High-resolution, genome-wide exploration of the DSB landscape
Upper: the Spo11-oligo map agrees with ssDNA microarray analysis (Buhler et al., PLoS Biology, 2007) but is higher resolution. Lower: Agreement of the Spo11-oligo map with direct DSB detection in sae2∆ genomic DNA (spo11yf = DSB deficient mutant; P=parental re-striction frag.; *=cross hyb.). Reproduced from Pan et al. Cell, 2011.
DSBs are more likely to occur in some genomic regions than others. To understand this non-random distribution, we are taking advantage of the Spo11-oligonucleotide complexes formed as a byproduct of DSB formation and processing. Each Spo11 oligo is a tag that records precisely where a break was made. We devised methods to purify, amplify, and deep-sequence the oligos, allowing us to quantitatively map DSBs across the genome at nucleotide resolution with high sensitivity (Pan et al., 2011).
Using this map, we demonstrated whole-chromosome variation in DSB frequency correlated with size; delineated large domains of elevated or reduced DSBs (subtelomeric domains, pericentromeric regions, and alternating high- and low-DSB interstitial regions); examined the influence of nucleosomes, sequence-specific DNA-binding proteins, and local DNA composition; and found evidence for asymmetry in nucleolytic Spo11-oligo release. An overarching theme is the view that the DSB landscape is shaped by combinatorial action of many factors. These factors operate hierarchically over many size scales, with the general trend that level in hierarchy is proportional to scale. Thus, for example, two DNA segments that are equally free of nucleosomes may have substantially different DSB probability depending on whether they lie in hotter or colder domains.
In current work, we are exploiting Spo11-oligo mapping in various S. cerevisiae mutants to understand the factors that influence DSB distributions, and in various Saccharomyces species to explore the evolutionary dynamics of the DSB landscape. We are also extending Spo11-oligo mapping to S. pombe, mouse, and other organisms.
Spore-autonomous fluorescent protein reporters
Spore autonomous fluorescent protein constructs
Composite image of S. cerevisiae tetrads carrying red, cyan, and green fluorescent reporters under the control of promoters with spore autonomous expression activity (i.e., that are principally expressed after prospore membrane formation). Only the spore that inherits one of the constructs becomes fluorescent. Distinct segregation patterns arise from different recombinant configurations of the reporters. Nonfluorescent spores are outlined by dashes. Cover image, Genetics, 2011. © 2011 Genetics Society of America. See also Thacker, D., Lam, I., Knop, M., Keeney, S. 2011. Genetics 189: 423-439.
Studies of meiotic recombination often rely on microdissecting the four products of a single meiosis and analyzing the configuration of genetic markers in viable spore clones. This type of analysis is powerful but laborious, and reliance on viable spores may introduce selection bias. To overcome these limitations, we developed new markers whose segregation can be scored without ascus dissection, even in inviable spores. Using previously described promoters that can drive fluorescent protein expression specifically in only those spores that inherit the reporter, we developed a versatile, portable set of “spore-autonomous” reporters that can be integrated essentially anywhere in the genome. Proof-of-principle experiments showed that different configurations can be used to quantify crossover frequency, crossover interference, gene conversion, crossover/noncrossover ratios, and chromosome missegregation.
Crossover formation is controlled such that each chromosome gets at least one crossover despite a low average number of crossovers per chromosome, and multiple crossovers on the same chromosome tend to be evenly and widely spaced. More DSBs are formed than crossovers and DSBs tend to be randomly placed relative to one another, so crossover control involves a decision by which a subset of DSBs enters a pathway that culminates in crossover formation, while all other DSBs follow a pathway(s) that generates primarily noncrossover products. To understand the logic of this decision, we examined recombination when breaks are reduced through the use of a series of yeast partial loss-of-function spo11 mutants (Martini et al., 2006). We found that crossovers tend to be maintained at the expense of noncrossovers, a phenomenon we refer to as “crossover homeostasis.” These findings defined a previously unsuspected manifestation of crossover control, i.e., that the crossover/noncrossover ratio can change to maintain crossovers. Our results support the hypothesis that an obligate crossover is a genetically programmed event tied to crossover interference.