Regulation of V(D)J Recombination by Arginine Methylation

National Jewish Health, Denver CO

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Abstract

DESCRIPTION (provided by applicant): V(D)J recombination is the hallmark of adaptive immunity. In T and B cells, a series of highly regulated somatic gene rearrangement events produce functional genes from gene segments. This process results in the expression of functional antigen receptors. Immunoglobulin (Ig) genes encode B cell receptors, while T cell receptors are encoded by Tcr loci. The central mechanisms necessary for the assembly of these genes are cleavage and ligation of variable (V), diversity (D, at a subset of antigen receptor loci), and Joining (J) segments by V(D)J recombination. V(D)J recombinase complexes comprise multiple proteins including Recombination activating genes 1 and 2 (Rag1 and Rag2), which recognize and cleave recombination signal sequences (RSS) when RSS are in an 'accessible' state. Here, we will determine the roles of Protein Arginine Methyltransferase 5 (PRMT5), methylated arginine, and its removal by epigenetic 'erasers' in this process. Accessibility of RSS for recombination is largely controlled by epigenetic mechanisms including DNA methylation and post-translational modifications of histones. The importance of each of these mechanisms has been documented in V(D)J recombination. However, far less is understood concerning the importance of arginine methylation and the identities of enzymes that add or remove methyl groups from arginine during lymphocyte development. We hypothesize that V(D)J recombination of T cell receptor (Tcra) loci is regulated by PRMT5, which dimethylates arginine symmetrically at multiple residues of histones H3 and H4, as well as some non-histone proteins. Provocatively, Prmt5 transcripts are expressed in developing thymocytes (and B cells), but not at stages that express the Rag1 gene and feature ongoing V(D)J recombination. These observations suggest that arginine methylation by PRMT5 inhibits V(D)J recombination. Indeed, depletion of Prmt5 mRNA in a pre-T cell line greatly increases mature TCR? expression on the plasma membrane. We predict that this mechanism regulates antigen receptor assembly in both T and B cells. Therefore, we will address functions of arginine methylation and PRMT5 in T and B cell lines that undergo efficient V(D)J recombination in vitro. Our evidence suggests that the loss of arginine methylation is an active process involving one or more epigenetic erasers, which are likely members of the Jumonji family of protein dioxigenases. We will use biochemical methods to identify candidate arginine demethylases in lymphocyte progenitors. Together, our experiments will address novel epigenetic mechanisms that control antigen receptor assembly during lymphocyte development, but are also important for gene regulation in a wide variety of contexts including cancer.