The goal of this project is to determine the biochemical mechanisms by which the essential histone-modifying PRMT5-MEP50 complex (protein arginine methyltransferase 5 - methylosome protein 50) guides early development. Arginine methylation of histones is carried out by a family of protein arginine methyltransferases (PRMTs) that have diverse roles. The primary focus of this proposal is PRMT5, the major Type II enzyme that catalyzes arginine mono- and symmetric-dimethylation (Rme1 and Rme2s). PRMT5-catalyzed histone H2A and H4 R3me2s is correlated with gene repression. PRMT5 is always found in complex with the WD-repeat protein MEP50 (also known as Wdr77 or androgen coreceptor p44). We recently solved the crystal structure of the Xenopus laevis PRMT5-MEP50 complex that provides insight into how MEP50 is required for PRMT5 histone methylation and showed that PRMT5-MEP50 is responsible for the abundant histone H2A and H4 R3 methylation in Xenopus eggs. Both PRMT5 and MEP50 are embryonic lethal when their expression is knocked out in mice or in frogs. We hypothesize that MEP50 targets egg and embryo chaperone-bound histones H2A and H4 for methylation by its partner PRMT5 to regulate early development.
Our specific aims are:
Aim 1) Test the hypothesis that MEP50 recognizes substrate for PRMT5. We will determine affinities for substrate by both MEP50 and PRMT5 using quantitative binding studies, perform co-crystallography with substrate peptides to identify domains of interaction, and assess the contribution of MEP50 substrate binding to PRMT5 catalysis using kinetic methyltransferase assays.
Aim 2) Test the hypothesis that PRMT5-MEP50 methylation of the maternal store of pre-deposition histones is regulated by histone chaperones and post-translational modifications. For this aim we will determine how histone chaperones and maternal histone acetylation and phosphorylation modulate PRMT5-MEP50's activity towards H2A and H4.
Aim 3) Test the hypothesis that PRMT5-MEP50 methylates histones H2A and H4 to regulate Xenopus early embryonic development. For this aim we will test the functional role of the maternal methylation and new zygotic methylation of histones by PRMT5-MEP50 in Xenopus laevis embryos. We will mimic the loss of maternally methylated histones through the overexpression of H2A and/or H4 R3Q mutants and probe the loss of zygotic PRMT5 and MEP50 by injection of antisense morpholino oligonucleotides. We will follow these treated embryos via phenotypic analysis, probe the coexistence of activation and repressive histone modifications with R3 methylation, and perform gene expression studies to probe the biological function of H2A and H4 R3 methylation. Our studies probe the biochemical mechanism of a major regulator of early embryonic development in a unique living vertebrate model organism. We have documented ability to effectively combine biochemical and biological studies that will allow us to dissect the function of this essential and highly conserve histone modifying complex. Our findings will inform future studies in both normal and cancerous mammalian cells.
Epigenetics is a phenomenon important for an overall increase in the complexity of the genome without changes in gene sequence. It has clinical significance for cancer diagnosis and therapies and other pathologies, and is explicitly connected with the process of pattern formation crucial for proper development of metazoans. Understanding the molecular phenomena underlying the establishment of the embryonic epigenetic code will promote greater understanding of pluripotency, stem cells, and development. Furthermore, the specific factors we target in this proposal, PRMT5 and MEP50, are misregulated in human diseases, including cancer. The information we develop from this study will potentially lead to improved diagnosis and treatment.
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