Date Published: May 10, 2019
Publisher: Public Library of Science
Author(s): William Rodgers, Jennifer N. Byrum, Destiny A. Simpson, Walker Hoolehan, Karla K. Rodgers, Albert Jeltsch.
RAG2 of the V(D)J recombinase is essential for lymphocyte development. Within the RAG2 noncore region is a plant homeodomain (PHD) that interacts with the modified histone H3K4me3, and this interaction is important for relieving inhibition of the RAG recombinase for V(D)J recombination. However, the effect of the noncore region on RAG2 localization and dynamics in cell nuclei is poorly understood. Here, we used cell imaging to measure the effect of mutating the RAG2 noncore region on properties of the full length protein. We measured GFP-labeled full length RAG2 (FL), the RAG2 core region alone (Core), and a T490A mutant in the noncore region, which has unique regulatory properties. This showed that FL, T490A, and Core localized to nuclear domains that were adjacent to DAPI-rich heterochromatin, and that contained the active chromatin marker H3K4me3. Within the RAG2-enriched regions, T490A exhibited greater colocalization with H3K4me3 than either FL or Core. Furthermore, colocalization of H3K4me3 with FL and T490A, but not Core, increased in conditions that increased H3K4me3 levels. Superresolution imaging showed H3K4me3 was distributed as puncta that RAG2 abutted, and mobility measurements showed that T490A had a significantly lower rate of diffusion within the nucleus than either FL or Core proteins. Finally, mutating Trp453 of the T490A mutant (W453A,T490A), which blocks PHD-dependent interactions with H3K4me3, abolished the T490A-mediated increased colocalization with H3K4me3 and slower mobility compared to FL. Altogether, these data show that Thr490 in the noncore region modulates RAG2 localization and dynamics in the pre-B cell nucleus, such as by affecting RAG2 interactions with H3K4me3.
V(D)J recombination generates antigen receptor (AR) genes in developing lymphocytes by a cut-and-paste recombination reaction from component genes in AR loci [1, 2]. The large array of gene combinations that follow from the V(D)J recombination is the basis for sequence diversity of the antigen binding receptors in the immune system. The V(D)J recombinase, which consists of the RAG1 and RAG2 proteins, function by creating DNA double strand breaks at select recombination signal sequences (RSS) that are located at the border of each gene segment in the AR loci. A critical step in this process is the selective binding of the RAG proteins to an RSS over the vast array of accessible DNA in the genome. Importantly, the genome also contains numerous RSS-like sites, where RAG binding and DNA cleavage activity can result in oncogenic chromosomal translocations [3–5]. Thus, properties that affect targeting of the RAG complex to the genome have important consequences regarding genomic integrity.
The V(D)J recombinase is composed of the proteins RAG1 and RAG2, which together function in the incision of specific genetic loci for assembly of AR genes [1, 2]. Enzymatic activity of the recombinase complex is restricted to RAG1, yet RAG2 is essential for relieving an autoinhibition of the recombinase through binding to H3K4me3 by a PHD region in the RAG2 noncore domain [11–13]. Since H3K4me3 populates sites of recombination in the AR locus, RAG2 noncore interactions with H3K4me3 will both target the recombinase to potential recombination sites, and activate the complex for V(D)J recombination. Previous studies show RAG2 is sequestered from transcriptionally inactive heterochromatin within the nucleus [8, 9], but the effect of altering the noncore domain on RAG2 localization and dynamics is not understood. Here, we used analytical imaging approaches to measure the role of the noncore domain in RAG2 localization and dynamics to elucidate its role in targeting RAG2 to chromatin in pre-B cell nuclei.