BACKGROUND: Genetic 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.
RESULTS: We 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 PRDM9(Dom2) 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.
CONCLUSIONS: These 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.