Mechanistic studies of epigenetic regulation by the H3 tail

Abstract

The N-terminal tail of histone H3 is heavily post-translationally modified. Numerous regulatory lysine residues are methylated, and these modifications play critical roles in the regulation of gene expression. Trimethylation at H3K27 (H3K27me3) is associated with transcriptional silencing of genes important in growth and development. Intriguingly, H3K27me3 is mutually exclusive with H3K36 tri-methylation on the same histone tail. Disruptions in this crosstalk result in aberrant H3K27/H3K36 methylation patterns and altered transcriptional profiles that have been implicated in tumorigenesis and other disease states. Despite their importance, the molecular details of this crosstalk are unclear. This thesis presents work revealing an unusual mode of allosteric enzyme modulation by its histone H3 substrate molecule. PRC2-mediated H3K27 methylation is sensitive to the chromatin environment, including the methylation state of H3K36 on the same histone tail.

We demonstrate that PRC2 is allosterically activated in cis by the unmodified side chain of H3K36, and that this activation results in a 5-fold increase in the kcat of its enzymatic activity catalyzing H3K27 methylation compared to activity on a substrate methylated at H3K36. Utilizing a photocrosslinking-MS strategy and histone methyltransferase activity assays with PRC2 mutants, we find that the catalytic subunit EZH2 contains a specific sensing pocket for H3K36 methylation state which allows the PRC2 complex to distinguish between modified and unmodified H3K36 residues, altering enzymatic activity accordingly to preferentially methylate the unmodified nucleosome substrate. Avenues for inhibition of PRC2 by targeting its interactions with the H3 tail are also explored. Beyond its interactions with polycomb machinery, H3K36 and other sections of the H3 tail may also have roles of epigenetic regulation of other cellular processes such as DNA damage response.