Methyltransferase activities of recombination initiator PRDM9: separating H3K4me3 and H3K36me3 with single amino acid mutations.

Max Coffey

Document Type

Article

Publication Date

Summer 2016

JAX Location

In: Student Reports, Summer 2016, Jackson Laboratory

Abstract

The zinc finger histone methyltransferase PRDM9 trimethylates histone H3 at Lysine 4 (H3K4) and Lysine 36 (H3K36), establishing hotspots –--1-2kb regions that host genetic recombination. While genetic recombination in mammals is known to depend on these epigenetic marks of PRDM9, the separate function of each PRDM9-induced trimethylation is unknown. We mutated the catalytic SET domain of full-length PRDM9 in an attempt to achieve trimethylation activity at H3K4 but not at H3K36. Previous experiments in the Paigen-Petkov Lab have demonstrated two such separation of function mutations in the isolated SET domain of PRDM9 in vitro. However, when induced in full-length PRDM9 in a mouse model, these mutations demonstrate a reduction of both H3K4me3 and H3K36me3 activity, with resulting sterility in homozygous mutants. This project explored these discrepant results by inducing the same single amino acid substitutions in full-length PRDM9, and testing the activity of the mutant protein in vitro. Both mutations in full-length PRDM9 showed markedly diminished H3K4me3 and no detectable H3K36me3---consistent with the in vivo results. We further explored the capability of single amino acid substitutions to separate catalytic functions in full-length PRDM9, by inducing a mutation present in a related protein, PRDM7. This PRDM7-mutation n the isolated PRDM9 set domain has demonstrated separation of H3K4me3 and H3K36me3 in vitro. The findings of this study demonstrate the importance of PRDM9’s non-catalytic domains and/or sensitive 3-dimensional structure to the maintenance of catalytic activity.

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