In mammals, meiotic recombination occurs at 1- to 2-kb genomic regions termed 
hotspots, whose positions and activities are determined by PRDM9, a DNA-binding 
histone methyltransferase. We show that the KRAB domain of PRDM9 forms complexes 
with additional proteins to allow hotspots to proceed into the next phase of 
recombination. By a combination of yeast-two hybrid assay, in vitro binding, and 
coimmunoprecipitation from mouse spermatocytes, we identified four proteins that 
directly interact with PRDM9's KRAB domain, namely CXXC1, EWSR1, EHMT2, and 
CDYL. These proteins are coexpressed in spermatocytes at the early stages of 
meiotic prophase I, the limited period when PRDM9 is expressed. We also detected 
association of PRDM9-bound complexes with the meiotic cohesin REC8 and the 
synaptonemal complex proteins SYCP3 and SYCP1. Our results suggest a model in 
which PRDM9-bound hotspot DNA is brought to the chromosomal axis by the action 
of these proteins, ensuring the proper chromatin and spatial environment for 
subsequent recombination events.