-specific maps of meiotic DSBs in the human genome. Results We report the maps of meiotic DSBs in five males: two homozygous for the most common PRDM9 allele (PRDM9A) and three heterozygous for the PRDM9A allele and for the less frequent PRDM9B or PRDM9C alleles. We find that PRDM9A and PRDM9B define similar DSB hotspots, whereas the PRDM9C allele has a distinct specificity. A comparison of DSB hotspot maps with linkage disequilibrium (LD)–based estimates of recombination rates in the human population indicates that the LD map is a superimposition of PRDM9 allele-specific DSB maps and that the contribution of individual maps is proportional to the PRDM9 allele frequency in modern Africans. In individuals with identical PRDM9 alleles, over 5% of DSB hotspots vary in strength, yet less than half of this variation could be explained by sequence variation at putative PRDM9 binding sites. We also find that PRDM9 heterozygosity affects hotspot strength. In human males, DSBs, like crossovers, occur more frequently at subtelomeric regions, and the crossover rate is directly proportional to our estimate of DSB frequency. This indicates that DSB initiation frequency is a major driver of the crossover rate in human males. We detect distinct signatures of GC-biased gene conversion and of recombination-coupled mutagenesis at DSB hotspots. In addition, DSB hotspots are enriched at the breakpoints of copy number variants that arise via homology-mediated mechanisms. Such variants may give rise to genomic disorders, and indeed, we find that meiotic DSBs defined by PRDM9A often coincide with disease-associated chromosomal breakpoints. Conclusions Our genome-wide recombination initiation maps in individual human males offer unprecedented resolution of recombination initiation sites defined by specific alleles of the PRDM9 protein. Our analysis indicates that DSB frequency is a primary determinant of crossover frequency, and that factors other than PRDM9 modulate the frequency of recombination initiation. We also find that meiotic DSB repair and subsequent recombination affect the genome sequence both locally and at the level of structural rearrangements. Taken together, these data provide a foundation for future studies of genetic recombination, meiosis, and genome stability. 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 human male meiotic recombination sites reveals their influence on genome evolution and genetic disease. [Also see Perspective by de Massy] Mapping recombination in individual human males Sperm 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

Rsx is a metatherian RNA with Xist-like properties in X-chromosome inactivation

In female (XX) mammals one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY)1. X-inactivation in eutherian mammals is mediated by the non-coding RNA Xist2. Xist is not found in metatherians3 and how X-inactivation is initiated in these mammals has been the subject of speculation for decades4. Using the marsupial Monodelphis domestica we identify Rsx (RNA-on-the-silent X), an RNA that exhibits properties consistent with a role in X-inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, where both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-inactivation in mammals and pose questions about the mechanisms by which X-inactivation is achieved in eutherians.In female (XX) mammals, one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY). X-chromosome inactivation in eutherian mammals is mediated by the non-coding RNA Xist. Xist is not found in metatherians (marsupials), and how X-chromosome inactivation is initiated in these mammals has been the subject of speculation for decades. Using the marsupial Monodelphis domestica, here we identify Rsx (RNA-on-the-silent X), an RNA that has properties consistent with a role in X-chromosome inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, in which both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-chromosome inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-chromosome inactivation in mammals and pose questions about the mechanisms by which X-chromosome inactivation is achieved in eutherians.

Integrated transcriptome analysis of mouse spermatogenesis

BackgroundDifferentiation of primordial germ cells into mature spermatozoa proceeds through multiple stages, one of the most important of which is meiosis. Meiotic recombination is in turn a key part of meiosis. To achieve the highly specialized and diverse functions necessary for the successful completion of meiosis and the generation of spermatozoa thousands of genes are coordinately regulated through spermatogenesis. A complete and unbiased characterization of the transcriptome dynamics of spermatogenesis is, however, still lacking.ResultsIn order to characterize gene expression during spermatogenesis we sequenced eight mRNA samples from testes of juvenile mice from 6 to 38 days post partum. Using gene expression clustering we defined over 1,000 novel meiotically-expressed genes. We also developed a computational de-convolution approach and used it to estimate cell type-specific gene expression in pre-meiotic, meiotic and post-meiotic cells. In addition, we detected 13,000 novel alternative splicing events around 40% of which preserve an open reading frame, and found experimental support for 159 computational gene predictions. A comparison of RNA polymerase II (Pol II) ChIP-Seq signals with RNA-Seq coverage shows that gene expression correlates well with Pol II signals, both at promoters and along the gene body. However, we observe numerous instances of non-canonical promoter usage, as well as intergenic Pol II peaks that potentially delineate unannotated promoters, enhancers or small RNA clusters.ConclusionsHere we provide a comprehensive analysis of gene expression throughout mouse meiosis and spermatogenesis. Importantly, we find over a thousand of novel meiotic genes and over 5,000 novel potentially coding isoforms. These data should be a valuable resource for future studies of meiosis and spermatogenesis in mammals.

Crystal structure of the Escherichia coli YcdX protein reveals a trinuclear zinc active site

Alexey Teplyakov, Galina Obmolova, Pavel P. Khil, Andrew J. Howard, R. Daniel Camerini-Otero, and Gary L. Gilliland Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, Rockville, Maryland Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland Center for Synchrotron Radiation Research and Instrumentation, Biological, Chemical and Physical Sciences Department, Illinois Institute of Technology, Chicago, Illinois

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA

Meiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method--single-stranded DNA (ssDNA) sequencing (SSDS)--that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.

Crystal structure of Escherichia coli protein ybgI, a toroidal structure with a dinuclear metal site

BackgroundThe protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.ResultsThe structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.ConclusionThe toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.