Tet2 recruits Ogt for B cell nuclear O-GlcNAcylation, active DNA demethylation and maturation of the antibody response
The maturation of the antibody response entails antibody somatic hypermutation (SHM), class-switch DNA recombination (CSR), plasma cell (PC) differentiation and generation of specific memory B cells (MBCs). SHM/CSR are driven by AID, encoded by Aicda, a potent DNA cytidine deaminase. PC differentiation requires Blimp-1, encoded by Prdm1, a key transcription factor. As we have shown, Aicda and Prdm1 expression is mediated by several epigenetic factors, such as histone deacetylase Sirt1 and non-coding miRNAs. Here, we identified and demonstrated the central role of active DNA demethylator Ten-eleven translocation (Tet) methylcytosine dioxygenase, Tet2/TET2, an epigenetic factor in embryonic cells and T cells development and differentiation, in modulating B cell-intrinsic Aicda/AID and Prdm1/Blimp-1 expression, and thereby promoting the maturation of mouse and human antibody responses. Tet proteins consist of Tet1, Tet2 and Tet3. As we show here, Tet1/Tet1, Tet2/Tet2 and Tet3/Tet3 are not or virtually not expressed in resting B cells. In B cells undergoing germinal center (GC) differentiation, Tet2/Tet2, not Tet3 nor Tet1 is induced for Aicda/AID and Prdm1/Blimp-1 expression. Tet2 not Tet3 B cell-specific (AicdaCreTet2fl/fl, AicdaCreTet3fl/fl) deletion impairs SHM/CSR, PC differentiation and the generation of antigen-specific, class-switched memory B cells. In B cells, Tet2 is recruited to Aicda and Prdm1 loci regulatory regions for active DNA demethylation by 5mC to 5hmC oxidation for AID and Blimp-1 expression in T-dependent and T-independent antibody responses. As we revealed here, in addition to its catalytic activity, Tet2 recruits O-linked N-acetylglucosamine transferase (Ogt) for Ogt-mediated histone H2B O-GlcNAcylation of Aicda and Prdm1 regulatory regions, as shown by impaired Ogt recruitment and subsequentially reduced H2B O-GlcNAcylation of these loci in AicdaCreTet2fl/fl B cells. In addition, Tet2-recruited Ogt O-GlcNAcylated Tet2 itself, a novel and important post-translational modification of Tet2 protein. The important role of Ogt is reflected in reduced AID and Blimp-1 expression and impaired SHM/CSR and PC differentiation by intrinsic AicdaCreOgtfl/fl B cell Ogt deletion. Transduction of Tet2-/- B cells with Tet2 catalytic site mutant (Tet2HxD-mut), putative Ogt-recruitment site deletion mutant (Tet2Ogt-Dmut) or dual mutant (Tet2HxD-mut/Ogt-Dmut) shows that Tet2-catalyzed 5mC to 5hmC oxidation and Tet2-recruited Ogt (histone H2B and Tet2) O-GlcNAcylation cooperate at Aicda and Prdm1 loci regulatory regions for overall Tet2 function. This is further supported by impaired AICDA/AID, PRDM1/BLIMP-1 expression, CSR and PC differentiation by combined inhibition of TET2 catalytic activity and OGT function, as well as Alphafold 3.0 3D-modeling of Tet2-Ogt complex at Aicda super-enhancer. Finally, B cell Tet2/TET2 activation by vitamin C and inhibition by fumarate indicate that Tet2/TET2 takes metabolic cues to promote maturation of the mouse and human antibody response.