R-loops are three-stranded, RNA–DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

Defective apoptotic death of activated macrophages has been implicated in the pathogenesis of rheumatoid arthritis (RA). However, the molecular signatures defining apoptotic resistance of RA macrophages are not fully understood. Here, global transcriptome profiling of RA macrophages revealed that the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) critically regulates diverse pathologic processes in synovial macrophages including the cell cycle, apoptosis, and proliferation. Transcriptomic analysis of NFAT5-deficient macrophages revealed the molecular networks defining cell survival and proliferation. Proinflammatory M1-polarizing stimuli and hypoxic conditions were responsible for enhanced NFAT5 expression in RA macrophages. An in vitro functional study demonstrated that NFAT5-deficient macrophages were more susceptible to apoptotic death. Specifically, CCL2 secretion in an NFAT5-dependent fashion bestowed apoptotic resistance to RA macrophages in vitro. Injection of recombinant CCL2 into one of the affected joints of Nfat5^+/– mice increased joint destruction and macrophage infiltration, demonstrating the essential role of the NFAT5/CCL2 axis in arthritis progression in vivo. Moreover, after intra-articular injection, NFAT5-deficient macrophages were more susceptible to apoptosis and less efficient at promoting joint destruction than were NFAT5-sufficient macrophages. Thus, NFAT5 regulates macrophage survival by inducing CCL2 secretion. Our results provide evidence that NFAT5 expression in macrophages enhances chronic arthritis by conferring apoptotic resistance to activated macrophages.

Objective To investigate the role of NF‐AT5, an osmoprotective transcription factor, in synovial hyperplasia and angiogenesis in patients with rheumatoid arthritis (RA). Methods The expression of NF‐AT5 in synovial tissue and synoviocytes from RA patients was examined by immunohistochemistry and Western blot analysis, respectively. Messenger RNA (mRNA) in RA synoviocytes and human umbilical vein endothelial cells (HUVECs) transfected with dummy small interfering RNA (siRNA) or NF‐AT5 siRNA were profiled using microarray technology. Assays to determine synoviocyte apoptosis and proliferation were performed in the presence of NF‐AT5 siRNA. VEGF₁₆₅‐induced angiogenesis was assessed by measuring the proliferation, tube formation, and wound migration of HUVECs. Experimental arthritis was induced in mice by injection of anti–type II collagen antibody. Results NF‐AT5 was highly expressed in rheumatoid synovium, and its activity was increased by proinflammatory cytokines, such as interleukin‐1β and tumor necrosis factor α. The mRNA profiling of synoviocytes and HUVECs transfected with NF‐AT5–targeted siRNA revealed 3 major changes in cellular processes associated with the pathogenesis of RA: cell cycle and survival, angiogenesis, and cell migration. Consistent with these results, NF‐AT5 knockdown in RA synoviocytes and HUVECs inhibited their proliferation/survival and impeded angiogenic processes in HUVECs. Mice with NF‐AT5 haploinsufficiency (NF‐AT5^+/–) developed a very limited degree of synovial proliferation, as seen on histologic analysis, and decreased angiogenesis, and they exhibited a nearly complete suppression of experimentally induced arthritis. Conclusion NF‐AT5 regulates synovial proliferation and angiogenesis in chronic arthritis.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by autoreactive B cells and dysregulation of many other types of immune cells including myeloid cells. Lupus nephritis (LN) is a common target organ manifestations of SLE. Tonicity-responsive enhancer-binding protein (TonEBP, also known as nuclear factor of activated T-cells 5 (NFAT5)), was initially identified as a central regulator of cellular responses to hypertonic stress and is a pleiotropic stress protein involved in a variety of immunometabolic diseases. To explore the role of TonEBP, we examined kidney biopsy samples from patients with LN. Kidney TonEBP expression was found to be elevated in these patients compared to control patients - in both kidney cells and infiltrating immune cells. Kidney TonEBP mRNA was elevated in LN and correlated with mRNAs encoding inflammatory cytokines and the degree of proteinuria. In a pristane-induced SLE model in mice, myeloid TonEBP deficiency blocked the development of SLE and LN. In macrophages, engagement of various toll-like receptors (TLRs) that respond to damage-associated molecular patterns induced TonEBP expression via stimulation of its promoter. Intracellular signaling downstream of the TLRs was dependent on TonEBP. Therefore, TonEBP can act as a transcriptional cofactor for NF-κB, and activated mTOR-IRF3/7 via protein-protein interactions. Additionally, TonEBP-deficient macrophages displayed elevated efferocytosis and animals with myeloid deficiency of TonEBP showed reduced Th1 and Th17 differentiation, consistent with macrophages defective in TLR signaling. Thus, our data show that myeloid TonEBP may be an attractive therapeutic target for SLE and LN.

NFκB is a central mediator of inflammation. Present inhibitors of NFκB are mostly based on inhibition of essential machinery such as proteasome and protein kinases, or activation of nuclear receptors; as such, they are of limited therapeutic use due to severe toxicity. Here we report an LPS-induced NFκB enhanceosome in which TonEBP is required for the recruitment of p300. Increased expression of TonEBP enhances the NFκB activity and reduced TonEBP expression lowers it. Recombinant TonEBP molecules incapable of recruiting p300 do not stimulate NFκB. Myeloid-specific deletion of TonEBP results in milder inflammation and sepsis. We discover that a natural small molecule cerulenin specifically disrupts the enhanceosome without affecting the activation of NFκB itself. Cerulenin suppresses the pro-inflammatory activation of macrophages and sepsis without detectable toxicity. Thus, the NFκB enhanceosome offers a promising target for useful anti-inflammatory agents.