In most sexually reproducing organisms, homologous recombination is a prominent feature of meiosis, which creates the genetic diversity of the meiotic products by promoting a safe exchange of DNA information between the paternal and maternal chromosomes. As importantly, the crossovers maintain a physical connection between the homologs which ensures their proper disjunction at the first meiotic division (55
). Therefore, understanding how the cells control the process of meiotic recombination is important because defects in the number or the localization of recombination events lead to failure in homolog disjunction or unviable gametes. For instance, errors in chromosome segregation are correlated with unusual crossover positions in many cases of human maternally derived trisomy 21 (27
). In particular, segregation defects are observed when a crossover occurs close to a telomere, probably because the structure is less efficient at providing a strong physical link between homologs (36
). Also, crossovers very close to the centromere lead to the premature loss of sister chromatid cohesion and nondisjunction at meiosis II (26
). Not surprisingly, then, the meiotic recombination process is tightly controlled from initiation to completion stages.
Once recombination is initiated, a decision is made to channel the early intermediates along a pathway ending in crossover formation or in noncrossover events. This step is influenced both by “crossover interference,” in which a crossover in one region makes it unlikely that another will occur nearby (18
), and by “crossover homeostasis,” which regulates the crossover/noncrossover ratio to ensure a minimal amount of crossover per chromosome (34
Before this stage, the frequency and the localization of the initiation events are the earliest determinants of how meiotic recombination events are distributed along the chromosomes. Extensive studies performed with Saccharomyces cerevisiae
have shown the heterogeneous distribution of the initiating DNA double-strand breaks (DSBs) along the chromosomes and the >100-fold variation in cleavage frequency from site to site (39
). These findings are consistent with the nonrandom distribution of recombination along the chromosomes observed in all organisms. The factors that determine whether a specific region is prone to DSB formation are not well understood at the molecular level. The primary sequence is not the main determinant of DSB formation since a reporter construct shows various DSB frequencies and recombination rates depending on where it is inserted into the genome (10
). Several rules regarding the distribution of DSBs have nevertheless been described. First, DSBs always occur in open chromatin regions, mainly in promoter-containing regions (4
). Second, DSBs are formed preferentially in the chromatin loops, as opposed to the loop basis in which cohesins are located (7
). Third, DSB frequencies are generally low in a 20-kb region around centromeres (9
) and the centromere itself has a strong inhibitory effect on meiotic recombination initiation (30
). Fourth, the rate of meiotic recombination is also usually low close to the natural chromosome ends (3
) and DSB frequency is very low up to 40 kb from a telomere (9
). This effect may be exaggerated by the fact that these DSB measurements were made with rad50S
Δ mutants, which accumulate unresected DSBs and in which DSB formation is specifically reduced in late-replicated regions (8
). Finally, another factor influencing DSB formation at one site is the proximity to another DSB site (11
), although this factor may not be a general rule (15
The catalytic activity for meiotic DSB formation is carried by the widely conserved Spo11 protein (5
). Besides Spo11, at least nine additional DSB proteins are absolutely required for DSB formation (reviewed in reference 24
). Recent cytological and chromatin immunoprecipitation (ChIP) analyses have begun to dissect the chromatin association of these proteins on meiotic nucleus spreads and their requirement for Spo11 association with DSB hot-spot regions (24
). However, how DSB sites are selected and how Spo11 is recruited to the chromatin of the DSB region remain to be understood.
To address these issues, we previously reported that a fusion between the Gal4 DNA binding domain and Spo11 (Gal4BD-Spo11) yields a protein that is able to introduce DSBs and stimulate meiotic recombination in the naturally cold GAL2
promoter, which contains Gal4 consensus binding sequences (CGGN11
CCG, where N11
represents 11 various nucleotides) (38
). Thus, the normal recruitment of Spo11 to chromatin can be bypassed by the Gal4BD moiety of the fusion protein. However, all the DSB proteins are still required for Gal4BD-Spo11 DSB formation at GAL2
, showing that they are all important for Spo11's ability to create a DSB and not only for the recruitment of Spo11 to the DSB site. Importantly, it was shown that Gal4BD-Spo11 also cleaves the three natural Spo11 DSB sites examined.
Here, to determine to what extent the genome-wide Gal4BD-Spo11 DSB profile was modified and to investigate the mechanisms that regulate meiotic DSB distribution, we have further exploited the in vivo properties of the Gal4BD-Spo11 protein to probe the entire genome. We show that DSB distribution is profoundly remodeled upon the introduction of strong new Gal4BD-Spo11 DSBs and that DSB cleavage at a subset of Gal4BD-Spo11-bound sites remains subject to repressing position effects. Globally, the modification of Spo11 cleavage sites leads to a genome-wide redistribution of meiotic double-strand breaks without decreasing the meiotic product viability.