Meiotic recombination generates new genetic variation and assures the proper segregation of chromosomes in gametes. PRDM9, a zinc finger protein with histone methyltransferase activity, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we measured activity of the Prdm9 Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst was introduced experimentally. Comparing these two strains, we find that haplotype differences at hotspots lead to qualitative and quantitative changes in PRDM9 binding and activity. Using Mus spretus as an outlier, we found most variants affecting PRDM9Cst binding arose and were fixed in M.m. castaneus, suppressing hotspot activity. Furthermore, M.m. castaneus×M.m. domesticus F1 hybrids exhibit novel hotspots, with large haplotype biases in both PRDM9 binding and chromatin modification. These novel hotspots represent sites of historic evolutionary erosion that become activated in hybrids due to crosstalk between one parent's Prdm9 allele and the opposite parent's chromosome. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion.

Meiotic recombination hotspots activated by PRDM9 are associated with the chromosomal axis and synaptonemal complex via their interaction with other proteins, including CDYL, EHMT2, EWSR1, and CXXC1.

BackgroundGenetic recombination plays an important role in evolution, facilitating the creation of new, favorable combinations of alleles and the removal of deleterious mutations by unlinking them from surrounding sequences. In most mammals, the placement of genetic crossovers is determined by the binding of PRDM9, a highly polymorphic protein with a long zinc finger array, to its cognate binding sites. It is one of over 800 genes encoding proteins with zinc finger domains in the human genome.ResultsWe report a novel technique, Affinity-seq, that for the first time identifies both the genome-wide binding sites of DNA-binding proteins and quantitates their relative affinities. We have applied this in vitro technique to PRDM9, the zinc-finger protein that activates genetic recombination, obtaining new information on the regulation of hotspots, whose locations and activities determine the recombination landscape. We identified 31,770 binding sites in the mouse genome for the PRDM9Dom2 variant. Comparing these results with hotspot usage in vivo, we find that less than half of potential PRDM9 binding sites are utilized in vivo. We show that hotspot usage is increased in actively transcribed genes and decreased in genomic regions containing H3K9me2/3 histone marks or bound to the nuclear lamina.ConclusionsThese results show that a major factor determining whether a binding site will become an active hotspot and what its activity will be are constraints imposed by prior chromatin modifications on the ability of PRDM9 to bind to DNA in vivo. These constraints lead to the presence of long genomic regions depleted of recombination.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0024-6) contains supplementary material, which is available to authorized users.

Developmental progress of germ cells through meiotic phases is closely tied to ongoing meiotic recombination. In mammals, recombination preferentially occurs in genomic regions known as hotspots; the protein that activates these hotspots is PRDM9, containing a genetically variable zinc-finger domain and a PR-SET domain with histone H3K4 trimethyltransferase activity. PRDM9 is required for fertility in mice, but little is known about its localization and developmental dynamics. Application of spermatogenic stage-specific markers demonstrates that PRDM9 accumulates in male germ-cell nuclei at pre-leptonema to early leptonema, but is no longer detectable in nuclei by late zygonema. By the pachytene stage, PRDM9-dependent histone H3K4 trimethyl marks on hotspots also disappear. PRDM9 localizes to nuclei concurrently with the deposition of meiotic cohesin complexes, but is not required for incorporation of cohesin complex proteins into chromosomal axial elements, or accumulation of normal numbers of RAD51 foci on meiotic chromatin by late zygonema. Germ cells lacking PRDM9 exhibit inefficient homology recognition and synapsis, with aberrant repair of meiotic DNA double-strand breaks and transcriptional abnormalities characteristic of meiotic silencing of unsynapsed chromatin. Together, these results on the developmental time course for nuclear localization of PRDM9 establish its direct window of function, and demonstrate the independence of chromosome axial element formation from the concurrent PRDM9-mediated activation of recombination hotspots.

In a previous study we demonstrated that Escherichia coli thymidylate synthase activity could be restored completely by incubating basically inactive mutants of this enzyme at room temperature with R(126)E, another inactive mutant [Maley, F., Pedersen-Lane, J., and Changchien, L.-M. (1995) Biochemistry 34, 1469-1474]. Since only one of the enzyme's two subunits possessed a functional active site and the restoration of activity could be titrated to be equivalent to that of the wild-type enzyme's specific activity, it was proposed that thymidylate synthase was a half-of-the-sites activity enzyme. We now provide additional support for this thesis by presenting an in-depth analysis of some conditions affecting the restoration of enzyme activity. For this purpose, we employed two mutants with marginal thymidylate synthase activity, Y(94)A and R(126)E. The parameters that were examined included pH, concentration of protein, temperature, and urea concentration, all of which influenced the rate of activity restoration. It was found, surprisingly, that by maintaining the amount of each protein constant, while increasing the volume of solution, the rate and total activity restored was greatly enhanced. Increasing the pH from 6.0 to 9.0 markedly increased the rate at which the optimal activity was restored, as did increasing the temperature from 4 to 40 degrees C. A similar effect was obtained when the incubation of the mutants was conducted at 4 degrees C in the presence of 1.5 M urea, a temperature at which activity is restored extremely slowly. Raising the pH to 9.0 resulted in an almost instantaneous restoration of activity at 4 degrees C. The manner in which thymidylate synthase activity is restored from the mutants in the presence of varying concentrations of ethanol, ethylene glycol, and glycerol suggests that changes in subunit interaction and enzyme conformation are in part responsible for the observed differences. Most significantly, at solution levels of 10%, ethanol was found to activate, while ethylene glycol inhibited slightly and glycerol was somewhat more inhibitory. At a concentration of 20%, ethanol inhibited rather strikingly, ethylene glycol was slightly more inhibitory than at 10%, and glycerol was strongly inhibitory. Since the net result of these findings is the suggestion that the restoration of thymidylate synthase activity is due to a separation of the mutant dimers into their respective subunits, followed by their recombination to an active heterodimer, evidence for this phenomenon was sought by separating the recombined dimers using nondenaturating polyacrylamide gel electrophoresis. Sequence analysis of the isolated homo- and heterodimers clearly demonstrated that the active enzyme is a product of subunit exchange, one that is very efficient relative to the wild-type enzyme, which did not exchange subunits unless denatured.