CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, noncanonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wildtype NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell typespecific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.Published versio

IκBα Nuclear Export Enables 4-1BB–Induced cRel Activation and IL-2 Production to Promote CD8 T Cell Immunity

Optimal CD8 T cell immunity is orchestrated by signaling events initiated by TCR recognition of peptide Ag in concert with signals from molecules such as CD28 and 4-1BB. The molecular mechanisms underlying the temporal and spatial signaling dynamics in CD8 T cells remain incompletely understood. In this study, we show that stimulation of naive CD8 T cells with agonistic CD3 and CD28 Abs, mimicking TCR and costimulatory signals, coordinately induces 4-1BB and cRel to enable elevated cytosolic cRel:IκBα complex formation and subsequent 4-1BB-induced IκBα degradation, sustained cRel activation, heightened IL-2 production and T cell expansion. NfkbiaNES/NES CD8 T cells harboring a mutated IκBα nuclear export sequence abnormally accumulate inactive cRel:IκBα complexes in the nucleus following stimulation with agonistic anti-CD3 and anti-CD28 Abs, rendering them resistant to 4-1BB induced signaling and a disrupted chain of events necessary for efficient T cell expansion. Consequently, CD8 T cells in NfkbiaNES/NES mice poorly expand during viral infection, and this can be overcome by exogenous IL-2 administration. Consistent with cell-based data, adoptive transfer experiments demonstrated that the antiviral CD8 T cell defect in NfkbiaNES/NES mice was cell intrinsic. Thus, these results reveal that IκBα, via its unique nuclear export function, enables, rather than inhibits 4-1BB-induced cRel activation and IL-2 production to facilitate optimal CD8 T cell immunity

Several Alphaherpesviruses Interact Similarly with the NF-κB Pathway and Suppress NF-κB-Dependent Gene Expression

ABSTRACT Alphaherpesvirus infection is associated with attenuation of different aspects of the host innate immune response that is elicited to confine primary infections at the mucosal epithelia. Here, we report that infection of epithelial cells with several alphaherpesviruses of different species, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), feline alphaherpesvirus 1 (FHV-1), and bovine alphaherpesvirus 1 (BoHV-1) results in the inactivation of the responses driven by the nuclear factor kappa B (NF-κB) pathway, considered a pillar of the innate immune response. The mode to interact with and circumvent NF-κB-driven responses in infected epithelial cells is seemingly conserved in human, feline, and porcine alphaherpesviruses, consisting of a persistent activation of the NF-κB cascade but a potent repression of NF-κB-dependent transcription activity, which relies on replication of viral genomes. However, BoHV-1 apparently deviates from the other investigated members of the taxon in this respect, as BoHV-1-infected epithelial cells do not display the persistent NF-κB activation observed for the other alphaherpesviruses. In conclusion, this study suggests that inhibition of NF-κB transcription activity is a strategy used by several alphaherpesviruses to prevent NF-κB-driven responses in infected epithelial cells. IMPORTANCE The current study provides a side-by-side comparison of the interaction of different alphaherpesviruses with NF-κB, a key and central player in the (proinflammatory) innate host response, in infected nontransformed epithelial cell lines. We report that all studied viruses prevent expression of the hallmark NF-κB-dependent gene IκB, often but not always via similar strategies, pointing to suppression of NF-κB-dependent host gene expression in infected epithelial cells as a common and therefore likely important aspect of alphaherpesviruses

Pseudorabies virus infection results in a broad inhibition of host gene transcription

Pseudorabies virus (PRV) is a porcine alphaherpesvirus that belongs to the Herpesviridae family. We showed earlier that infection of porcine epithelial cells with PRV triggers activation of the nuclear factor kappa B (NF-kappa B) pathway, a pivotal signaling axis in the early immune response. However, PRV-induced NF-kappa B activation does not lead to NF-kappa B-dependent gene expression. Here, using electrophoretic mobility shift assays (EMSAs), we show that PRV does not disrupt the ability of NF-kappa B to interact with its kappa B target sites. Assessing basal cellular transcriptional activity in PRV-infected cells by quantitation of prespliced transcripts of constitutively expressed genes uncovered a broad suppression of cellular transcription by PRV, which also affects the inducible expression of NF-kappa B target genes. Host cell transcription inhibition was rescued when viral genome replication was blocked using phosphonoacetic acid (PAA). Remarkably, we found that host gene expression shutoff in PRV-infected cells correlated with a substantial retention of the NF-kappa B subunit p65, the TATA box binding protein, and RNA polymerase II-essential factors required for (NF-kappa B-dependent) gene transcription-in expanding PRV replication centers in the nucleus and thereby away from the host chromatin. This study reveals a potent mechanism used by the alphaherpesvirus PRV to steer the protein production capacity of infected cells to viral proteins by preventing expression of host genes, including inducible genes involved in mounting antiviral responses. IMPORTANCE Herpesviruses are highly successful pathogens that cause lifelong persistent infections of their host. Modulation of the intracellular environment of infected cells is imperative for the success of virus infections. We reported earlier that a DNA damage response in epithelial cells infected with the alphaherpesvirus pseudorabies virus (PRV) results in activation of the hallmark proinflammatory NF-kappa B signaling axis but, remarkably, that this activation does not lead to NF-kappa B-induced (proinflammatory) gene expression. Here, we report that PRV-mediated inhibition of host gene expression stretches beyond NF-kappa B-dependent gene expression and in fact reflects a broad inhibition of host gene transcription, which correlates with a substantial recruitment of essential host transcription factors in viral replication compartments in the nucleus, away from the host chromatin. These data uncover a potent alphaherpesvirus mechanism to interfere with production of host proteins, including proteins involved in antiviral responses. Herpesviruses are highly successful pathogens that cause lifelong persistent infections of their host. Modulation of the intracellular environment of infected cells is imperative for the success of virus infections

A Novel Pathway Links Oxidative Stress to Loss of Insulin Growth Factor-2 (<i>IGF2</i>) Imprinting through NF-κB Activation

<div><p>Genomic imprinting is the allele-specific expression of a gene based on parental origin. Loss of imprinting(LOI) of Insulin-like Growth Factor 2 (<i>IGF2</i>) during aging is important in tumorigenesis, yet the regulatory mechanisms driving this event are largely unknown. In this study oxidative stress, measured by increased NF-κB activity, induces LOI in both cancerous and noncancerous human prostate cells. Decreased expression of the enhancer-blocking element CCCTC-binding factor(CTCF) results in reduced binding of CTCF to the H19-ICR (imprint control region), a major factor in the allelic silencing of <i>IGF2</i>. This ICR then develops increased DNA methylation. Assays identify a recruitment of the canonical pathway proteins NF-κB p65 and p50 to the CTCF promoter associated with the co-repressor HDAC1 explaining gene repression. An IκBα super-repressor blocks oxidative stress-induced activation of NF-κB and <i>IGF2</i> imprinting is maintained. <i>In vivo</i> experiments using IκBα mutant mice with continuous NF-κB activation demonstrate increased <i>IGF2</i> LOI further confirming a central role for canonical NF-κB signaling. We conclude CTCF plays a central role in mediating the effects of NF-κB activation that result in altered imprinting both <i>in vitro</i> and <i>in vivo</i>. This novel finding connects inflammation found in aging prostate tissues with the altered epigenetic landscape.</p></div

Increased binding of NF-κB subtypes p50 and p65 to the human CTCF promoter.

<p>(A) Schema showing the location of 14 putative NF-κB binding sites in CTCF promoter region relative to the CTCF transcription start site. These were identified using the JASPA database. Binding of NF-κB was assessed across all of these putative NF-κB binding sites. (B) ChIP-based qPCR for NF-κB binding demonstrates H₂O₂ exposure induces significant recovery of both p50 and p65 at CTCF region 11–13. Other binding sites evaluated did not show alterations in binding. HDAC binding also increases consistent with repression of the target gene. Values are expressed as mean+/−S.D * <i>P</i><0.05 (t-test), compared with IgG controls.</p

Oxidative stress results in CTCF reduction, loss of CTCF binding to and DNA hypermethylation across the H19-ICR region.

<p>(A) Oxidative stress represses CTCF protein expression in prostate cells. Whole lysates were extracted from treated cells (PPC1, left panel; 9E6/E7, right panel) and analyzed by Western blot analysis using an anti-CTCF antibody and tubulin as loading control. The blots were quantified using ImageJ and are shown as percentage of control. The CTCF decrease was statistically significant when calculated from three independent Western blots. (B) Oxidative stress decreases mRNA expression of <i>CTCF</i> in prostate cells. The mRNA levels of <i>CTCF</i> were measured using RT-qPCR. Values are expressed as mean+/−S.D. of three independent experiments measured in duplicates. * <i>P</i><0.05 (t-test). (C) ChIP to evaluate CFCF binding demonstrates H₂O₂ exposure reduces recovery of CTCF at the H19-ICR. Values are expressed as mean+/−S.D * <i>P</i><0.05 (t-test) and compared with IgG controls. (D) Oxidative stress induces progressive hypermethylation of the H19-ICR in PPC1 cells. Density of the circles represents fold change in methylation compared to the no treatment control. The ICR region is ∼2 kb upstream of H19 transcription start site and encompasses all CTCF binding site 6.</p

An IκBα super-repressor blocks NF-κB activity and <i>IGF2</i> LOI.

<p>(A) IκBα super repressor blocks oxidative stress-induced NF-κB activity. An IκBα super-repressor was utilized that contains mutant IκBα resistant to phosphorylation and degradation, thus blocking canonical NF-κB activation. Stable integration of this super-repressor or empty vector control was performed in PPC1 and 9E6/E7. These cells were then transduced with an NF-κB reporter for 48 hr and then treated with H₂O₂ for 6 hr. Values are expressed as mean+/−S.D. of three independent experiments. ** <i>P</i><0.01 (t-test). (B) IκBα super-repressor inhibits oxidative stress-induced decreases in CTCF protein expression. Western blot of protein lysates were analyzed and quantitated. (C) IκBα super-repressor inhibits oxidative stress-induced decreases in CTCF mRNA expression. The mRNA levels of <i>CTCF</i> were measured using RT-qPCR in the cells. (D) IκBα super-repressor blocks loss of <i>IGF2</i> imprinting. FLuPE analysis of RNA was performed to evaluate allele-specific expression.</p

Oxidative stress induces nuclear translocation and subtype-specific activation of NF-κB in prostate cells.

<p>PPC1 and 9E6/E7 cells were treated with H₂O₂ for the times indicated, then used for extraction of either whole cell lysates or cytosol and nuclear fractions. (A) Induction of p50 and repression of IκBα with oxidative stress. NF-κB member protein levels were analyzed by Western blot using antibodies to specific NF-κB subtypes. Tubulin and LaminB1 were utilized as loading controls. (B) NF-κB DNA binding increases after exposure to H₂O₂. NF-κB DNA binding activity was analyzed by EMSA. NF-κB binding in whole cell extracts prepared from indicated cell samples was analyzed using a Igκ-κB probe. The location of NF-κB is indicated by the arrow. Free probe is not shown. (C) Supershift analysis of DNA-binding activity of NF-κB indicates p50 and p65 binding (arrows). For supershift analysis, 1 µg of antibodies against p65, c-Rel, p50 and RelB subunits, or control IgG, were included in the binding reaction prior to EMSA.</p