Differential effects of two catalytic mutations on full-length PRDM9 and its isolated PR/SET domain reveal a case of pseudo-modularity

PRDM9 is a DNA-binding histone methyltransferase that designates and activates recombination hotspots in mammals by locally trimethylating lysines 4 and 36 of histone H3. In mice, we recently reported two independently produced point mutations at the same residue, glu360pro ( Prdm9 EP ) and glu360lys ( Prdm9 EK ), which severely reduce its H3K4 and H3K36 methyltransferase activities in vivo. Prdm9 EP is slightly less hypomorphic than Prdm9 EK , but both mutations reduce both the number and amplitude of PRDM9-dependent H3K4me3 and H3K36me3 peaks in spermatocytes. While both mutations cause infertility with complete meiotic arrest in males, Prdm9 EP , but not Prdm9 EK , is compatible with some female fertility. When we tested the effects of these mutations in vitro , both Prdm9 EP and Prdm9 EK abolished H3K4 and H3K36 methyltransferase activity in full-length PRDM9. However, in the isolated PRDM9 PR/SET domain, these mutations selectively compromised H3K36 methyltransferase activity, while leaving H3K4 methyltransferase activity intact. The difference in these effects on the PR/SET domain versus the full-length protein show that PRDM9 is not an intrinsically modular enzyme; its catalytic domain is influenced by its tertiary structure and possibly by its interactions with DNA and other proteins in vivo . These two informative mutations illuminate the enzymatic chemistry of PRDM9, and potentially of PR/SET domains in general, reveal the minimal threshold of PRDM9-dependent catalytic activity for female fertility, and potentially have some practical utility for genetic mapping and genomics.