key: cord-0952779-6hkdmc9o
authors: Burns, Veronica Elizabeth; Kerppola, Tom Klaus
title: Keap1 moderates the transcription of virus induced genes through G9a-GLP and NFкB p50 recruitment, and H3K9me2 deposition
date: 2022-02-10
journal: bioRxiv
DOI: 10.1101/2022.02.08.479619
sha: 0802b8fb9265598076df5e90032e1c5c4df22e5c
doc_id: 952779
cord_uid: 6hkdmc9o

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.

Genes that are induced upon virus infection protect organisms from pathogenesis. Excess transcription of virus induced genes has deleterious effects. Elucidation of the factors and molecular mechanisms that moderate the transcription of virus induced genes is necessary to understand how homeostasis is maintained during viral infections.

Keap1 (Kelch-like ECH-associated protein) deletion can cause spontaneous inflammation in several mouse organs as well as reduce experimentally induced inflammatory responses. Germline Keap1 deletion combined with conditional Nrf2 deletion in keratinocytes causes kidney inflammation that is associated with hydronephrosis and polyuria (1) . Conditional Keap1 deletion in Tregs causes lung and liver inflammation (2) . Conversely, conditional Keap1 deletions in Clara cells, in macrophages and granulocytes, in thymocytes, and in renal tubule cells reduce experimentally induced inflammatory responses (3) (4) (5) (6) . Since Keap1 fl alleles can be hypomorphic and their deletion is frequently inefficient, conditional Keap1 deletion may not produce loss of function phenotypes. The pro-and anti-inflammatory effects of Keap1 deletions suggest that Keap1 influences immune functions through multiple mechanisms. To characterize the roles of Keap1 in immune regulation it is important to identify the cellular and molecular processes that mediate Keap1 effects on immune functions. Keap1 depletion can enhance or reduce the induction of cytokine transcription in cultured cells. Keap1 depletion in RAW 264.7 and THP-1 macrophages/monocytes enhances Il6 induction by LPS (7) . Similarly, Keap1 depletion in human primary monocyte derived macrophages enhances inflammatory cytokine induction by Mycobacterium avium infection (8) . These increases in cytokine induction correlate with increases in IKK/ levels and phosphorylation (7, 8) . In contrast, conditional Keap1 deletion in mouse bone marrow derived macrophages reduces cytokine induction by LPS and IFN (9) . This reduction of cytokine induction correlates with an increase in Nrf2. The contrasting effects of Keap1 depletion on cytokine induction in different macrophages could be due to indirect effects of Keap1 depletion, or they could be due to differences in the immune exposures of the macrophages prior to Keap1 depletion. To identify the direct and innate effects of Keap1 on cytokine transcription, it is important to determine how Keap1 regulates transcription in cells that have not been altered by prior immune exposures.

Fibroblasts respond rapidly to virus infection and serve as sentinel cells of the immune system (10) .

Lymphocytic choriomeningitis virus infection in mice induces immune response genes in the fibroblasts of many organs (11) . Influenza virus infection in mice induces changes in lung fibroblast gene expression in regions of interstitial inflammation (12) . Fibroblasts are a major constituent of the tumor microenvironment, which influences immune responses to tumor initiation, tumor growth, and immune checkpoint inhibitor therapeutics.

Mouse embryo fibroblasts (MEFs) can be used to study mechanisms of gene regulation in naïve cells that have not been exposed to immune responses. Understanding of the mechanisms for regulation of the transcription of virus induced genes in MEFs provides a basis for the investigation of transcription regulation in other cells that control immune functions.

Sendai virus infection induces Keap1 to bind cytokine genes in MEFs (13) . The levels of cytokine transcripts are higher in virus infected Keap1-/-MEFs than in MEFs with intact Keap1, suggesting that Keap1 moderates cytokine induction. To elucidate how Keap1 moderates the transcription of virus induced genes, it is necessary to determine the effects of Keap1 on transcription factor binding and on chromatin modifications, and their reciprocal effects on Keap1 binding to chromatin. Keap1 binding to chromatin was discovered on Drosophila polytene chromosomes (14, 15) . Keap1 binds to Drosophila genomic loci that encompass genes that are associated with innate immunity as well as other functions.

It is important to compare the effects of virus infection on Keap1 binding to different classes of genes.

The G9a (encoded by Ehmt2) and GLP (encoded by Ehmt1) lysine methyltransferases catalyze histone H3 lysine 9 dimethylation (H3K9me2) (16) . They have overlapping and interdependent activities (17) . They are required for the differentiation and functions of both innate and adaptive immune cells (18) (19) (20) . G9a and GLP are associated with the repression of cytokine transcription (21) (22) (23) . It is important to determine the effects of virus infection on G9a and GLP binding to virus induced genes and the relationships between Keap1 and G9a-GLP recruitment.

Many pharmacological inhibitors of G9a and GLP have been developed (24) (25) (26) (27) . These compounds inhibit G9a and GLP lysine methyltransferase activities through different mechanisms and with different potencies. G9a

and GLP can methylate substrates other than H3, and some functions of G9a and GLP do not require their lysine methyltransferase activities (18, 28) . It is important to determine if H3K9me2 deposition or other G9a and GLP activities moderate regulatory protein binding to chromatin, or the transcription of virus induced genes. NFB p50 (encoded by Nfkb1) can both repress and activate cytokine transcription, whereas NFB p65 (encoded by RelA) activates transcription (29) (30) (31) (32) (33) . Conditional Nfkb1 deletion in dendritic cells or in follicular B cells of bone marrow chimeric mice causes autoimmunity (34, 35) . Heterozygous loss of function mutations in NFKB1 are associated with both sporadic and familial common variable immunodeficiency (36, 37) . Many of these patients present with autoinflammatory or autoimmune complications (38, 39) . It is important to identify the differences in NFB p50 and NFB p65 regulation that can contribute to their distinct effects on immune functions, and to establish the effects of Keap1 on NFB p50 and NFB p65 binding to different genes in virus infected and in uninfected cells.

Nrf2. Some electrophiles reduce cytokine induction and autoinflammatory reactions (40) . Dimethylfumarate is used to treat psoriasis and multiple sclerosis. The potential roles of Keap1 in electrophile effects on immunomodulatory gene transcription need to be evaluated.

We tested the hypothesis that Sendai virus infection induces Keap1 to bind specific genes, and that Keap1 moderates the transcription of those genes. We examined the relationships between Keap1 and G9a-GLP recruitment and H3K9me2 deposition at virus induced genes, and the interdependence of their effects on virus induced transcription. We investigated the roles of lysine methyltransferase activity in G9a-GLP effects on transcription and on recruitment of other proteins at virus induced genes by using several structurally dissimilar G9a-GLP inhibitors. We compared the effects of Keap1 on NFB p50 and NFB p65 binding to different genes and to proximal and distal elements. We also compared the effects of Keap1 and of electrophiles on transcription and on NFB subunit recruitment at virus induced genes. We found that Keap1, G9a, GLP and NFB p50 regulated recruitment of each other, and moderated transcription at virus induced genes through H3K9me2 deposition.

We compared the effects of Keap1 on the transcription of genes that were induced by Sendai virus infection and of uninduced genes in MEFs. We measured the transcript levels in MEFs with intact Keap1 and in MEFs with Keap1-/-deletions (Fig. 1A) . To avoid the indirect effects of Keap1-/-deletions that result from constitutive Nrf2 activation, we focused on the effects of Keap1-/-deletions in MEFs that also carried Nrf2-/deletions (Keap1-/-MEFs). We measured transcript levels at different times after virus infection to characterize the effects of Keap1 on the primary transcriptional response.

Virus infection induced Ifnb1, Tnf and Il6 transcription more rapidly and to higher levels in Keap1-/-MEFs than in MEFs with intact Keap1 (Fig. 1A) . The higher transcript levels were observed beginning at 2 h after virus infection. The peak Ifnb1, Tnf and Il6 transcript levels were 3-to 8-fold higher on average in 7 different sets of Keap1-/-MEFs relative to MEFs with intact Keap1. Keap1 reduced the levels of virus induced transcripts at the earliest times after infection that were tested by mechanisms that were independent of Nrf2, consistent with direct moderation of the transcription of virus induced genes by Keap1.

We examined the effects of Keap1 on cell cycle associated gene transcription because Keap1 interacts with DNA replication factors, and because the stability of Keap1 retention in the nucleus varies at different stages of the cell cycle (41) . The basal levels of Cdkn1a and Ccng1 transcripts were higher in Keap1-/-MEFs than in MEFs with intact Keap1 (Fig. 1A) . Virus infection induced Cdkn1a transcription in two of the three independent Keap1-/-MEFs that were tested. In contrast, virus infection did not induce Cdkn1a transcription in MEFs with intact Keap1. 

We compared the effects of virus infection on Keap1 binding to the promoter regions of cytokine and cell cycle associated genes. Virus infection induced Keap1 to bind Ifnb1, Tnf and Il6 in the 7 independent MEFs with intact Keap1 that were tested, and not in Keap1-/-MEFs (Fig. 1B, 1C) . The Keap1 ChIP signals at these genes were 11-to 14-fold higher, on average, in virus infected MEFs than in uninfected MEFs. We determined that the ChIP signals reflected Keap1 binding to virus induced genes by using antibodies that recognized different epitopes in Keap1 and by employing several independent criteria to establish the validity of the ChIP signals. Since Keap1 bound to these genes upon virus infection, and since the levels of these transcripts were higher in Keap1-/-MEFs, we inferred that Keap1 moderated their transcription by binding to the genes upon virus infection.

Keap1 bound to Cdkn1a and Ccng1 in uninfected MEFs. Virus infection reduced Keap1 binding to these genes in the 4 independent MEFs that were tested (Fig. 1B, 1C) . The Keap1 ChIP signals at Cdkn1a and Ccng1 were 21% and 23% lower, on average, in virus infected MEFs than in uninfected MEFs. The differences in the effects of virus infection on Keap1 binding to Ifnb1, Tnf and Il6 versus Cdkn1a and Ccng1 suggest that virus infection regulated Keap1 binding to different genes by selective mechanisms.

G9a and GLP bind to cytokine genes in virus infected MEFs with intact Keap1, but not in Keap1-/-MEFs (13). 

intact Keap1 at the genes that were tested (Fig. 1B, 1C) . Thus, the requirement for Keap1 was unique to G9a-GLP and NFB p50 recruitment among the chromatin binding proteins that were tested.

Inhibitors of G9a and GLP lysine methyltransferase activities enhance cytokine transcription and augment Keap1 binding at cytokine genes (13, 23) . We measured the effects of G9a-GLP inhibitors on virus induced gene transcription in MEFs with intact Keap1 and MEFs with Keap1-/-deletions to investigate the relationships between Keap1 and G9a-GLP in the moderation of virus induced gene transcription. The G9a-GLP inhibitor BIX01294 (42) was added to the MEFs one hour before virus infection to focus on the direct effects of G9a-GLP inhibition.

BIX01294 enhanced Ifnb1, Tnf, and Il6 transcription in the 4 independent MEFs with intact Keap1 that were tested ( Fig. 2A , left column). The peak Ifnb1, Tnf and Il6 transcript levels were 2.3-to 2.4-fold higher on average in the MEFs with intact Keap1 that were cultured with BIX01294 than in the same MEFs that were cultured with vehicle.

In contrast, BIX01294 did not increase the peak Ifnb1, Tnf, or Il6 transcript levels in Keap1-/-MEFs ( Fig 

We compared the effects of G9a-GLP lysine methyltransferase inhibitors on Keap1 binding to virus induced and to uninduced genes in virus infected and in uninfected MEFs. BIX01294 augmented Keap1 binding to Ifnb1, Tnf and Il6 upon virus infection in the 5 independent MEFs with intact Keap1 that were tested (Fig. 2B, 3B ). The Keap1 ChIP signals at these genes were 1.9-to 2.1-fold higher, on average, in virus infected MEFs that were cultured with BIX01294 than in the same MEFs that were cultured with vehicle. BIX01294 augmented the low Keap1 ChIP signals at these genes in uninfected MEFs, but these signals remained near the IgG background. The augmentation of Keap1 binding by BIX01294 was detected by antibodies that were raised against different regions of Keap1. 

We compared BIX01294 effects on NFB p50 and NFB p65 binding to different genes in MEFs with intact Keap1 and in Keap1-/-MEFs. BIX01294 augmented viral induction of NFB p50 binding to Ifnb1, Tnf and Il6 in the 5 independent MEFs with intact Keap1 that were tested (Fig. 2B, 3A) . The p50 ChIP signals were 1.6-to 1.9-fold higher, on average, in virus infected MEFs with intact Keap1 that were cultured with BIX01294 than they were in the same MEFs that were cultured with vehicle. The effects of BIX01294 on NFB p50 binding to Cdkn1a and Ccng1 correlated with BIX01294 effects on Keap1 binding in the same MEFs (Fig. 2B, 3A) . BIX01294 did not affect the low p50 ChIP signals at the genes examined in Keap1-/-MEFs. Since BIX01294 had parallel effects on NFB p50 and Keap1 binding to the genes that were examined, and since Keap1 was essential for NFB p50 binding, it is likely that the effects of BIX01294 on NFB p50 binding were mediated by BIX01294 effects on Keap1 binding to these genes.

BIX01294 augmented NFB p65 binding to the genes that were examined in virus infected MEFs with intact Keap1 (Fig. 2B ). In contrast, BIX01294 did not augment viral induction of NFB p65 binding to these genes in Keap1-/-MEFs. Thus, Keap1 was specifically required for BIX01294 to augment NFB p65 binding to these genes, whereas Keap1 was not required for viral induction of NFB p65 binding. The requirement for Keap1 in the augmentation of NFB p65 binding by BIX01294 suggests that G9a-GLP moderates NFB p65 binding only when G9a-GLP binding to the same genes is facilitated by Keap1.

BIX01294 inhibited virus induced H3K9me2 deposition in MEFs with intact Keap1 (Fig. 2B, 3A) . In contrast, BIX01294 had no effect on basal H3K9me2 in uninfected MEFs or in Keap1-/-MEFs. BIX01294 did not affect the H3 or H3K27me3 ChIP signals in any of the MEFs (Fig. 3A) . The effects of BIX01294 on Keap1 and NFB p50 binding and on transcription correlated with BIX01294 effects on H3K9me2 deposition at virus induced genes (Ifnb1, Tnf and Il6), but not at uninduced genes (Cdkn1a and Ccng1).

We compared the effects of five G9a-GLP inhibitors on Keap1 and on NFB p50 binding to virus induced genes. MS012 interacts with the peptide binding sites of G9a and GLP (27) . MS012 augmented Keap1 and NFB p50 binding to Ifnb1, Tnf and Il6 in the three independent MEFs with intact Keap1 that were tested (Fig. 3A) . The Keap1 ChIP signals at these genes were 2.3-to 2.7-fold higher, and the p50 signals were 1.9-to 2.0-fold higher, in virus infected MEFs that were cultured with MS012 than in the same MEFs that were cultured with vehicle.

MS012 had parallel effects on Keap1 and on NFB p50 binding to different genes in virus infected MEFs (Fig. 3A) .

MS012 did not affect the low p50 ChIP signals in Keap1-/-MEFs. MS012 inhibited virus induced H3K9me2 deposition in MEFs with intact Keap1 (Fig. 3A) . MS012 augmented Keap1 and NFB p50 binding and inhibited H3K9me2 deposition at a lower concentration than BIX01294. The parallel effects of BIX01294 and MS012 both on Keap1 and NFB p50 binding, and on H3K9me2 deposition indicate that these compounds augmented Keap1 and NFB p50 binding by inhibiting G9a-GLP lysine methyltransferase activities at virus induced genes.

BRD4770 inhibits lysine methyltransferases by competition with S-adenosyl methionine and its overall structure differs from those of the other G9a-GLP inhibitors that were tested (26) . BRD4770 augmented Keap1 and NFB p50 binding and inhibited H3K9me2 deposition at virus induced genes in MEFs with intact Keap1 (Fig. 3A ).

BRD4770 reduced H3K27me3 at these genes both in MEFs with intact Keap1 and in Keap1-/-MEFs, suggesting that it inhibited lysine methyltransferases other that G9a-GLP independently of Keap1. UNC0638 and UNC0642

augmented Keap1 and NFB p50 binding and inhibited H3K9me2 deposition at virus induced genes with different efficiencies in different experiments. These differences correlated with differences in their inhibition of H3K9me2 deposition in different MEFs (Fig. 4) . Other compounds, including dexamethasone and tert-butylhydroquinone (tBHQ) did not augment Keap1 or NFB p50 binding to virus induced genes, and had no effect on H3K9me2 deposition (Fig. 6B) . Thus, structurally dissimilar G9a-GLP lysine methyltransferase inhibitors augmented Keap1 and NFB p50 binding, and inhibited H3K9me2 deposition at virus induced genes.

We compared the effects of structurally dissimilar G9a-GLP inhibitors on virus induced gene transcription.

BIX01294, MS012 and BRD4770 enhanced Ifnb1, Tnf and Il6 transcription in virus infected MEFs with intact Keap1 (Fig. 3B ). MS012 enhanced these transcript levels 2.1-to 2.3-fold on average in the two independent MEFs with intact Keap1 that were tested. BRD4770 enhanced transcription in Keap1-/-MEFs, whereas UNC0638 and UNC0642 inhibited transcription in Keap1-/-MEFs (Fig. 3B) . These effects could be due to the inhibition of other lysine methyltransferases, as suggested by the reduction of H3K27me3 by BRD4770 (Fig. 3A) . BIX01294, MS012

and BRD4770 had little effect on Cdkn1a or Ccng1 transcription and did not increase viral M gene transcript accumulation. The preferential enhancement of virus induced gene transcription by BIX01294, MS012 and BRD4770 in MEFs with intact Keap1 suggests that G9a-GLP inhibition counteracted the ability of Keap1 to moderate the transcription virus induced genes.

We investigated if Keap1 was required for NFB p50 to bind the Ccl2 enhancer, which is located at a distance from the Ccl2 promoter. Virus infection induced Keap1 to bind the Ccl2 promoter and NFB p50 to bind the Ccl2 enhancer in MEFs with intact Keap1 (Fig. 4) . A low level of NFB p50 bound to the Ccl2 promoter. Virus infection did not induce NFB p50 to bind the Ccl2 enhancer or promoter in Keap1-/-MEFs. Keap1 was required for NFB p50 to bind the Ccl2 enhancer, though Keap1 binding was detected mainly at the Ccl2 promoter.

We examined the effects of G9a-GLP inhibitors on Keap1 and NFB p50 binding to the Ccl2 promoter and enhancer. The G9a-GLP inhibitors augmented Keap1 binding to the Ccl2 promoter and NFB p50 binding to the Ccl2 enhancer upon virus infection (Fig. 4) . The G9a-GLP inhibitors augmented NFB p50 binding to the Ccl2 enhancer in virus infected MEFs with intact Keap1, and not in Keap1-/-MEFs. The augmentation of Keap1 and NFB p50 binding to the Ccl2 promoter and enhancer by different G9a-GLP inhibitors correlated with the inhibition of virus induced H3K9me2 deposition by these compounds (Fig. 4) .

To determine if G9a-GLP inhibitors affected the stability of Keap1 interactions in cells, we compared Keap1 retention in MEFs that were cultured with BIX01294 or with vehicle, followed by incubation with detergents.

Stronger Keap1 binding to chromatin is predicted to increase Keap1 retention in MEFs. Differences in Keap1 retention in cells have been interpreted to reflect differences in Keap1 binding to chromatin and to other protein complexes (41, 43) .

After culture with vehicle, almost all Keap1 was released from the MEFs during incubations with 0.1% and 0.5% Triton X-100 (Fig. 5A) . In contrast, after culture with BIX01294, most of Keap1 was retained in the MEFs during incubations with 0.1% and 0.5% Triton X-100. One fourth of Keap1 was retained in the latter MEFs during incubation with 1% SDS. Most of Keap1 and histone H3 were released together from MEFs that were cultured with BIX01294 (Fig. 5A) . Culture with BIX01294 did not affect cJun, lamin B1 or histone H3 retention in MEFs during incubations with the detergents that were tested (Fig. 5A) . The stabilization of Keap1 retention in MEFs that were cultured with BIX01294 suggests that BIX01294 strengthened Keap1 interactions in MEFs After culture with vehicle, all NFB p50 was released from the MEFs during incubations with 0.1% and 0.5% Triton X-100 (Fig. 5A ). After culture with BIX01294, 10% of NFB p50 was retained in the MEFs during incubations with 0.1% and 0.5% Triton X-100. BIX01294 did not affect the amount of NFB p65 that was retained in MEFs.

However, S536 phosphorylation increased NFB p65 retention, and the electrophoretic mobility of retained NFB p65 was altered by culture with BIX01294 (Fig. 5A) .

The concentration of BIX01294 and the time of culture that stabilized Keap1 retention in MEFs were similar to the conditions that augmented Keap1 binding to specific genes and inhibited H3K9me2 deposition (Fig. 5B, 2B) . We examined if tBHQ affected Keap1 binding to virus induced genes. Culture with tBHQ did not alter Keap1 binding to these genes upon virus infection (Fig. 6B) . BIX01294 augmented Keap1 binding to Ifnb1, Tnf and Il6 in MEFs that were cultured with tBHQ, suggesting that BIX01294 augmented Keap1 binding to these genes by mechanisms that were unrelated to electrophile responses. The independent effects of Keap1 and of tBHQ on Ifnb1 and Tnf transcription, and the lack of tBHQ effects on Keap1 binding to virus induced genes suggest that Keap1 and tBHQ moderated their transcription through independent mechanisms.

We compared the effects of Keap1 and of tBHQ on NFB subunit binding to virus induced genes. Keap1 was required for NFB p50 to bind virus induced genes, whereas tBHQ had no effect on NFB p50 binding to these genes (Fig. 6B ). In contrast, tBHQ inhibited NFB p65 binding to these genes both in MEFs with intact Keap1 and in Keap1-/-MEFs, whereas Keap1 did not affect NFB p65 binding to these genes in the absence of BIX01294.

Moreover, tBHQ blocked the augmentation of NFB p65 binding by BIX01294, whereas it had no effect on the augmentation of NFB p50 binding in the same MEFs. Both Keap1 and tBHQ attenuated virus induced gene transcription and altered the compositions of NFB complexes that bound to virus induced genes, but the molecular mechanisms for these effects were distinct.

These experiments have identified a regulatory circuit that moderates the transcription virus induced genes. Virus infection actuated this circuit by inducing Keap1 to bind virus induced genes. The parallel actuation of mechanisms that activate and that moderate the transcription of virus induced genes can shape the timing and the amplitude of virus induced gene transcription in response to signals that modulate their expression.

H3K9me2 and other histone modifications that correlate with reduced transcription in CD34+ progenitors and monocyte subsets (44) . The increased H3K9me2 correlates with reduced cytokine induction in vitro in PBMCs from vaccinated subjects. Vaccination also reduces chromatin accessibility at cytokine, chemokine and other antiviral response genes in classical monocytes. Chromatin accessibility was reduced within one day after vaccination, suggesting that this is an acute response to vaccination. It is possible that the mechanisms whereby Keap1, G9a- Keap1 was required for G9a-GLP and NFB p50 recruitment and for H3K9me2 deposition both within virus induced genes and in regions flanking these genes. The molecular mechanisms whereby Keap1 facilitated G9a-GLP and NFB p50 recruitment remain unclear. Keap1 forms complexes with NFB p50 and NFB p65 that can bind chromatin in living cells, suggesting that they can be recruited in concert. G9a and GLP co-precipitate with NFB subunits from cell extracts, suggesting that they can also be recruited in concert (22, 45) . It is plausible that initial The depletion or inhibition of several other lysine methyltransferases and demethylases influences innate immune response gene transcription. Jmjd3 demethylase deletion alters the transcription of a subset of LPS induced genes in mouse macrophages by mechanisms that do not correlate with H3K27me3 levels (46) . Kdm2b deletion and KDM5 inhibitors have opposite effects on the transcription of LPS and interferon response genes in mouse macrophages versus human cancer cells that do not correlate with the H3K4me3 levels at these genes (47, 48) . Ezh2 deletion and inhibitors reduce LPS and poly(I:C) induction of pro-inflammatory genes in mouse macrophages, and they enhance CXCL10 transcription in tumor cell lines through mechanisms that are unrelated to H3K27me2 deposition at these genes (49, 50) The effects of Keap1 mutations in mice and in human cancers have been attributed to changes in Nrf2 activity (2) (3) (4) (5) (6) 51) . The observations that Keap1 can regulate immunomodulatory gene transcription directly as well as indirectly suggest that the physiological functions of Keap1 can be mediated by many mechanisms (7, 8, 13) .

Compounds that alter Keap1 moderation of virus induced gene transcription could be beneficial in the prophylaxis and treatment of infectious diseases and immune disorders. 

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A) Virus infection induces higher levels of cytokine transcription in Keap1-/-MEFs than in MEFs with intact Keap1

The levels of the transcripts indicated in the graphs were measured by RT-qPCR at the times after virus infection indicated on the bottom graph. The line graphs show the results (mean±2*SD) of a representative experiment in which Keap1+/+ Nrf2-/-#3 and Keap1-/-Nrf2-/-#4 MEFs were compared. The # after each genotype reflects a MEF population that was isolated from an independent embryo. The reproducibility of Keap1 effects on transcript levels were

Virus infection has opposite effects on Keap1 binding to cytokine versus cell cycle genes, and Keap1 is required for G9a and GLP to bind and to deposit H3K9me2 upon virus infection

MEFs with Keap1-/-deletions (red bars), each with Nrf2-/-deletions, were infected with mock (solid bars) or

The levels of Keap1, G9a, GLP and IRF3 binding, and of H3K9me2 were measured 6 hours after infection at the genes indicated in the graphs using the antibodies indicated. The bar graphs show the results (mean±2*SD) of a representative experiment in which Keap1+/+ Nrf2-/-#2 and Keap1-/-Nrf2-/-#4 MEFs were compared. The reproducibility of Keap1 effects on G9a, GLP and IRF3 binding, and on H3K9me2 were evaluated in 2-5 independent sets of MEFs by two-factor ANOVA analyses

MEFs with intact Keap1 (blue bars) and MEFs with Keap1-/-deletions (red bars), each with Nrf2-/-deletions, were infected with mock

Chromatin was isolated. The levels of NFB p50, NFB p65, IRF3 and cJun binding were measured 6 hours after infection at the genes indicated in the graphs using the antibodies indicated at the bottom. The bar graphs show the results (mean±2*SD) of a representative experiment in which Keap1+/+ Nrf2-/-#1 and Keap1-/-Nrf2-/-#3 MEFs were compared. The reproducibility of Keap1 effects on NFB p50 and NFB p65 binding were evaluated in 4-6 independent sets of MEFs by two-factor ANOVA analyses

GLP and NFB p50 binding, on H3K9me2 deposition, and on transcription at cytokine versus cell cycle associated genes. The blue arrows indicate effects that are required for binding or for deposition. The red arcs and bars indicate effects that moderate transcription. Dotted lines and ovals indicate differences between different genes and MEFs

After culture, the MEFs were incubated in RIPA buffer, and the proteins that were retained in the MEFs were analyzed by immunoblotting. The images show Keap1 C and Vinculin immunoblots of the proteins that were retained in the MEFs. The images show the results from a representative

The diagram illustrates the stabilization of Keap1 retention in MEFs by G9a-GLP inhibition

MEFs with intact Keap1 and MEFs with Keap1-/-deletions, each with Nrf2-/-deletions, were cultured with vehicle (left graphs) or with 50 M tBHQ (right graphs) for 24 hours before virus infection. The levels of the transcripts indicated in the graphs were measured at the times after virus infection indicated at the bottom. The graphs show the results of a representative experiment in which Keap1+/+ Nrf2-/-#2 and Keap1-/-Nrf2-/-#2 MEFs were compared. The reproducibility of Keap1 effects on transcription in MEFs that were cultured with tBHQ or with vehicle were evaluated in two independent sets of MEFs by two-factor ANOVA analyses

each with Nrf2-/-deletions, were cultured with 50 M tBHQ , 50 M tBHQ and 20 M BIX01294, or vehicle for 24 hours before infection. The levels of Keap1, NFB p50 and NFB p65 binding were measured 6 hours after mock (solid bars) or virus (striped bars) infection at the genes indicated in the graphs using the antibodies indicated at the bottom. The graphs show the results of a representative experiment in which Keap1+/+ Nrf2-/-#7 and Keap1-/-Nrf2-/-#1 MEFs were compared. The reproducibility of tBHQ and BIX01294+tBHQ effects on Keap1, NFB p50, and NFB p65 binding were evaluated in two independent sets of MEFs by two-factor ANOVA analyses

The diagram depicts effects of tBHQ and of BIX01294 on Keap1, NFB p50 and NFB p65 binding, on H3K9me2 deposition, and on transcription at virus induced genes. The blue arrows indicate effects that are required for binding or for deposition. The red arcs and bars indicate effects that inhibit or moderate binding, deposition