Regulation and recognition of H3K79me

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Project Summary/Abstract Patterns of dynamic post-translational modifications on histone proteins of nucleosomes regulate local and global eukaryotic gene expression by recruiting specialized effector proteins. Mono-, di-, and tri- methylation of histone H3 lysine 79 (H3K79me1/2/3) is a hallmark of actively transcribed genes and is central to many fundamental biological processes. Importantly, aberrant hypermethylation of H3K79 is found in the Mixed Lineage Leukemia 1-rearranged (MLL-r) leukemias which are resistant to treatment. New potent treatments for these leukemias are desperately needed, but the underlying mechanisms of H3K79me regulation and recognition are poorly understood. Dot1L is the sole methyltransferase responsible for establishing H3K79me. It is stimulated by mono-ubiquitylation of histone H2B (H2Bub). In cells, Dot1L is found in complex with partner proteins AF9 and AF10, which are known to upregulate H3K79me levels. However, very little is known about their effects on Dot1L binding affinity and activity on H2Bub-modified nucleosomes. In Aim 1, I will compare the binding affinity and activity of Dot1L + AF9 and Dot1L + AF10 on nucleosomes containing H2Bub and H3K79me1/2/3 modifications using biochemical techniques. To guide future structure-based leukemia therapies, I will also determine the cryo-EM structure of Dot1L + AF9 + AF10 on H2Bub nucleosome. H3K79me2/3 is known to regulate fundamental biological processes, yet no H3K79me effector proteins have been identified. H3K79 is located within the globular core of the nucleosome and may require the use of nucleosomal substrates as bait in pulldown experiments to identify H3K79me binding proteins. However, no pulldown studies have used H3K79me-modified nucleosomes to identify binding proteins. In Aim 2, I will identify H3K79me binding proteins using H3K79me2/3-modified nucleosomes as bait in protein pulldowns from mammalian nuclear extract. I will identify potential hits using mass-spectrometry based proteomics and validate them for specific H3K79me binding using a biochemical technique. Finally, I will study the mechanism of H3K79me recognition by determining a cryo-EM structure of a high-affinity H3K79me binding protein on nucleosome. The outcomes from these proposed experiments in Aims 1 and 2 will form a basis for guiding the design of drug-based leukemia therapies targeting Dot1L partner proteins and will reveal how H3K79 methyl marks are recognized by effector proteins to modulate gene expression.