Title: Elucidating the Biological Function of PWWP-Domain Containing Protein Complexes
Description: In eukaryotes, nuclear DNA is folded with histone proteins in the form of chromatin, and this structure plays a critical role in multiple biological processes, including development, DNA damage repair, and aging. Post-translational modifications of histones, such as acetylation and methylation, are essential regulators of chromatin structure and function. Consequently, misregulation of these post-translational modifications has causal roles in numerous diseases, including multiple types of cancer. However, the mechanisms that direct the localization of histone-modifying enzymes and regulate their activities are not fully understood. This thesis focuses on the characterization of a class of proteins containing the PWWP domain. This domain is often present in chromatin proteins, and it is predicted to recognize methylated histones based on structural analysis. Here, we have demonstrated that the PWWP domain proteins in fission yeast bind to methylated histones. Additionally, we have shown that proteins with this domain form complexes with diverse histone modifying activities to regulate multiple cellular processes.

Methylation of histone H4 lysine 20 (H4K20me) is essential for the activation of a DNA damage checkpoint, which blocks the progression of cell cycle to allow sufficient time for DNA damage repair. In fission yeast, only the enzyme Set9 catalyzes H4K20me, and the mechanisms that underlie the regulation of this protein are poorly characterized. Here we showed that Set9 forms a stable complex with the PWWP domain containing protein Pdp1. The PWWP domain of Pdp1 binds to H4K20me, demonstrating that the PWWP domain constitutes a novel methyl-lysine recognition motif. Moreover, the binding of PWWP domain to methylated H4K20 plays a critical role in regulating Set9 activity, thus facilitating higher degrees of H4K20 methylation. Histone H3K9 methylation is critical for heterochromatin assembly in diverse organisms. The RNAi pathway is required for the formation of pericentric heterochromatin, although the exact role that RNAi plays in heterochromatin assembly remains a topic of significant debate.

We discovered that a separate PWWP domain protein, Pdp3, forms a stable complex with the H3K14 histone acetyltransferase Mst2. Interestingly eliminating the enzymatic activity of the Pdp3-Mst2 complex obviates the requirement for the RNAi machinery in pericentric heterochromatin functions. Furthermore we demonstrated that one function of RNAi during heterochromatin assembly is to exclude the Pdp3-Mst2 complex, thus maintaining low levels of RNA polymerase II localization to pericentric regions in order to retain the parental histone modification patterns for its passage through generations. Altogether, my results have firmly demonstrated that the PWWP domain is a novel class of methyl-lysine binding motifs. Moreover, in fission yeast the PWWP domain proteins form stable complexes with other chromatin proteins to regulate diverse cellular processes.