Tom Klaus Kerppola

Cells must control genes that are induced by virus infection to mitigate deleterious consequences of inflammation. We investigated the mechanisms whereby Keap1 moderates the transcription of genes that are induced by Sendai virus infection in mouse embryo fibroblasts (MEFs). Keap1−/− deletions increased the transcription of virus induced genes independently of Nrf2. Keap1 moderated early virus induced gene transcription. Virus infection induced Keap1 to bind Ifnb1, Tnf and Il6, and reduced Keap1 binding at Cdkn1a and Ccng1. Virus infection induced G9a‐GLP and NFκB p50 recruitment, and H3K9me2 deposition. Keap1−/− deletions eliminated G9a‐GLP and NFκB p50 recruitment, and H3K9me2 deposition, but they did not affect NFκB p65, IRF3 or cJun recruitment. G9a‐GLP inhibitors (BIX01294, MS012, BRD4770) enhanced virus induced gene transcription in MEFs with intact Keap1, but not in MEFs with Keap1−/− deletions. G9a‐GLP inhibitors augmented Keap1 binding to virus induced genes in infected MEFs, and to cell cycle genes in uninfected MEFs. G9a‐GLP inhibitors augmented NFκB subunit recruitment in MEFs with intact Keap1. G9a‐GLP inhibitors stabilized Keap1 retention in permeabilized MEFs. G9a‐GLP lysine methyltransferase activity was required for Keap1 to moderate transcription, and it moderated Keap1 binding to chromatin. The interdependent effects of Keap1 and G9a‐GLP on the recruitment of each other and on the moderation of virus induced gene transcription constitute a feedback circuit. Keap1 and the electrophile tBHQ reduced virus induced gene transcription through different mechanisms, and they regulated the recruitment of different NFκB subunits. Characterization of the mechanisms whereby Keap1, G9a‐GLP and NFκB p50 moderate virus induced gene transcription can facilitate the development of immunomodulatory agents.

Cells must moderate transcription that is induced by virus infection to mitigate deleterious consequences of inflammation. We investigated the mechanisms whereby Keap1 moderates the transcription of genes that are induced by Sendai virus infection in mouse embryo fibroblasts (MEFs). Virus infection induced Keap1 to bind Ifnb1, Tnf and Il6 , and reduced Keap1 binding at Cdkn1a and Ccng1 . Keap1 was required for G9a and GLP to bind and to deposit H3K9me2 at these genes upon virus infection. Keap1 moderated the transcription of genes that were induced by virus infection in concert with G9a, GLP, and NFкB p50 recruitment. G9a-GLP lysine methyltransferase activity was required for Keap1 to moderate the transcription of virus induced genes. G9a-GLP inhibitors enhanced the transcription of virus induced genes, and they augmented Keap1 and NFкB p50 recruitment, in parallel with the inhibition of H3K9me2 deposition. The interdependent effects of Keap1 and G9a-GLP on transcription and on the recruitment of each other constitute a feedback circuit that moderates the transcription of virus induced genes. G9a-GLP inhibitors augmented Keap1 binding to different genes in virus infected and in uninfected MEFs, whereas they inhibited H3K9me2 deposition that was induced by virus infection selectively. G9a-GLP inhibitors stabilized Keap1 retention in permeabilized MEFs and augmented Keap1 binding to specific genes in parallel. Keap1 was required for NFкB p50 recruitment, and for the augmentation of NFкB binding by G9a-GLP inhibitors. Keap1 and the electrophile tBHQ attenuated virus induced gene transcription through independent mechanisms, and they regulated the recruitment of different NFкB subunits. Importance: Excess and maladaptive immune responses to virus infections are a major contributing factor to the morbidity and mortality of COVID-19 and other diseases. Conversely, inadequate immune responses to vaccines and pathogens by individuals with suppressed immune function expose them to infections. Currently available drugs that enable therapeutic management of immune responses have low specificity and can blunt beneficial immune functions. The molecular mechanisms that moderate the transcription of genes that are induced by virus infection are incompletely understood. Characterization of the mechanisms whereby Keap1, G9a-GLP and NFкB p50 moderate virus induced gene transcription in mouse embryo fibroblasts represents the first step toward the identification of new targets for therapeutic agents that can modulate immune responsiveness.

Proinflammatory cytokine gene transcription must be moderated to avoid the pathological consequences of excess cytokine production. The relationships between virus infection and the mechanisms that moderate cytokine transcription are incompletely understood. We investigated the influence of Keap1 on cytokine gene induction by Sendai virus infection in mouse embryo fibroblasts. Virus infection induced Keap1 binding to the Ifnb1, Tnf, and Il6 genes. Keap1 moderated viral induction of their transcription by mechanisms that did not require Nrf2. Keap1 was required for NF-κB p50 recruitment, but not for NF-κB p65 or IRF3 recruitment, to these genes. Keap1 formed complexes with NF-κB p50 and NF-κB p65, which were visualized using bimolecular fluorescence complementation analysis. These bimolecular fluorescence complementation complexes bound chromosomes in live cells, suggesting that Keap1 could bind chromatin in association with NF-κB proteins. Keap1 was required for viral induction of G9a-GLP lysine methyltransferase binding and H3K9me2 modification at cytokine genes. G9a-GLP inhibitors counteracted transcription repression by Keap1 and enhanced Keap1 and NF-κB recruitment to cytokine genes. The interrelationships among Keap1, NF-κB, and G9a-GLP recruitment, activities, and transcriptional effects suggest that they form a feedback circuit, which moderates viral induction of cytokine transcription. Nrf2 counteracted Keap1 binding to cytokine genes and the recruitment of NF-κB p50 and G9a-GLP by Keap1. Whereas Keap1 has been reported to influence cytokine expression indirectly through its functions in the cytoplasm, these findings provide evidence that Keap1 regulates cytokine transcription directly in the nucleus. Keap1 binds to cytokines genes upon virus infection and moderates their induction by recruiting NF-κB p50 and G9a-GLP. Key Points Keap1 moderates cytokine induction upon virus infection by binding to chromatin. Keap1 is required for NF-κB p50 and G9a-GLP recruitment, which represses transcription. Keap1 forms complexes with NF-κB p50 and NF-κB p65 that bind chromatin in living cells. Visual Abstract

Innate immunity requires a balance of positive and negative regulators of cytokine transcription. Keap1 deletion in mice alters innate immunity and inflammation. We investigated the influence of Keap1 on cytokine gene induction by Sendai virus infection in mouse embryo fibroblasts (MEFs). Keap1 bound to the Ifnb1, Tnf and Il6 promoters upon virus infection, and moderated viral induction of their transcription. Keap1 was required for viral induction of NFκB p50 and G9a-GLP lysine methyltransferase binding to these genes. Keap1 formed BiFC complexes with NFκB p50 that were localized to the nuclei in a subset of cells. Nrf2 counteracted viral induction of Keap1 binding to the promoters, and the effects of Keap1 on NFκB p50 and on G9a-GLP recruitment. Lysine methyltransferase inhibitors enhanced viral induction of transcription of the genes that were bound by Keap1 only in MEFs with intact Keap1, and not in Keap1-/- MEFs. They also enhanced NFκB p50 and NFκB p65 recruitment to these genes only in MEFs with intact Keap1, whereas they inhibited G9a-GLP recruitment. The reciprocal effects of Keap1 and of G9a-GLP lysine methyltransferase activity on chromatin binding by each other constitute a feedback circuit that moderates viral induction of cytokine transcription. Summary: Virus infection induces Keap1 binding to cytokine promoters, which recruits NFκB p50 and G9a-GLP and moderates their transcription.

Background and Purpose: To further the development of new agents for the treatment of adrenocortical carcinoma (ACC), we characterized the molecular and cellular mechanisms of cytotoxicity by the adrenalytic compound ATR‐101 (PD132301‐02).Experimental Approach: We compared the effects of ATR‐101, PD129337, and ABC transporter inhibitors on cholesterol accumulation and efflux, on cortisol secretion, on ATP levels, and on caspase activation in ACC‐derived cell lines. We examined the effects of these compounds in combination with methyl‐β‐cyclodextrin or exogenous cholesterol to determine the roles of altered cholesterol levels in the effects of these compounds.Key Results: ATR‐101 caused cholesterol accumulation, ATP depletion, and caspase activation within 30 minutes after addition to ACC‐derived cells, whereas PD129337 did not. Suppression of cholesterol accumulation by methyl‐β‐cyclodextrin or exogenous cholesterol, prevented ATP depletion and caspase activation by ATR‐101. ATR‐101 blocked cholesterol efflux and cortisol secretion, suggesting that it inhibited ABCA1, ABCG1, and MDR1 transporters. Combinations of ABCA1, ABCG1, and MDR1 inhibitors were also cytotoxic. Combinations of ATR‐101 with inhibitors of ABCG1, MDR1, or mitochondrial functions had increased cytotoxicity. Inhibitors of steroidogenesis reduced ATP depletion by ATR‐101, whereas U18666A enhanced cholesterol accumulation and ATP depletion together with ATR‐101. ATR‐101 repressed ABCA1, ABCG1, and IDOL transcription by mechanisms that were distinct from the mechanisms that caused cholesterol accumulation.Conclusions and Implications: Inhibition of multiple ABC transporters and the consequent accumulation of cholesterol mediated the cytotoxicity of ATR‐101. Compounds that replicate these effects in tumours are likely to be useful in the treatment of ACC.

Adrenocortical carcinoma (ACC) generally has poor prognosis. Existing treatments provide limited benefit for most patients with locally advanced or metastatic tumors. We investigated the mechanisms for the cytotoxicity, xenograft suppression, and adrenalytic activity of ATR-101 (PD132301-02), a prospective agent for ACC treatment. Oral administration of ATR-101 inhibited the establishment and impeded the growth of ACC-derived H295R cell xenografts in mice. ATR-101 induced H295R cell apoptosis in culture and in xenografts. ATR-101 caused mitochondrial hyperpolarization, reactive oxygen release, and ATP depletion within hours after exposure, followed by cytochrome c release, caspase-3/7 activation, and membrane permeabilization. The increase in mitochondrial membrane potential occurred concurrently with the decrease in cellular ATP levels. When combined with ATR-101, lipophilic free radical scavengers suppressed the reactive oxygen release, and glycolytic precursors prevented the ATP depletion, abrogating ATR-101 cytotoxicity. ATR-101 directly inhibited F1F0-ATPase activity and suppressed ATP synthesis in mitochondrial fractions. ATR-101 administration to guinea pigs caused oxidized lipofuscin accumulation in the zona fasciculata layer of the adrenal cortex, implicating reactive oxygen release in the adrenalytic effect of ATR-101. These results support the development of ATR-101 and other adrenalytic compounds for the treatment of ACC.

Interactions among transcription factors control their physiological functions by regulating their binding specificities and transcriptional activities. We implement a strategy to visualize directly the genomic loci that are bound by multi-protein complexes in single cells in Drosophila. This method is based on bimolecular fluorescence complementation (BiFC) analysis of protein interactions on polytene chromosomes. Drosophila Keap1 (dKeap1)-CncC complexes localized to the nucleus and bound chromatin loci that were not bound preferentially by dKeap1 or CncC when they were expressed separately. dKeap1 and CncC binding at these loci was enhanced by phenobarbital, but not by tert-butylhydroquinone (tBHQ) or paraquat. Endogenous dKeap1 and CncC activated transcription of the Jheh (Jheh1, Jheh2, Jheh3) and dKeap1 genes at these loci, whereas CncC alone activated other xenobiotic response genes. Ectopic dKeap1 expression increased CncC binding at the Jheh and dKeap1 gene loci and activated their transcription, whereas dKeap1 inhibited CncC binding at other xenobiotic response gene loci and suppressed their transcription. The combinatorial chromatin-binding specificities and transcriptional activities of dKeap1-CncC complexes mediated the selective activation of different sets of genes by different xenobiotic compounds, in part through feed-forward activation of dKeap1 transcription.

Embryonic stem (ES) cells express pluripotency-associated genes and repress differentiation-inducible genes. The activities of these genes are coordinately reversed during differentiation. The changes in the transcriptome upon conditional KAP1 knockout in ES cells overlapped with the changes during embryoid body formation. KAP1 repressed differentiation-inducible genes and derepressed pluripotency-associated genes in ES cells. KAP1 formed complexes with polycomb repressive complexes 1 (PRC1) through an interaction that was mediated by the KAP1 coiled-coil region. KAP1 and PRC1 bound cooperatively at the promoters of differentiation-inducible genes and repressed their transcription. In contrast, KAP1 bound the transcribed and flanking sequences of pluripotency-associated genes, did not enhance PRC1 binding, and derepressed their transcription. KAP1 had opposite effects on differentiation-inducible and pluripotency-associated gene transcription both in ES cells and in differentiating embryoid bodies. The region of KAP1 that mediated the interaction with PRC1 was required for KAP1 enhancement of PRC1 binding and for KAP1 repression of transcription at differentiation-inducible promoters. This region of KAP1 was not required for KAP1 suppression of PRC1 binding or for KAP1 derepression of transcription at pluripotency-associated promoters. The opposite effects of KAP1 on the transcription of differentiation-inducible versus pluripotency-associated genes contributed to the reciprocal changes in their transcription during differentiation.

Ubiquitin-family peptide conjugation regulates the functions and stabilities of many proteins. Numerous cellular proteins are modified by covalent conjugation of ubiquitin-family peptides to specific lysine residues. These modifications provide a flexible means for regulating the properties of the substrate proteins. Because ubiquitin can be conjugated to substrate proteins at many different sites and in many topological configurations, these modifications have the potential to confer a wide range of functional states to the modified proteins. Ubiquitin conjugation is typically detected by immunoprecipitation of a putative substrate protein followed by immunoblotting to detect ubiquitin conjugated to the substrate. However, this assay cannot be used to detect ubiquitin conjugates in live cells. It is also difficult to determine the subcellular distribution of a specific ubiquitin conjugate using this approach. To visualize ubiquitin conjugates in live cells, we have developed the ubiquitin-mediated fluorescence complementation assay, which is based on the association of fragments of fluorescent proteins when ubiquitin fused to one fragment is conjugated to a substrate protein fused to a complementary fragment. This protocol focuses on the visualization of ubiquitin conjugated in cultured mammalian cells, but it can be adapted to any cell type or aerobically grown organism that can be genetically modified to express the fusion proteins.