RAG2 interactions with H3K4me3 are regulated by Thr490 proximal to the RAG2 PHD region

Autor: William A. Rodgers, Jennifer Byrum, Karla Rodgers
Rok vydání: 2018
Předmět:
Zdroj: The Journal of Immunology. 200:103.23-103.23
ISSN: 1550-6606
0022-1767
Popis: Association of RAG2 with the epigenetic marker H3K4me3 through a plant homeodomain (PHD) in the RAG2 non core domain is important for relieving auto inhibition of the RAG recombinase activity for V(D)J recombination. However, little is known regarding the properties of RAG2 that regulate its PHD-dependent interactions with H3K4me3. Accordingly, we measured the localization and dynamics of an N-terminal GFP fusion protein of full length RAG2 (GFP-FL); a T490A mutant of full length RAG2 (GFP-T490A) that does not undergo a regulatory phosphorylation proximal to the PHD region of RAG2; and GFP-labeled core domain of RAG2 (GFP-Core). The proteins were expressed in RAG2−/− pro-B cells, and the H3K4me3 was labeled using monoclonal antibody. Cross-correlation analysis showed that GFP-FL and GFP-Core exhibited similar colocalization with H3K4me3, which was significantly less than colocalization of GFP-T490A with H3K4me3. Blocking H3K4 demethylation using 2,4-pyridinedicarboxylic acid (PDA) caused GFP-FL/H3K4me3 colocalization to approach that of GFP-T490A and H3K4me3, whereas GFP-Core/H3K4me3 colocalization was unchanged. Super resolution imaging showed that RAG2 interactions with H3K4me3 were restricted to interfaces with H3K4me3-enriched puncta, and that GFP-T490A exhibited greater overlap with the H3K4me3-enriched puncta than GFP-FL. Finally, measurement of fluorescence recovery after photobleaching demonstrated that GFP-T490A had a lower mobile fraction and rate of diffusion than GFP-FL in conditions where RAG2 interactions with H3K4me3 were enhanced. These data suggest that RAG2 interactions with H3K4me3 are regulated by Thr490 proximal to the PHD region, such as by a phosphoryation/dephosphorylation cycle.
Databáze: OpenAIRE
Externí odkaz: