Abstract
Abstract RAG2 is essential for V(D)J recombination in developing lymphocytes, yet its localization and dynamics in cell nuclei is poorly understood. Here, we used single-cell imaging to interrogate RAG2 interactions with transcriptionally active chromatin containing H3K4me3. We measured GFP-labeled full length RAG2 (FL), the RAG2 core region alone (Core), and a T490A mutant of RAG2. Each RAG2 construct localized to nuclear regions that were adjacent to DAPI-rich heterochromatin, where the T490A RAG2 exhibited greater colocalization with H3K4me3 than either FL or Core. Furthermore, mobility measurements showed that T490A had a significantly lower rate of diffusion within the nucleus than either FL or Core proteins. Finally, mutating W453 in the T490A mutant (W453A, T490A), which blocks interactions between the RAG2 PHD region and H3K4me3, decreased the RAG2 colocalization with H3K4me3 and increased its mobility to values similar to that of FL. Altogether, these data show that Thr490 modulates RAG2 localization and dynamics in the pre-B cell nucleus, such as by affecting PHD-dependent interactions between RAG2 and H3K4me3.
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