Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.

38 Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme into the tricarboxylic acid (TCA) cycle. Here we show that 39 PARK7/DJ-1, a key familial Parkinson's disease (PD) gene, is a pacemaker controlling PDH activity in CD4 regulatory 40 T cells (Tregs). DJ-1 bound to PDH-E1 beta (PDHB), inhibiting the phosphorylation of PDH-E1 alpha (PDHA), thus 41 promoting PDH activity and oxidative phosphorylation (OXPHOS). Dj-1 depletion impaired Treg proliferation and 42 cellularity maintenance in older mice, increasing the severity during the remission phase of experimental autoimmune 43 encephalomyelitis (EAE). The compromised proliferation and differentiation of Tregs in Dj-1 knockout mice were caused 44 via regulating PDH activity. These findings provide novel insight into the already complicated regulatory machinery of 45 the PDH complex and demonstrate that the DJ-1-PDHB axis represents a potent target to maintain Treg homeostasis, 46 which is dysregulated in many complex diseases. 47 48 9 Bonifati, V. et al. Mutations in the DJ-1 gene associated with autosomal recessive early-onset 789 parkinsonism. Science 299, 256-259, 792 11 van der Brug, M. P. et al. RNA binding activity of the recessive parkinsonism protein DJ-1 supports 793 involvement in multiple cellular pathways.

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme into the tricarboxylic acid (TCA) cycle. Here we show that PARK7/DJ-1, a key familial Parkinson's disease (PD) gene, is a pacemaker controlling PDH activity in CD4 regulatory T cells (Tregs). DJ-1 bound to PDH-E1 beta (PDHB), inhibiting the phosphorylation of PDH-E1 alpha (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Dj-1 depletion impaired Treg proliferation and cellularity maintenance in older mice, increasing the severity during the remission phase of experimental autoimmune encephalomyelitis (EAE). The compromised proliferation and differentiation of Tregs in Dj-1 knockout mice were caused via regulating PDH activity. These findings provide novel insight into the already complicated regulatory machinery of the PDH complex and demonstrate that the DJ-1-PDHB axis represents a potent target to maintain Treg homeostasis, which is dysregulated in many complex diseases.