Role of a RAG1-DCAF1(VprBP)-Ezh2 axis in B cell development - PROJECT SUMMARY B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates from RAG (recombination activating gene)-mediated V(D)J recombination that assembles antigen receptor genes during lymphocyte development. However, V(D)J recombination is not always benign, as aberrant rearrangement can contribute to genomic instability and cancer in lymphocytes. We previously showed that the amino-terminal 215 residues of RAG1, which is dispensable for RAG endonuclease activity, constrains V(D)J recombination by recruiting Vpr Binding Protein (VprBP) and its associated Cullin4-DDB1- Rbx1 (CRL4) E3 ubiquitin ligase complex to mediate timely RAG1 degradation. Interestingly, VprBP (also called DCAF1) was recently found to stabilize the histone-lysine N-methyltransferase Enhancer of zeste homolog 2 (Ezh2), which catalyzes the addition of methyl groups to histone H3 at lysine 27 (H3K27) by the Polycomb Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) is generally considered to promote gene silencing. In mice, B lineage-specific loss of Ezh2 expression arrests B cell development at the pro-B to pre-B cell transition, and selectively impairs rearrangement of the distal immunoglobulin (Ig) VHJ558 gene family. This phenotype resembles that observed in mice lacking VprBP in B cells. Truncating the first 215 residues of RAG1 in mice to eliminate the VprBP binding site (R1215 mice) confers a less severe phenotype, but nevertheless impairs short-range Ig D-J rearrangement for reasons that remain unclear. These observations lead us to hypothesize that VprBP stabilizes Ezh2 in B cells to promote PRC2 formation, H3K27 methylation, transcriptional programs, and immune repertoire in early B lineage cells, and through VprBPs association with full-length RAG1, specifically regulates H3K27 methylation within the Igh locus to regulate short-range Ig D-J rearrangement. This hypothesis will be tested by experiments described in two broad Aims. In Aim I of this project, we will: (a) establish the concordance of the phenotypes presented by mice lacking VprBP or Ezh2 in the B lineage when rescued by enforced Bcl2 expression; (b) compare Ezh2, total H3, and H3K27me3 protein levels in wild-type and VprBP-deficient B cells, using Ezh2-deficient B cells as a negative control; and (c) establish VprBP-dependent Ezh2 association with PRC2 subunits EED and SUZ12 in primary B cells. In Aim II of this project, we will use RNA, immune repertoire, and H3K27me3 “Cut&Run” sequencing approaches to analyze and compare the transcriptome, Ig gene usage, and localization of H3K27me3 marks in sorted pro-B and pre-B cells from wild-type, VprBP-deficient, Ezh2-deficient, and R1215-expressing B cells. Establishing that the RAG1-VprBP-Ezh2-H3K27me3 axis regulates V(D)J recombination would be conceptually innovative, as it provides a means for the RAGs to locally modify Ig gene accessibility and expression within antigen receptor loci by recruiting VprBP and regulating Ezh2 activity.
