The interplay between chromosome structure and meiotic integrity

Sexually reproducing organisms employ a specialized cell division called meiosis to form unique, haploid gametes from a diploid precursor cell. Upon fertilization, two opposite-sex gametes fuse to create a zygote that will develop into the offspring. Fundamental to meiosis is the formation and repair of programmed DNA double-strand breaks (DSBs) during prophase. These DSBs are repaired using the homologous chromosome (homolog) as a template for homologous recombination, which creates linkages between the homologs that are essential for faithful segregation. Following prophase, two successive rounds of chromosome segregation ensue. The first, meiosis I (MI), segregates homologs and the second, meiosis II (MII), segregates sister chromatids. Recombination is dependent on chromosome structure, and events that alter the DNA landscape will impact meiotic fidelity and, in turn, genomic integrity in gametes and future offspring. Thus understanding the relationship between chromosome structure and meiosis can help gain insight into human fertility and underlying causes of genetic disorders. It was the goal of this thesis to investigate factors known to regulate chromosome structure and determine their influence on meiosis in the budding yeast Saccharomyces cerevisiae and mice. In addition, we tested a novel method to search for new mammalian meiotic factors that may impact fertility. In Paper I, we identified a role for the conserved Structural Maintenance of Chromosomes (SMC) 5/6 complex during meiotic recombination in budding yeast. smc5/6 complex mutants experienced a DSB-dependent segregation block, suggesting that the defect was caused by recombination. Consistent with this notion, Smc6-deficient cells accumulated high levels of recombination intermediates, particularly between sister chromatids, which is normally not seen in the wild type. Return-to-function studies indicated that the Smc5/6 complex was most crucial during resolution of recombination intermediates. These results suggest that the Smc5/6 complex works primarily in the resolution of recombination structures formed outside of homolog-directed pathways during meiosis. We characterized a role for DNA topoisomerases Top2 and Top3 during meiosis in S. cerevisiae by using meiosis-specific mutants in Paper II. Cells deficient for either Top2 or Top3 experienced a segregation block. While top3 cells were rescued completely by removing recombination, the top2 mutant was only partially rescued. This suggests that Top3 mainly functions during meiotic recombination. In contrast, the data indicates that Top2 has a role outside of recombination. In line with this idea, some of the segregation defects in cells lacking Top2 seemed to arise from break-independent sister entanglements. Since Top2 is known to be important in resolving sister chromatid intertwinings during mitosis to facilitate proper segregation, it is likely that it plays a similar role during meiosis. The CCCTC-binding factor (CCTF) is an architectural protein essential for proper genome structure and function in higher eukaryotes. In Paper III, we created a testes- specific ctcf mouse mutant strain (cctf-cKO) in order to study the function of CTCF during gamete (sperm in males) formation in mice. CTCF-deficient mice completed meiosis and sperm specialization without any major abnormalities, though mice were infertile and had low sperm counts. Sperm from the ctcf-cKO had chromatin compaction defects, most likely due to lack of sperm-specific compaction factors. These findings indicate that CTCF is essential for proper chromatin organization during spermiogenesis and suggest that infertility in ctcf-cKO mice was a result of the chromatin defects in the sperm. Using a method called phylogenetic profiling in Paper IV, we showed that new meiotic factors can be discovered by clustering proteins according to their function

Human disease locus discovery and mapping to molecular pathways through phylogenetic profiling

Genes with common profiles of the presence and absence in disparate genomes tend to function in the same pathway. By mapping all human genes into about 1000 clusters of genes with similar patterns of conservation across eukaryotic phylogeny, we determined that sets of genes associated with particular diseases have similar phylogenetic profiles. By focusing on those human phylogenetic gene clusters that significantly overlap some of the thousands of human gene sets defined by their coexpression or annotation to pathways or other molecular attributes, we reveal the evolutionary map that connects molecular pathways and human diseases. The other genes in the phylogenetic clusters enriched for particular known disease genes or molecular pathways identify candidate genes for roles in those same disorders and pathways. Focusing on proteins coevolved with the microphthalmia-associated transcription factor (MITF), we identified the Notch pathway suppressor of hairless (RBP-Jk/SuH) transcription factor, and showed that RBP-Jk functions as an MITF cofactor

Inhibition of the Smc5/6 Complex during Meiosis Perturbs Joint Molecule Formation and Resolution without Significantly Changing Crossover or Non-crossover Levels

<div><p>Meiosis is a specialized cell division used by diploid organisms to form haploid gametes for sexual reproduction. Central to this reductive division is repair of endogenous DNA double-strand breaks (DSBs) induced by the meiosis-specific enzyme Spo11. These DSBs are repaired in a process called homologous recombination using the sister chromatid or the homologous chromosome as a repair template, with the homolog being the preferred substrate during meiosis. Specific products of inter-homolog recombination, called crossovers, are essential for proper homolog segregation at the first meiotic nuclear division in budding yeast and mice. This study identifies an essential role for the conserved Structural Maintenance of Chromosomes (SMC) 5/6 protein complex during meiotic recombination in budding yeast. Meiosis-specific <i>smc5/6</i> mutants experience a block in DNA segregation without hindering meiotic progression. Establishment and removal of meiotic sister chromatid cohesin are independent of functional Smc6 protein. <i>smc6</i> mutants also have normal levels of DSB formation and repair. Eliminating DSBs rescues the segregation block in <i>smc5/6</i> mutants, suggesting that the complex has a function during meiotic recombination. Accordingly, <i>smc6</i> mutants accumulate high levels of recombination intermediates in the form of joint molecules. Many of these joint molecules are formed between sister chromatids, which is not normally observed in wild-type cells. The normal formation of crossovers in <i>smc6</i> mutants supports the notion that mainly inter-sister joint molecule resolution is impaired. In addition, return-to-function studies indicate that the Smc5/6 complex performs its most important functions during joint molecule resolution without influencing crossover formation. These results suggest that the Smc5/6 complex aids primarily in the resolution of joint molecules formed outside of canonical inter-homolog pathways.</p></div

The Smc6 protein localizes to meiotic chromosomes during prophase in a cohesin- dependent manner.

<p>Cells were isolated at the indicated time points and surface-spread to detect epitope-tagged proteins Smc6 (Smc6-13Myc, green) and Rec8 (Rec8-3HA, red) using standard immunofluorescence techniques. DNA visualized with DAPI shown in grey. (<b>A</b>) Wild type (CB1361). (<b>B</b>) <i>rec8Δ</i> (CB1411). (<b>C</b>) Enlarged image of a representative cell from the wild type (CB1361) at the 4 h time point. Solid arrows indicate an Smc6 site at a weaker-staining Rec8 site. Dashed arrows indicate a strong Smc6 site on top of a strong Rec8 site.</p

Smc5/6 mutations cause a recombination-dependent segregation block without affecting meiotic progression.

<p>(<b>A</b>) Set-up of the soft-shift method. Cells were shifted to non-permissive temperature (33°C) upon completion of pre-meiotic S phase as judged from FACS profiles shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#pgen.1003898.s002" target="_blank">Figure S2</a>. (<b>B</b>) Meiotic time courses for wild-type (CB1017), <i>smc6-56</i> (CB1032), <i>smc5-mn</i> (CB1872), <i>nse4-mn</i> (CB1511) and <i>nse2-mn</i> (CB2053) strains. At indicated times, cells were fixed and stained with DAPI to determine their nuclear content. Percent of MI+MII cells shown at left, percent of cells with one DNA mass outside four empty spores shown at right. Inset picture illustrates the “one DNA mass outside of four empty spores” phenotype, scale bar = 1 µm. Graphs represent a single synchronous meiotic time course. N = 200. (<b>C</b>) Meiotic spindle formation in wild type (CB1017) and <i>smc6-56</i> (CB1032). Fixed whole cells were stained with an anti-α-tubulin antibody (green) and DAPI (red). The images represent spindle morphology in wild type and <i>smc6-56</i> at prophase (pro), metaphase I (meta I), metaphase II (meta II), anaphase II (ana II) and after completing sporulation (spores). Meiotic progression demonstrated by plotting the fraction of cells with a single tubulin focus remaining at each time point on an inverted y-axis. N = 200. (<b>D</b>) Meiotic progression determined as percent of nuclei with full or partial Zip1 axes analyzed on meiotic spreads at indicated times. Picture demonstrates full Zip1 axes shown in green, scale bar = 2 µm. Dark blue line shows Zip1 axis formation when in the absence of <i>NDT80</i> function. N = 100. (<b>E</b>) <i>smc6-56</i> (CB1346) and wild-type (CB46) cells undergoing meiosis under soft-shift conditions were isolated and surface-spread to detect Zip1 (green) and epitope-tagged Rec8 (red). DNA was visualized with DAPI (gray). Scale bar = 1.8 µm. (<b>F</b>) Meiotic progression in <i>spo11Δ</i> (CB1302), <i>spo11Δ smc6-56</i> (CB1301), <i>spo11Δ sm5-mn</i> (CB1754), <i>spo11Δ nse4-mn</i> (CB1510) and <i>spo11Δ nse2-mn</i> (CB2067) shown in percent of MI+MII cells. N = 200.</p

The <i>smc6-56</i> mutant accumulates unresolved joint molecules but forms normal levels of recombination products.

<p>Analysis of recombination measured at the ectopic <i>URA3-ARG4</i> interval on chromosome III (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#pgen.1003898.s005" target="_blank">Figure S5</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#pgen.1003898-Sourirajan1" target="_blank">[84]</a> in strains containing the estradiol-inducible <i>NDT80</i> allele (<i>NDT80-IN</i>) under soft-shift conditions. <i>NDT80-IN</i> was induced at 7 h with 1 µM β-estradiol (+ED, arrow). Blue curves indicate <i>NDT80-IN SMC6</i> (CB2096); red <i>NDT80-IN smc6-56</i> (CB2097). (<b>A</b>) Representative Southern blot used to detect JMs after digesting with <i>XmnI</i> and probing for a region in <i>ARG4</i>. P1, P2 and JM regions based on expected sizes. (<b>B</b>) Representative Southern blot used to detect CO/NCO products at the same interval after digesting with <i>XhoI/EcoRI</i> and probing for <i>HIS4</i> sequences. P2, CO and NCO regions based on expected sizes. (<b>C</b>) Quantifications of total JM levels, NCOs and COs in percent of total DNA from blots illustrated in (A) and (B). Dotted line and arrow indicate time of <i>NDT80-IN</i> induction. Plots represent mean ± standard deviation from three independent experiments. (<b>D</b>) Two-dimensional analysis of JM species. DNA from <i>NDT80-IN SMC6</i> and <i>NDT80-IN smc6-56</i> undergoing meiosis under soft-shift conditions was isolated and subjected to two-dimensional electrophoresis as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#s5" target="_blank">Materials and Methods</a>. The interpretive panel shows the assumed identity of JM species after probing for a region which recognizes both homologs. JMs between P1×P1 are the result of random breaks and are therefore expected to give a broader signal than P2×P2 and P2×P1. Dashed line, P2×P2 IS-JM; solid line, P2×P1 IH-JM; dotted line, P1×P1 IS-JM. The enlarged panels are enhanced images of the JM region from the designated time point. Ratio of IH-JMs/ΣJMs and IS-JMs/ΣJMs given below relevant images and were calculated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#pgen.1003898.s009" target="_blank">Figure S9</a>. (<b>E</b>) Quantification of total JMs in percent of total DNA from the gels represented in (D). Arrow denotes <i>NDT80</i> induction. Curves represent one experiment, with similar results from a second independent experiment presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#pgen.1003898.s007" target="_blank">Figure S7A</a>.</p

Model for the role of the Smc5/6 complex during meiosis.

<p>Schematic diagram depicting the result of having non-functional Smc6 protein before and/or after <i>NDT80</i> induction as indicated (red = non-permissive/non-functional, green = permissive/functional) and described in the text.</p

The Smc5/6 complex performs its most critical functions at the time of joint molecule resolution.

<p>Meiotic progression and cell viability were determined following temperature shifts in strains carrying an inducible <i>NDT80</i> allele (<i>NDT80-IN</i>) under the control of estradiol (ED). Meiotic progression given as percent of MI+MII cells in <i>NDT80-IN SMC6</i> (CB1753) and <i>NDT80-IN smc6-56</i> (CB1723) at the indicated time points. N = 200. For viability assessment, cells were collected at the indicated time points, sonicated briefly, diluted to the desired concentration, spread onto YPD plates and grown at permissive temperature for 3 days. Viability is given in percent as determined by the number of colony-forming units divided by the total number of cells plated. (<b>A</b>) Cells were accumulated in an <i>ndt80</i> arrest at permissive temperature until 7 h when 1 µM β-estradiol (+ED, arrow) was added and cultures were shifted to non-permissive temperature. (<b>B</b>) Cell viability for cells undergoing meiosis under the same conditions described for (A) at 0 h and 24 h after meiotic induction. (<b>C</b>) Cells were accumulated in <i>ndt80</i> arrest under soft-shift conditions at non-permissive temperature until 7 h when 1 µM β-estradiol (+ED, arrow) was added and cultures were shifted to permissive temperature. (<b>D</b>) Cell viability for cells undergoing meiosis under the same conditions described for (C) at 0 h and 24 h after meiotic induction.</p

Sister chromatid cohesion, Rec8 dynamics and DSB repair are largely unchanged in <i>smc6-56</i> mutants.

<p>(<b>A</b>) Sister chromatid separation of chromosome V. Percent of sister chromatid separation 35 Kb away from the centromere (CenV) in wild type (CB1197) and <i>smc6-56</i> (CB1248) is shown in solid lines. Percent separation 50 Kb away from the right telomere (TelV) in wild type (CB1427) and <i>smc6-56</i> (CB1426) indicated in dotted lines. N = 200. (<b>B</b>) Percent of cells with full Rec8 protein axes in wild type (CB46) and <i>smc6-56</i> (CB1346). Rec8 axes were assessed by detecting epitope-tagged Rec8 (Rec8-3HA) on chromosome spreads using standard immunofluorescence techniques, picture illustrates a cell with full Rec8 axes shown in red, scale bar indicates 2 µm. N = 100. (<b>C</b>) DSB repair at the <i>HIS4LEU2</i> hotspot on chromosome III in wild type (CB1183) and <i>smc6-56</i> (CB1303). The curves represent mean break levels at the two DSB sites at the indicated time points. The Southern blot shown is representative for the three used for quantifications, DSB species were identified according to their size. (<b>D</b>) Cumulative DSB levels at the <i>HIS4LEU2</i> hotspot in <i>rad50S</i> (CB2059) and <i>rad50S smc6-56</i> (CB2060). The Southern blot shown is representative for those used to quantify DSBs in the <i>rad50S</i> background. Plots in (C) and (D) represent mean ± standard deviation from three independent experiments. All experiments were run under soft-shift conditions.</p