Concept, Design and Implementation of a Cardiovascular Gene-Centric 50 K SNP Array for Large-Scale Genomic Association Studies

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a “cosmopolitan” tagging approach to capture the genetic diversity across ∼2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions

Meiotic Recombination Initiation in and around Retrotransposable Elements in <i>Saccharomyces cerevisiae</i>

<div><p>Meiotic recombination is initiated by large numbers of developmentally programmed DNA double-strand breaks (DSBs), ranging from dozens to hundreds per cell depending on the organism. DSBs formed in single-copy sequences provoke recombination between allelic positions on homologous chromosomes, but DSBs can also form in and near repetitive elements such as retrotransposons. When they do, they create a risk for deleterious genome rearrangements in the germ line via recombination between non-allelic repeats. A prior study in budding yeast demonstrated that insertion of a Ty retrotransposon into a DSB hotspot can suppress meiotic break formation, but properties of Ty elements in their most common physiological contexts have not been addressed. Here we compile a comprehensive, high resolution map of all Ty elements in the rapidly and efficiently sporulating <i>S. cerevisiae</i> strain SK1 and examine DSB formation in and near these endogenous retrotransposable elements. SK1 has 30 Tys, all but one distinct from the 50 Tys in S288C, the source strain for the yeast reference genome. From whole-genome DSB maps and direct molecular assays, we find that DSB levels and chromatin structure within and near Tys vary widely between different elements and that local DSB suppression is not a universal feature of Ty presence. Surprisingly, deletion of two Ty elements weakened adjacent DSB hotspots, revealing that at least some Ty insertions promote rather than suppress nearby DSB formation. Given high strain-to-strain variability in Ty location and the high aggregate burden of Ty-proximal DSBs, we propose that meiotic recombination is an important component of host-Ty interactions and that Tys play critical roles in genome instability and evolution in both inbred and outcrossed sexual cycles.</p></div

Chromatin structures of Ty elements.

<p>(A) Preferential MNase cleavage of chromatin in nucleosome-depleted regions (NDR) and linkers between nucleosomes. (B–C) MNase sensitivity of regions in and around Ty<i>_CGR1-SCW11</i> (B) and Ty<i>_PEX25-CAR1</i> (C). Intact meiotic nuclei were treated with 0, 2.5×10⁻⁵, or 5×10⁻⁵ units of MNase per µg of DNA (lanes 1–3) and purified genomic DNA (N, for naked DNA) from vegetative cells was treated with 1.6×10⁻⁴ units per µg DNA (lane 4), then MNase cleavage patterns were determined by Southern blotting and indirect end-labeling. Genomic DNA prepared from meiotic <i>sae2Δ</i> cells is a marker for DSB positions (lane 5). Profiles of lanes 1 (−MNase), 3 (+MNase), and 5 (DSBs) are shown to the right of each blot. Red bars on ORF maps indicate probe positions.</p

Location of Ty elements in SK1.

<p>The insertion sites and orientations of SK1 Ty elements are shown in comparison to S288C Tys (chromosomal coordinates are from S288C). Fragmented arrowheads indicate partial Ty elements. Open circles show centromeres. Dashed circle highlights the only Ty shared between the two strains.</p

Location of Ty elements in SK1.

a<p>The coordinates of Ty insertion sites are based on the S288C reference genome (the June 2008 assembly from the Saccharomyces Genome Database). When a Ty insertion site exhibits a 5-bp duplication, the third and fourth bp are used as the start and end coordinates, respectively.</p>b<p>Although the family of Ty<i>_URA3</i> and Ty<i>_YER137C-RTR1</i> could not be determined by established criteria <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003732#pgen.1003732-Kim1" target="_blank">[11]</a> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003732#pgen.1003732.s001" target="_blank">Figure S1D</a>), these Tys were classified as Ty1 by Gabriel et al. (2006).</p>c<p>The presence (+) or absence (−) of tRNA in the same intergenic region with a Ty is indicated.</p>d<p>The target site sequence duplicated on the same strand with a Ty is indicated. N.D. indicates that the presence or absence of sequence duplication was not determined. “–” indicates that sequence duplication was not observed.</p>e<p>Ty element is inserted in a novel SK1 LTR. The insertion site of the LTR is indicated.</p>f<p>Ty<i>_{YBL108W-YBL107C}</i> and Ty<i>_{TEL03L-YCL073C}</i> are located in subtelomeric regions, which are enriched with repeated sequences and are dynamic among strains <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003732#pgen.1003732-Louis1" target="_blank">[65]</a>. Since chromosome ends are not well defined in the SK1 genome assembly, it remains to be determined which chromosome end(s) carry these Tys.</p>g<p>Ty<i>_{TEL03L-YCL073C}</i> is the same as <i>YCLWTy5-1</i> in S288C.</p>h<p>A full-length Ty<i>_{EXG2-YDR262W-1}</i> and a fragmented Ty<i>_{EXG2-YDR262W-2}</i> of >1 kb in size are located adjacent to each other.</p>i<p>Ty<i>_OMS1-HIM1</i> is a ∼1-kb fragmented Ty.</p>j<p>Ty_{NCE103-YNL035C-1} is disrupted by Ty_{NCE103-YNL035C-2}.</p

Meiotic DSBs in and around Ty elements.

<p>(A) Spo11 oligo densities around Ty elements. For each SK1 Ty, Spo11 oligo densities (hits per million mapped reads (hpM) per kb) were determined in the indicated window of adjacent sequence. Sites where Tys are present in S288C but absent in SK1 serve as controls. Bars are means and standard deviations; the dashed line is the genome average; p values are from Wilcoxon rank sum tests. For comparison, the internal Spo11 oligo density averaged across all Ty elements was 6.7 hpM/kb, approximately 30–40-fold lower than the mean for these flanking regions. (B) Spo11 oligo densities around Ty elements in different types of intergenic regions. (C–F) Physical detection of DSBs. (Left) Spo11 oligo distribution from Pan et al. (2011) and maps of ORFs (blue-filled polygons) and tRNA genes (horizontal bars). (Right) Southern blots of genomic DNA isolated from <i>spo11-Y135F</i>, <i>sae2Δ</i> and <i>dmc1Δ</i> strains at 6 hrs after entry into meiosis. Red numbers are DSB frequencies within the bracketed regions in each lane (% of total hybridization signal in the lane); quantification is provided separately for each lane, representing independent cultures. Red bars, probe positions; P, unbroken (parental) restriction fragments; asterisks, cross hybridizing bands. Flanking restriction sites plus internal sites used to generate genomic DNA markers (run on the same gels; not shown) are indicated: NcoI (N), BsaXI (XI), PpuMI (MI), Bsu36I (Bs), BglII (Bg), BspHI (HI), BamHI (B), ApaLI (Ap), SnaBI (Sn), NdeI (Nd). In (F), the inset shows a more exposed contrast of the phosphorimager signal for the region indicated by the dashed line. (G) Quantitative agreement between Spo11 oligo counts and DSB frequencies at hotspots near Ty elements in <i>dmc1Δ</i> mutants. DSB values are the means of the two independent cultures shown in panels C–F.</p

Deleting Ty elements increases DSB formation nearby.

<p>(A–D) Genomic DNA was isolated from meiotic cultures of a <i>dmc1Δ</i> strain containing the full complement of SK1 Tys and <i>dmc1Δ</i> strains in which either Ty<i>_EST3-FAA3</i> or Ty<i>_CGR1-SCW11</i> was deleted. DSBs were detected by Southern blotting and indirect end-labeling. Figures are labeled as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003732#pgen-1003732-g004" target="_blank">Figures 4C–F</a>. Circled lower case roman numerals indicate hotspots discussed in the text. Red numerals are DSB frequencies within the bracketed regions in each of two independent cultures, corrected where appropriate for differences in transfer efficiency for the parental fragments (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003732#s4" target="_blank">Materials and Methods</a>). Blots were stripped and rehybridized to probes from separate loci to serve as loading controls (lower panels). (A,B) DSBs around the Ty<i>_EST3-FAA3</i> insertion site, probed from either side. (C,D) DSBs around the Ty<i>_CGR1-SCW11</i> insertion site, probed from either side. (E) DSBs at the <i>YCR048W</i> hotspot (control locus) in the same samples as in panels A–D.</p

Structural and functional characterization of the Spo11 core complex

54 p.-8 fig.Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomyces cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex–duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.MSKCC core facilities are supported by National Cancer Institute (NCI) Cancer Center support grant no. P30 CA08748. The SEC–LS/UV/RI instrumentation was supported by NIH Award Number 1S10RR023748-01. Work in the S.K. laboratory was supported principally by the Howard Hughes Medical Institute and in part by NIH grant no. R35 GM118092(S.K.). Work in the J.M.B. laboratory was funded by NCI grant no. R01-CA0777373(J.M.B.). C.C.B. was supported in part by funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (European Research Council grant agreement no. 802525) and from the Fonds National de la Recherche Scientifique (FNRS MIS-Ulysse grant no. F.6002.20) (C.C.B.).Peer reviewe

Genomic and chromatin features shaping meiotic double-strand break formation and repair in mice

<p>The SPO11-generated DNA double-strand breaks (DSBs) that initiate meiotic recombination occur non-randomly across genomes, but mechanisms shaping their distribution and repair remain incompletely understood. Here, we expand on recent studies of nucleotide-resolution DSB maps in mouse spermatocytes. We find that trimethylation of histone H3 lysine 36 around DSB hotspots is highly correlated, both spatially and quantitatively, with trimethylation of H3 lysine 4, consistent with coordinated formation and action of both PRDM9-dependent histone modifications. In contrast, the DSB-responsive kinase ATM contributes independently of PRDM9 to controlling hotspot activity, and combined action of ATM and PRDM9 can explain nearly two-thirds of the variation in DSB frequency between hotspots. DSBs were modestly underrepresented in most repetitive sequences such as segmental duplications and transposons. Nonetheless, numerous DSBs form within repetitive sequences in each meiosis and some classes of repeats are preferentially targeted. Implications of these findings are discussed for evolution of PRDM9 and its role in hybrid strain sterility in mice. Finally, we document the relationship between mouse strain-specific DNA sequence variants within PRDM9 recognition motifs and attendant differences in recombination outcomes. Our results provide further insights into the complex web of factors that influence meiotic recombination patterns.</p