Molecular Basis of Histone Methylation by PRMT5

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Project Summary/Abstract The dynamic writing and erasing of histone post-translational modifications on nucleosomes regulate eukaryotic gene expression by tuning chromatin organization and recruiting chromatin-binding proteins. The methylation of arginines can activate or repress transcription depending on the histone residue and its methylation state. Protein arginine methyltransferase 5 (PRMT5), along with its obligate binding partner Methylosome protein 50 (MEP50), is the primary complex for the symmetric dimethylation of arginine across all eukaryotes. In addition, PRMT5- MEP50 can either activate or repress the transcription of several genes, depending on which residue the enzyme modifies. PRMT5-MEP50 catalyzes methylation on four histone residues, namely histone H2A-Arg3 (H2AR3), H3-Arg2 (H3R2), H3-Arg8 (H3R8), and H4-Arg3 (H4R3). Due to PRMT5-MEP50’s diverse roles in transcription regulation, PRMT5 is overexpressed in several cancers as it regulates the transcription of several metastasis suppressor genes and epithelial-mesenchymal transition activating genes. Despite PRMT5-MEP50’s importance in gene expression, very little is known of how PRMT5-MEP50 methylates histone and/or nucleosome substrates. However, recent work has revealed that PRMT5’s specificity is regulated by (1) recognition of cytosolic H2A-H2B dimers to methylate H2AR3 and (2) being able to preferentially methylate histone H4 in the presence of substrate adaptor Coordinator of PRMT5 (COPR5). Despite these findings, molecular determinants towards this specificity are still unknown. Using a combination of biochemical and structural approaches, I will investigate the mechanism of histone specificity and activity by the PRMT5-MEP50 complex. In Aim 1, I will determine contributions towards PRMT5-MEP50’s recognition of H2A-H2B dimers by quantifying the activity and binding of PRMT5-MEP50 on various histone H2A-containing substrates. To provide molecular detail of this recognition, I will determine the structure of PRMT5-MEP50 bound to H2A-H2B dimers using cryo- electron microscopy (cryo-EM). While screening substrates of H2A methylation, I discovered that PRMT5- MEP50 activity is stimulated by ubiquitination of histone H2BK120 (H2BK120-Ub). I will probe in vivo relevance of this crosstalk by siRNA knockdowns. I will then reveal the mechanism of this activation by quantifying activity and binding of PRMT5-MEP50 in the presence of H2BK120-Ub and resolving the EM structure of PRMT5- MEP50 bound to H2A-H2BK120-Ub dimers. In Aim 2, I will elucidate the function of COPR5 and the PRMT5- MEP50-COPR5 complex. My preliminary data revealed that COPR5 does not bind to nucleosomes and cannot recruit PRMT5-MEP50 to the nucleosome, conflicting previous speculations of COPR5’s function. Therefore, I will identify COPR5’s preferred histone-containing substrate and quantify COPR5’s binding and contribution to the enzymatic activity of PRMT5-MEP50. Finally, I will solve the structure of PRMT5-MEP50-COPR5 bound to its histone substrate by cryo-EM. Together, this proposal will construct a molecular framework of PRMT5- MEP50’s substrate specificity to aid in structure-based drug design, by revealing substrate-specific interactions.