Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair.

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

G9a Inhibition Promotes Neuroprotection through GMFB Regulation in Alzheimer's Disease.

Epigenetic alterations are a fundamental pathological hallmark of Alzheimer's disease (AD). Herein, we show the upregulation of G9a and H3K9me2 in the brains of AD patients. Interestingly, treatment with a G9a inhibitor (G9ai) in SAMP8 mice reversed the high levels of H3K9me2 and rescued cognitive decline. A transcriptional profile analysis after G9ai treatment revealed increased gene expression of glia maturation factor ß (GMFB) in SAMP8 mice. Besides, a H3K9me2 ChIP-seq analysis after G9a inhibition treatment showed the enrichment of gene promoters associated with neural functions. We observed the induction of neuronal plasticity and a reduction of neuroinflammation after G9ai treatment, and more strikingly, these neuroprotective effects were reverted by the pharmacological inhibition of GMFB in mice and cell cultures; this was also validated by the RNAi approach generating the knockdown of GMFB/Y507A.10 in Caenorhabditis elegans. Importantly, we present evidence that GMFB activity is controlled by G9a-mediated lysine methylation as well as we identified that G9a directly bound GMFB and catalyzed the methylation at lysine (K) 20 and K25 in vitro. Furthermore, we found that the neurodegenerative role of G9a as a GMFB suppressor would mainly rely on methylation of the K25 position of GMFB, and thus G9a pharmacological inhibition removes this methylation promoting neuroprotective effects. Then, our findings confirm an undescribed mechanism by which G9a inhibition acts at two levels, increasing GMFB and regulating its function to promote neuroprotective effects in age-related cognitive decline.

Gcn5- and Bre1-mediated Set2 degradation promotes chronological aging of Saccharomyces cerevisiae.

Loss of transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3) contributes to shorter lifespans in eukaryotes. However, the molecular mechanism of the decline of H3K36me3 during aging remains poorly understood. Here, we report that the degradation of the methyltransferase Set2 is the cause of decreased H3K36me3 levels during chronological aging in budding yeast. We show that Set2 protein degradation during cellular senescence and chronological aging is mainly mediated by the ubiquitin-conjugating E2 enzyme Ubc3 and the E3 ligase Bre1. Lack of Bre1 or abolishment of the ubiquitination stabilizes Set2 protein, sustains H3K36me3 levels at the aging-related gene loci, and upregulates their gene expression, thus leading to extended chronological lifespan. We further illustrate that Gcn5-mediated Set2 acetylation is a prerequisite for Bre1-catalyzed Set2 polyubiquitination and proteolysis during aging. We propose that two sequential post-translational modifications regulate Set2 homeostasis, suggesting a potential strategy to target the Gcn5-Bre1-Set2 axis for intervention of longevity.

Dynamic Phosphorylation of G9a Regulates its Repressive Activity on Chromatin Accessibility and Mitotic Progression.

Phosphorylation of Ser10 of histone H3 (H3S10p), together with the adjacent methylation of Lys9 (H3K9me), has been proposed to function as a 'phospho-methyl switch' to regulate mitotic chromatin architecture. Despite of immense understanding of the roles of H3S10 phosphorylation, how H3K9me2 are dynamically regulated during mitosis is poorly understood. Here, it is identified that Plk1 kinase phosphorylates the H3K9me1/2 methyltransferase G9a/EHMT2 at Thr1045 (pT1045) during early mitosis, which attenuates its catalytic activity toward H3K9me2. Cells bearing Thr1045 phosphomimic mutant of G9a (T1045E) show decreased H3K9me2 levels, increased chromatin accessibility, and delayed mitotic progression. By contrast, dephosphorylation of pT1045 during late mitosis by the protein phosphatase PPP2CB reactivates G9a activity and upregulates H3K9me2 levels, correlated with decreased levels of H3S10p. Therefore, the results provide a mechanistic explanation of the essential of a 'phospho-methyl switch' and highlight the importance of Plk1 and PPP2CB-mediated dynamic regulation of G9a activity in chromatin organization and mitotic progression.

SETD3 Methyltransferase Regulates PLK1 Expression to Promote In Situ Hepatic Carcinogenesis.

Background: The development of a new strategy to overcome chemoresistance to hepatocellular carcinoma (HCC) treatment is a long-standing issue. We have previously found that upregulated SETD3 levels are closely correlated with HCC. This study aims to explore the mechanism underlying how upregulation of SETD3 promotes liver carcinogenesis. Methods: RNA-Sequencing analysis was used to explore the correlation of SETD3 with regulatory targets. In vitro assays including cell proliferation and migration were performed to study the oncogenic roles of SETD3 and PLK1. Western blotting, immunohistochemical staining, and blood biochemical assays were performed to examine protein expression or pathological index in tumor tissues and mice liver tissues. Luciferase reporter system and chromatin immunoprecipitation assays were used to explore the mechanism. Results: We revealed that SETD3 regulates gene expression in subgroups, including cell division, cell proliferation, and cell cycle, in hepatocellular tumor cells. We found that SETD3 upregulation is associated with elevated PLK1 level in both hepatic tumor cells and clinical liver tissues. We further showed that overexpression of SETD3 promoted tumor cell proliferation and migration, whereas inhibition of PLK1 activity attenuated these phenotypes caused by SETD3. By taking advantage of the Sleep Beauty transposase system, we confirmed that upregulated mouse Setd3 promoted hepatic carcinogenesis in situ, but knockdown of mouse Plk1 mitigated Setd3-promoted tumorigenesis in mice. Mechanistically, we showed that SETD3 could be recruited to the promoter of PLK1 gene to facilitate PLK1 transcription. Conclusions: Our data demonstrate that elevated SETD3 may promote HCC by enhancing PLK1 expression, which suggests that SETD3 may act as a potential drug target combined with PLK1 inhibition to treat HCC.

Tyrosine phosphorylation tunes chemical and thermal sensitivity of TRPV2 ion channel.

Transient receptor potential vanilloid 2 (TRPV2) is a multimodal ion channel implicated in diverse physiopathological processes. Its important involvement in immune responses has been suggested such as in the macrophages' phagocytosis process. However, the endogenous signaling cascades controlling the gating of TRPV2 remain to be understood. Here, we report that enhancing tyrosine phosphorylation remarkably alters the chemical and thermal sensitivities of TRPV2 endogenously expressed in rat bone marrow-derived macrophages and dorsal root ganglia (DRG) neurons. We identify that the protein tyrosine kinase JAK1 mediates TRPV2 phosphorylation at the molecular sites Tyr(335), Tyr(471), and Tyr(525). JAK1 phosphorylation is required for maintaining TRPV2 activity and the phagocytic ability of macrophages. We further show that TRPV2 phosphorylation is dynamically balanced by protein tyrosine phosphatase non-receptor type 1 (PTPN1). PTPN1 inhibition increases TRPV2 phosphorylation, further reducing the activation temperature threshold. Our data thus unveil an intrinsic mechanism where the phosphorylation/dephosphorylation dynamic balance sets the basal chemical and thermal sensitivity of TRPV2. Targeting this pathway will aid therapeutic interventions in physiopathological contexts.

All the cells in our body have a membrane that separates their interior from the outside environment. However, studded across this barrier are numerous ion channels which allow the cell to sense and react to changes in its surroundings. This includes the ion channel TRPV2, which opens in response to mechanical pressure, certain chemical signals, or rising temperature levels. Many types of cell express TRPV2, including cells in the nervous system, muscle, and the immune system. However, despite being extensively studied, it is still not clear how TRPV2 opens and closes upon encountering high temperatures. In particular, previous work suggested that TRPV2 only responds when a cell's surroundings reach around 52°C, which is a much higher temperature than cells inside our body normally encounter, even during a fever. To help resolve this mystery, Mo, Pang et al. studied TRPV2 in neurons responsible for sending sensory information and in immune cells called macrophages which had been extracted from rodents and grown in the laboratory. They found that when the cells were bathed in solutions containing magnesium ions, their TRPV2 channels were more sensitive to a number of different cues, including temperature. Further biochemical experiments showed that magnesium ions do not directly affect TRPV2, but increase the activity of another protein called JAK1. The magnesium ions caused JAK1 to attach specialized structures called phosphorylation tags to TRPV2. This modification (known as phosphorylation) made the channel more sensitive, allowing it to open in response to temperatures as low as 40°C. Mo, Pang et al. found that inhibiting JAK1 reduced the activity of TRPV2. Conversely, inhibiting the enzyme that removes the phosphorylation tags, called PTPN1, increased the channel's activity. They also discovered that when JAK1 was blocked, macrophages were less able to 'eat up' bacteria, which is one of their main roles in the immune system. Taken together these experiments advance our understanding of how TRPV2 becomes active. The balance between the phosphorylation by JAK1 and the dephosphorylation by PTPN1 controls the temperature at which TRPV2 opens. Since TRPV2 contributes to several biological functions, including the development of the nervous system, the maintenance of heart muscles, and inflammation, these findings will be important to scientists in a broad range of fields.

Derivation of feeder-free human extended pluripotent stem cells.

Human extended pluripotent stem cells (EPSCs), with bidirectional chimeric ability to contribute to both embryonic and extraembryonic lineages, can be obtained and maintained by converting conventional pluripotent stem cells using chemicals. However, the transition system is based on inactivated mouse fibroblasts, and the underlying mechanism is not clear. Here we report a Matrigel-based feeder-free method to convert human embryonic stem cells and induced pluripotent stem cells into EPSCs and demonstrate the extended pluripotency in terms of molecular features, chimeric ability, and transcriptome. We further identify chemicals targeting glycolysis and histone methyltransferase to facilitate the conversion to and maintenance of feeder-free EPSCs. Altogether, our data not only establish a feeder-free system to generate human EPSCs, which should facilitate the mechanistic studies of extended pluripotency and further applications, but also provide additional insights into the transitions among different pluripotent states.

Set2-mediated H3K36 methylation states redundantly repress the production of antisense transcripts: role in transcription regulation.

Methyltransferase Set2-mediated methylation of histone H3 lysine 36 (H3K36), which involves the addition of up to three methyl groups at this site, has been demonstrated to function in many chromatin-coupled events. The methylation of H3K36 is known to recruit different chromatin effector proteins, affecting transcription, mRNA splicing and DNA repair. In this study, we engineered two yeast set2 mutants that lack H3K36 mono/dimethylation (H3K36me1/2) and trimethylation (H3K36me3), respectively, and characterized their roles in the production of antisense transcripts under nutrient-rich conditions. Using our new bioinformatics identification pipeline analysis, we are able to identify a larger number of antisense transcripts in set2∆ cells than has been published previously. We further show that H3K36me1/2 or H3K36me3 redundantly repressed the production of antisense transcripts. Moreover, gene ontology (GO) analysis implies that H3K36me3-mediated antisense transcription might play a role in DNA replication and DNA damage repair, which is independent of regulation of the corresponding sense gene expression. Overall, our results validate a coregulatory mechanism of different H3K36 methylation states, particularly in the repression of antisense transcription.

O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity.

Pathological TDP-43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP); however, how TDP-43 aggregation and function are regulated remain poorly understood. Here, we show that O-GlcNAc transferase OGT-mediated O-GlcNAcylation of TDP-43 suppresses ALS-associated proteinopathies and promotes TDP-43's splicing function. Biochemical and cell-based assays indicate that OGT's catalytic activity suppresses TDP-43 aggregation and hyperphosphorylation, whereas abolishment of TDP-43 O-GlcNAcylation impairs its RNA splicing activity. We further show that TDP-43 mutations in the O-GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP-43 overexpression in Drosophila motor neurons. We finally demonstrate that O-GlcNAcylation of TDP-43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. Our findings suggest that O-GlcNAcylation might be a target for the treatment of TDP-43-linked pathogenesis.

The methyltransferase SETD3-mediated histidine methylation: Biological functions and potential implications in cancers.

SETD3 belongs to a family of SET-domain containing proteins. Recently, SETD3 was found as the first and so-far the only known metazoan histidine methyltransferase that catalyzes actin histidine 73 (His73) methylation, a pervasive modification which was discovered more than 50 years ago. In this review, we summarize some recent advances in SETD3 research, focusing on structural properties, substrate-recognition features, and physiological functions. We particularly highlight potential pathological relevance of SETD3 in human cancers and raise some questions to promote discussion about this novel histidine methyltransferase.

Rph1 coordinates transcription of ribosomal protein genes and ribosomal RNAs to control cell growth under nutrient stress conditions.

Coordinated regulation of ribosomal RNA (rRNA) synthesis and ribosomal protein gene (RPG) transcription by eukaryotic RNA polymerases (RNAP) is a key requirement for growth control. Although evidence for balance between RNPI-dependent 35S rRNA production and RNAPII-mediated RPG transcription have been described, the molecular basis is still obscure. Here, we found that Rph1 modulates the transcription status of both rRNAs and RPGs in yeast. We show that Rph1 widely associates with RNAPI and RNAPII-transcribed genes. Deletion of RPH1 remarkably alleviates cell slow growth caused by TORC1 inhibition via derepression of rRNA and RPG transcription under nutrient stress conditions. Mechanistically, Rim15 kinase phosphorylates Rph1 upon rapamycin treatment. Phosphorylation-mimetic mutant of Rph1 exhibited more resistance to rapamycin treatment, decreased association with ribosome-related genes, and faster cell growth compared to the wild-type, indicating that Rph1 dissociation from chromatin ensures cell survival upon nutrient stress. Our results uncover the role of Rph1 in coordination of RNA polymerases-mediated transcription to control cell growth under nutrient stress conditions.

Old factors, new players: transcriptional regulation of autophagy.

Macroautophagy/autophagy is a catabolic process that allows cells to adapt to environmental changes and maintain energy homeostasis. This multistep process is regulated at several levels, including transcriptionally regulating autophagy-related (ATG) gene expression through the action of transcription regulators. Very recently, Wen et al. and we have provided more evidence that two well-known transcription factors regulate different ATG genes to control either nonselective or selective forms of autophagy, respectively. Under nitrogen-starvation conditions, the Spt4-Spt5 complex derepresses ATG8 and ATG41 expression and upregulates bulk autophagy activity. By contrast, under glucose-starvation conditions, the Paf1 complex (the polymerase-associated factor 1 complex, Paf1C) specifically modulates expression of ATG11 and ATG32 to regulate mitophagy. These studies suggest the potential existence of other transcription regulators yet to be discovered that function in the regulation of diverse autophagy pathways.Abbreviations: AMPK: AMP-activated protein kinase; ATG: autophagy-related; NELF: negative elongation factor; Paf1C/PAF1C: polymerase-associated factor 1 complex; RNAP II: RNA polymerase II; Rpd3L: Rpd3 large complex.

The Paf1 complex transcriptionally regulates the mitochondrial-anchored protein Atg32 leading to activation of mitophagy.

Mitophagy is a critical process that safeguards mitochondrial quality control in order to maintain proper cellular homeostasis. Although the mitochondrial-anchored receptor Atg32-mediated cargo-recognition system has been well characterized to be essential for this process, the signaling pathway modulating its expression as a contribution of governing the mitophagy process remains largely unknown. Here, bioinformatics analyses of epigenetic or transcriptional regulators modulating gene expression allow us to identify the Paf1 complex (the polymerase-associated factor 1 complex, Paf1C) as a transcriptional repressor of ATG genes. We show that Paf1C suppresses glucose starvation-induced autophagy, but does not affect nitrogen starvation- or rapamycin-induced autophagy. Moreover, we show that Paf1C specifically regulates mitophagy through modulating ATG32 expression. Deletion of the genes encoding two core subunits of Paf1C, Paf1 and Ctr9, increases ATG32 and ATG11 expression and facilitates mitophagy activity. Although Paf1C is required for many histone modifications and gene activation, we show that Paf1C regulates mitophagy independent of its positive regulatory role in other processes. More importantly, we also demonstrate the mitophagic role of PAF1C in mammals. Overall, we conclude that Paf1C maintains mitophagy at a low level through binding the promoter of the ATG32 gene in glucose-rich conditions. Dissociation of Paf1C from ATG32 leads to the increased expression of this gene, and mitophagy induction upon glucose starvation. Thus, we uncover a new role of Paf1C in modulating the mitophagy process at the transcriptional level. ABBREVIATIONS: AMPK: AMP-activated protein kinase; ATP5F1A: ATP synthase F1 subunit alpha; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: chlorophenylhydrazone; DFP: chelator deferiprone; GFP: green fluorescent protein; H2B-Ub1: H2B monoubiquitination; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; KD: kinase dead; OPTN, optineurin; Paf1: polymerase-associated factor 1; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RT-qPCR: real-time quantitative PCR; SD-N: synthetic dropout without nitrogen base; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; WT: wild-type; YPD: yeast extract peptone dextrose; YPL: yeast extract peptone lactate.

A methylation-phosphorylation switch determines Plk1 kinase activity and function in DNA damage repair.

Polo-like kinase 1 (Plk1) is a crucial regulator of cell cycle progression; but the mechanism of regulation of Plk1 activity is not well understood. We present evidence that Plk1 activity is controlled by a balanced methylation and phosphorylation switch. The methyltransferase G9a monomethylates Plk1 at Lys209, which antagonizes phosphorylation of T210 to inhibit Plk1 activity. We found that the methyl-deficient Plk1 mutant K209A affects DNA replication, whereas the methyl-mimetic Plk1 mutant K209M prolongs metaphase-to-anaphase duration through the inability of sister chromatids separation. We detected accumulation of Plk1 K209me1 when cells were challenged with DNA damage stresses. Ablation of K209me1 delays the timely removal of RPA2 and RAD51 from DNA damage sites, indicating the critical role of K209me1 in guiding the machinery of DNA damage repair. Thus, our study highlights the importance of a methylation-phosphorylation switch of Plk1 in determining its kinase activity and functioning in DNA damage repair.

MiR-15b and miR-322 inhibit SETD3 expression to repress muscle cell differentiation.

SETD3 is a member of SET-domain containing methyltransferase family, which plays critical roles in various biological events. It has been shown that SETD3 could regulate the transcription of myogenic regulatory genes in C2C12 differentiation and promote myoblast determination. However, how SETD3 is regulated during myoblast differentiation is still unknown. Here, we report that two important microRNAs (miRNAs) could repress SETD3 and negatively contribute to myoblast differentiation. Using microRNA (miRNA) prediction engines, we identify and characterize miR-15b and miR-322 as the primary miRNAs that repress the expression of SETD3 through directly targeting the 3'-untranslated region of SETD3 gene. Functionally, overexpression of miR-15b or miR-322 leads to the repression of endogenous SETD3 expression and the inhibition of myoblast differentiation, whereas inhibition of miR-15b or miR-322 derepresses endogenous SETD3 expression and facilitates myoblast differentiation. In addition, knockdown SETD3 in miR-15b or miR-322 repressed myoblasts is able to rescue the facilitated differentiation phenotype. More interestingly, we revealed that transcription factor E2F1 or FAM3B positively or negatively regulates miR-15b or miR-322 expression, respectively, during muscle cell differentiation, which in turn affects SETD3 expression. Therefore, our results establish two parallel cascade regulatory pathways, in which transcription factors regulate microRNAs fates, thereby controlling SETD3 expression and eventually determining skeletal muscle differentiation.

USP2a Supports Metastasis by Tuning TGF-ß Signaling.

TGF-ß has been demonstrated to promote tumor metastasis, and the regulatory mechanisms are poorly understood. Here, we report the role of USP2a in promoting metastasis by facilitating TGF-ß-triggered signaling. USP2a interacts with TGFBR1 and TGFBR2 upon TGF-ß stimulation and removes K33-linked polyubiquitin chains from Lys502 of TGFBR1, promoting the recruitment of SMAD2/3. Simultaneously, TGFBR2 phosphorylates Ser207/Ser225 of USP2a, leading to the disassociation of SMAD2/3 from TGFBR1. The phosphorylation of USP2a and SMAD2 is positively correlated in human tumor biopsies, and USP2a is hyper-phosphorylated in lung adenocarcinomas with lymph node invasion. Depletion or pharmacologic inhibition of USP2a dampens TGF-ß-triggered signaling and metastasis. Our findings have characterized an essential role of USP2a as a potential target for treatment of metastatic cancers.

Pathological hydrogen peroxide triggers the fibrillization of wild-type SOD1 via sulfenic acid modification of Cys-111.

Amyotrophic lateral sclerosis (ALS) involves the abnormal posttranslational modifications and fibrillization of copper, zinc superoxide dismutase (SOD1) and TDP-43. However, how SOD1-catalyzed reaction product hydrogen peroxide affects amyloid formation of SOD1 and TDP-43 remains elusory. 90% of ALS cases are sporadic and the remaining cases are familial ALS. In this paper, we demonstrate that H2O2 at pathological concentrations triggers the fibrillization of wild-type SOD1 both in vitro and in SH-SY5Y cells. Using an anti-dimedone antibody that detects sulfenic acid modification of proteins, we found that Cys-111 in wild-type SOD1 is oxidized to C-SOH by pathological concentration of H2O2, followed by the formation of sulfenic acid modified SOD1 oligomers. Furthermore, we show that such SOD1 oligomers propagate in a prion-like manner, and not only drive wild-type SOD1 to form fibrils in the cytoplasm but also induce cytoplasm mislocalization and the subsequent fibrillization of wild-type TDP-43, thereby inducing apoptosis of living cells. Thus, we propose that H2O2 at pathological concentrations triggers the fibrillization of wild-type SOD1 and subsequently induces SOD1 toxicity and TDP-43 toxicity in neuronal cells via sulfenic acid modification of Cys-111 in SOD1. Our Western blot and ELISA data demonstrate that sulfenic acid modified wild-type SOD1 level in cerebrospinal fluid of 15 sporadic ALS patients is significantly increased compared with 6 age-matched control patients. These findings can explain how H2O2 at pathologic concentrations regulates the misfolding and toxicity of SOD1 and TDP-43 associated with ALS, and suggest that sulfenic acid modification of wild-type SOD1 should play pivotal roles in the pathogenesis of sporadic ALS.

Induction of INKIT by Viral Infection Negatively Regulates Antiviral Responses through Inhibiting Phosphorylation of p65 and IRF3.

The transcription factors p65 and IRF3 play key roles in the induction of cellular antiviral responses. Phosphorylation of p65 and IRF3 is required for their activity and constitutes a key checkpoint. Here we report that viral infection induced upregulation of INKIT, an inhibitor for NF-κB and IRF3 that restricted innate antiviral responses by blocking phosphorylation of p65 and IRF3. INKIT overexpression inhibited virus-induced phosphorylation of p65 and IRF3 and expression of downstream genes. In contrast, knockdown or knockout of INKIT had the opposite effect: Inkit-/- mice produced elevated levels of type I interferons and proinflammatory cytokines and were more resistant to lethal viral infection compared to wild-type. INKIT interacted with IKKα/ß and TBK1/IKKɛ, impairing the recruitment and phosphorylation of p65 and IRF3. Viral infection induced IKK-mediated phosphorylation of INKIT at Ser58, resulting in its dissociation from the IKKs. Our findings thus uncover INKIT as a regulator of innate antiviral responses.