PROJECT SUMMARY/ABSTRACT. Epigenetics of the autoantibody response in systemic lupus. Like “mature” antibody responses to viruses and bacteria, the lupus autoantibody response requires B cell class- switch DNA recombination (CSR), somatic hypermutation (SHM) and plasma cell differentiation. As we have shown, epigenetic factors, including histone modifiers (such as Sirt1) and inhibitors (such as butyrate) modulate B cell expression of AID (gene: AICDA/Aicda) and Blimp-1 (PRDM1/Prdm1), which are critically for CSR/SHM and plasma cell differentiation, respectively. Towards a better definition of the epigenetic landscape of lupus B cells, we hypothesize that Tet2, a key epigenetic factor, mediates the lupus autoantibody response, as prompted the B cell-intrinsic role of Tet2 in CSR/SHM and plasma cell differentiation (our recent findings). We argue that Tet2 is induced by the stimuli that induce B cell CSR/SHM and plasma cell differentiation, and it is upregulated in lupus B cells. We also argue that Tet2 boosts transcription of Aicda and Prdm1 through its Fe2+ and a- ketoglutarate (a-KG)-dependent catalytic activity for DNA demethylation as well as its non-catalytic function, i.e., recruiting Ogt (encoded by X-linked OGT/Ogt) to these loci to effect histone glycosylation. Finally, we contend that B cell Tet2 transduces hormonal, nutritional and metabolic cues into epigenetic changes to modulate autoantibody responses, as Tet2 transcription could be upregulated by estrogen (E2, our preliminary data) and vitamin A, and Tet2 is activated by vitamin C, but inhibited by fumarate (a metabolite and a-KG competitor) – as shown by us, E2 boosts AID expression, which would enhance females’ antibody and autoantibody responses. With extensive experience in and commitment to the mechanistic understanding of human and mouse lupus autoantibody responses, we are uniquely poised to test our hypotheses using molecular B cell biology systems, cutting-edge epigenetic tools (hydroxylmethyl DNA analysis, bisulfite and oxidative bisulfite conversion, ChIP and ATAC-Seq), genetically modified mice (TgAicda-creTet2fl/fl, Tet2HxD-mut/HxD-mut and Tet2Ogt-mut/Ogt-mut knockin mice, TgAicda-creOgtfl/fl, Tet2HxD-mut/HxD-mutTgAicda-creOgtfl/fl, MRL/Lpr TgAicda-creTet2fl/fl, MRL/Lpr Tet2HxD-mut/HxD-mut and MRL/Lpr TgAicda-creOgtfl/fl), proprietary humanized H-Mice® and Lupus-H-Mice® models. Aim 1 addresses human and mouse B cell differentiation stage-specific (resting, activated, plasma and memory cell) regulation of Tet2, Tet2 protein stability, E2 upregulation of Tet2 transcripts and underlying mechanisms. Aim 2 addresses the B cell- intrinsic role of Tet2 in promoting AID and Blimp-1 expression, the underlying mechanisms (active DNA demethylation and Ogt-mediated histone glycosylation), and potentiation effect of Tet2 inducer vitamin A and activator vitamin C, and suppressive effect of Tet2 catalytic inhibitor (fumarate, TET-IN-C35 or Bobcat339) alone or combined with Ogt ablati...