Nucleolar localization of RAG1 modulates V(D)J recombination activity Ryan M. Brechta,b, Catherine C. Liub, Helen A. Beilinsonb, Alexandra Khitunc,d, Sarah A. Slavoffa,c,d, and David G. Schatza,b,1 aDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06529; bDepartment of Immunobiology, Yale School of Medicine, New Haven, CT 06511; cDepartment of Chemistry, Yale University, New Haven, CT 06520; and dChemical Biology Institute, Yale University, West Haven, CT 06516 Contributed by David G. Schatz, January 15, 2020 (sent for review December 2, 2019; reviewed by Marcus R. Clark and Michael R. Lieber) V(D)J recombination assembles and diversifies Ig and T cell receptor interactions of RAG. RAG1, the major agent of DNA binding genes in developing B and T lymphocytes. The reaction is initiated by and cleavage, is a 1,040 amino acid (aa) protein that is largely the RAG1-RAG2 protein complex which binds and cleaves at discrete insoluble and difficult to extract from the nucleus (17, 18). As gene segments in the antigen receptor loci. To identify mechanisms such, much of the biochemical and structural characterization of that regulate V(D)J recombination, we used proximity-dependent RAG1 has been done on a truncated “core” version spanning biotin identification to analyze the interactomes of full-length and residues 384 to 1,008. While core RAG1 retains catalytic activity, truncated forms of RAG1 in pre-B cells. This revealed an association of its in vivo recombination efficiency and fidelity are reduced com- RAG1 with numerous nucleolar proteins in a manner dependent on pared to full-length RAG1 (FLRAG1) and its binding to the ge- amino acids 216 to 383 and allowed identification of a motif required nome is more promiscuous (19–24). The evolutionarily conserved for nucleolar localization. Experiments in transformed pre-B cell lines 383 aa N-terminal domain (NTD) missing from core RAG1 is and cultured primary pre-B cells reveal a strong correlation between predicted to harbor multiple zinc-binding motifs including a Really disruption of nucleoli, reduced association of RAG1 with a nucleolar Interesting New Gene (RING) domain (aa 287 to 351) capable of marker, and increased V(D)J recombination activity. Mutation of the – RAG1 nucleolar localization motif boosts recombination while re- ubiquitylating multiple targets, including RAG1 itself (23, 25 27). moval of the first 215 amino acids of RAG1, required for efficient Although this ubiquitylation activity has been characterized in vitro, egress from nucleoli, reduces recombination activity. Our findings its in vivo relevance to V(D)J recombination remains unclear. Also IMMUNOLOGY AND INFLAMMATION indicate that nucleolar sequestration of RAG1 is a negative regulatory contained within the NTD is a region (aa 1 to 215) that mediates mechanism in V(D)J recombination and identify regions of the RAG1 interactionwithDCAF1,causingdegradationofRAG1inaCRL4- N-terminal region that control nucleolar association and egress. dependent manner (28, 29). The NTD also contributes to chromatin binding and genomic targeting of the RAG complex (20, 24). V(D)J recombination | RAG1 | nucleolus | B cell development | Despite a growing body of evidence highlighting the importance proximity-dependent biotin identification of RAG1’s NTD, our understanding of its functional contribu- tion to V(D)J recombination is far from complete. In addition, vast diversity of molecular specificity is needed to mediate because of its low-level expression, microscopy of FLRAG1 in a Arecognition and interaction between host and pathogen in cellular context has been extremely limited, leaving many ques- the jawed vertebrate adaptive immune system. This diversity is cre- tions unanswered regarding RAG1 localization and trafficking. ated in part by combinatorial gene rearrangements carried out in developing B and T cells during the process of V(D)J recombination, Significance in which discrete V (variable), D (diversity), and J (joining) gene segments are stochastically combined to form a functional antigen Vertebrate immune systems can respond to many infections and receptor gene (1). V(D)J recombination is initiated by Recombina- insults. This ability relies on a diverse binding repertoire of an- tion Activating Gene (RAG) proteins 1 and 2 (2, 3). Together, tigen receptors. Antigen receptor diversity is created through a RAG1/2 carry out V(D)J recombination as a heterotetramer that process called V(D)J recombination in which arrayed gene seg- binds and cleaves DNA at specific recombination signal sequences ments are shuffled to form functional receptors. This process (RSSs) flanking exon segments. After cleavage, these exon seg- introduces breaks in chromosomal DNA catalyzed by the RAG1- ments are chaperoned into the nonhomologous end joining DNA RAG2 protein complex and requires strict regulation to guard repair pathway by the RAG complex (reviewed in refs. 4–6). genome integrity. Here we demonstrate a mode of RAG1 regu- While the RAG proteins enable the generation of a diverse B and lation by nucleolar sequestration. RAG1’s nucleolar localization is T cell receptor repertoire, the DNA breaks generated during dynamically regulated and is disrupted during a B cell’s transition V(D)J recombination are inherently genotoxic and can lead to to a prorecombination state, leading to increased recombination. harmful translocations and subsequent lymphocytic malignancies (7–10). In light of this, understanding the mechanisms underlying Author contributions: R.M.B., C.C.L., H.A.B., S.A.S., and D.G.S. designed research; R.M.B., C.C.L., H.A.B., and A.K. performed research; R.M.B. contributed new reagents/analytic RAG targeting and regulation are of great interest. tools; R.M.B., C.C.L., H.A.B., A.K., and S.A.S. analyzed data; and R.M.B., C.C.L., and Regulation of RAG2 at the protein level has been well char- D.G.S. wrote the paper. acterized. Spatial regulation of RAG2 away from the nuclear pe- Reviewers: M.R.C., University of Chicago; and M.R.L., University of Southern California. riphery is thought to contribute to allelic exclusion and the ordered The authors declare no competing interest. β rearrangement of the TCR loci (11), while CDK2-dependent Published under the PNAS license. degradation of RAG2 limits V(D)J recombination to the G 1 Data deposition: The mass spectrometry proteomics data have been deposited to the phase of the cell cycle (12, 13). In contrast, little is known re- publicly accessible ProteomeXchange Consortium via the PRIDE partner repository with garding the regulation and localization of the RAG1 protein, with the dataset identifier PXD016221 (DOI: 10.6019/PXD016221). most work focusing on RAG1 transcriptional regulation (14–16). 1To whom correspondence may be addressed. Email: [email protected]. Identifying proteins involved in the targeting, regulation, and This article contains supporting information online at https://www.pnas.org/lookup/suppl/ repair of RAG-mediated DNA breaks has been hindered by the doi:10.1073/pnas.1920021117/-/DCSupplemental. lack of methodologies amenable to probing the protein–protein www.pnas.org/cgi/doi/10.1073/pnas.1920021117 PNAS Latest Articles | 1of10 Downloaded by guest on September 23, 2021 Many proteins are regulated by their localization or seques- Results tration within distinct cellular compartments. The nucleolus is a Biotin Identification Identifies Multiple Nucleolar Proteins Proximal phase-separated, nonmembrane bound nuclear organelle that is to Full-Length RAG1. To identify RAG1-associated proteins, we the site of ribosome biogenesis. However, recent efforts to map used proximity-dependent biotin identification (BioID) (37), which the nucleolar proteome have revealed a plethora of proteins with makes use of a promiscuous Escherichia coli biotin ligase (BirM) roles beyond canonical nucleolar processes, including DNA re- to biotinylate lysine residues on proximal proteins. We generated pair and apoptosis (30–32). Further work has also shown the various truncations of RAG1 fused to BirM (Fig. 1A) and tested nucleolus as a dynamic hub capable of regulating protein func- these fusion proteins for biotinylation and recombination activity tion in response to specific stimuli, including DNA double-strand in HEK293T and the commonly used pre-B cell model system, breaks (DSBs) (33–35). Abelson murine leukemia virus-transformed (vAbl) cells (SI More than 20 y ago, RAG1 was reported to localize to the Appendix, Fig. S1). vAbl cells are developmentally arrested at the nucleolus when overexpressed in a nonlymphoid cell line (36). pre-B stage via expression of a constitutively active form of the We are not aware of subsequent studies to determine whether Abelson kinase. Upon addition of the Abelson kinase inhibitor this occurs at physiological levels of RAG1 expression in its STI-571, the cells exit cell cycle in the G1 phase and activate normal cellular context or whether it might have functional RAG expression and Igκ locus recombination (38). We utilized a relevance. Here, we demonstrate that RAG1 harbors a nucle- doxycycline-inducible system to express the RAG1-BioID con- olar localization signal (NoLS) motif in its NTD and that structs in stably retrovirally transduced vAbl cells, allowing us to RAG1 function is regulated by nucleolar localization. During Ig initiate V(D)J recombination and RAG1 interactome labeling (Ig) κ-gene recombination and in response to nucleolar stress, synchronously by addition of STI-571, doxycycline, and biotin. we observe that RAG1 egresses from the nucleolus and forms After 24 h of labeling, cells were lysed and biotinylated proteins small, bright puncta in a manner dependent on aa 1 to 215. were enriched, digested, and the resulting peptides analyzed by These findings delineate a repressive function for nucleolar liquid chromatography tandem mass spectrometry (LC-MS/MS). localization of RAG1 and set the stage for further work ex- We collected interactome data on four constructs in duplicate amining the role of the nucleolus in the regulation of RAG1 (39) (Fig.