κB Sites Research Articles

Tumor suppressor NME1/NM23-H1 modulates DNA binding of NF-κB RelA.

The dimeric NF-κB family of transcription factors activates transcription by binding sequence-specifically to DNA response elements known as κB sites, located within the promoters and enhancers of their target genes. While most NF-κB remain inactive in the cytoplasm of unstimulated cells, a small amount of RelA, one of its members, persists in the nucleus, ensuring low-level expression of genes essential for homeostasis. Several cofactors have been identified that aid in DNA binding of RelA. In this study, we identify NME1 (nucleoside diphosphate kinase 1) as a cofactor that enhances RelA's ability to bind κB sites within the promoters of a subset of its target genes, promoting their expression under both unstimulated and stimulated conditions. Depletion of NME1 influences activation or repression of several genes that are unresponsive to TNFα, despite containing κB sites in their promoters but not in clusters. This suggests that clustering of kB sites may be necessary for RelA-dependent transcription complex assembly. NME1 appears to act as a cofactor for other transcription factors to regulate these genes. NME1 does not directly contact κB DNA but interacts with RelA, with this interaction being further strengthened in the presence of κB DNA. Notably, NME1 alone has a marginal effect in enhancing RelA's DNA binding, suggesting that NME1 likely cooperate with other cofactors to regulate DNA binding and transcription through RelA. These observations underscore the intricate assembly of transcription complexes centered on NF-κB.

Tumor suppressor NME1/NM23-H1 modulates DNA binding of NF-κB RelA.

The dimeric NF-κB family of transcription factors activates transcription by binding sequence-specifically to DNA response elements known as κB sites, located within the promoters and enhancers of their target genes. While most NF-κB remain inactive in the cytoplasm of unstimulated cells, a small amount of RelA, one of its members, persists in the nucleus, ensuring low-level expression of genes essential for homeostasis. Several cofactors have been identified that aid in DNA binding of RelA. In this study, we identify NME1 (nucleoside diphosphate kinase 1) as a cofactor that enhances RelA's ability to bind κB sites within the promoters of a subset of its target genes, promoting their expression under both unstimulated and stimulated conditions. Depletion of NME1 influences activation or repression of several genes that are unresponsive to TNFα, despite containing κB sites in their promoters but not in clusters. This suggests that clustering of kB sites may be necessary for RelA-dependent transcription complex assembly. NME1 appears to act as a cofactor for other transcription factors to regulate these genes. NME1 does not directly contact κB DNA but interacts with RelA, with this interaction being further strengthened in the presence of κB DNA. Notably, NME1 alone has a marginal effect in enhancing RelA's DNA binding, suggesting that NME1 likely cooperate with other cofactors to regulate DNA binding and transcription through RelA. These observations underscore the intricate assembly of transcription complexes centered on NF-κB.

Cooperativity among clustered κB sites within promoters and enhancers dictates transcriptional specificity of NF-κB-RelA along with specific cofactors.

The functional role of weak DNA binding sites for transcription factor (TF) recruitment and gene expression remains largely unknown. Our study reveals that the weak NF-κB DNA binding sites, which are abundant in gene promoters and enhancers, appear in clusters and exhibit minimal to undetectable NF-κB binding activity in isolation in vitro, yet they play prominent roles in gene regulation within native context in cells. We found nuclear concentration of RelA/p65, the predominant NF-κB, is approximately 0.2 µM in stimulated cells, challenging the idea that these weak κB sites operate through mass action-dependent binding mechanisms. Through proteomic analysis, we identified a range of nuclear factors, including various other TFs, interacting with RelA at these κB-sites. ChIP-seq, RNA-seq and phase-separated condensation analyses suggest these additional TFs, referred to as the cofactors of NF-κB, facilitate dynamic recruitment of NF-κB to clustered κB sites of specific target genes. Overall, our findings demonstrate the collective contribution of both strong and weak κB sites in occupancy of NF-κB at the promoters and enhancers, with the recruitment facilitated by a variety of cofactors. This congregation of multiple factors forming larger dynamic complexes appearing as a transcription condensate is likely to be common to all transcriptional programs.

Transient interactions modulate the affinity of NF-κB transcription factors for DNA

The dimeric nuclear factor kappa B (NF-κB) transcription factors (TFs) regulate gene expression by binding to a variety of κB DNA elements with conserved G:C-rich flanking sequences enclosing a degenerate central region. Toward defining mechanistic principles of affinity regulated by degeneracy, we observed an unusual dependence of the affinity of RelA on the identity of the central base pair, which appears to be noncontacted in the complex crystal structures. The affinity of κB sites with A or T at the central position is ~10-fold higher than with G or C. The crystal structures of neither the complexes nor the free κB DNAs could explain the differences in affinity. Interestingly, differential dynamics of several residues were revealed in molecular dynamics simulation studies, where simulation replicates totaling 148 μs were performed on NF-κB:DNA complexes and free κB DNAs. Notably, Arg187 and Arg124 exhibited selectivity in transient interactions that orchestrated a complex interplay among several DNA-interacting residues in the central region. Binding and simulation studies with mutants supported these observations of transient interactions dictating specificity. In combination with published reports, this work provides insights into the nuanced mechanisms governing the discriminatory binding of NF-κB family TFs to κB DNA elements and sheds light on cancer pathogenesis of cRel, a close homolog of RelA.

Modeling the regulation of the Il12b locus by NF-κB dimers

Abstract The transcription factor NF-κB is composed of a family, which regulates the expression of various genes in immune and inflammatory responses. The NF-κB family consists of five members: RelA/p65, RelB, c-Rel, p50/p105, and p52/p100, which control the transcription of target genes by binding to regulatory κB sites in homo-or heterodimer forms. Unlike the widely known RelA, the mechanisms related to the regulation of target genes by c-Rel is less understood. Interleukin 12b(Il12b), which encodes the p40 subunit of IL12 and IL23, is one example of gene that depend on c-Rel for proper activation in macrophages. The differences in intrinsic binding properties of c-Rel- and RelA-containing dimers at various regulatory sites around Il12b can be a key to understand the mechanism of c-Rel-containing dimers’ role in Il12b regulation. Using c-Rel and Il12b reporter mice with fluorescence live-cell imaging, we observe the single-cell dynamics of nuclear c-Rel and Il12b gene expression in bone marrow-derived macrophages induced by NF-κB activating ligands. We analyze the live-cell imaging data and extract quantitative features of nuclear c-Rel and Il12b expression from the time-series data. For additional mechanistic insights of the Il12b gene regulation by NF-κB dimers, we propose a mathematical model of dynamic gene regulation which includes actions of RelA- and c-Rel-containing dimers at κB sites proximal and distal to the Il12b locus. Our combined experimental and mathematical modeling approach will reveal quantitative insights that may provide a more comprehensive understanding of regulatory mechanisms underlying Il12b gene expression employed by NF-κB dimers.

Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation.

The mammalian NF-κB p52:p52 homodimer together with its cofactor Bcl3 activates transcription of κB sites with a central G/C base pair (bp), while it is inactive toward κB sites with a central A/T bp. To understand the molecular basis for this unique property of p52, we have determined the crystal structures of recombinant human p52 protein in complex with a P-selectin(PSel)-κB DNA (5'-GGGGTGACCCC-3') (central bp is underlined) and variants changing the central bp to A/T or swapping the flanking bp. The structures reveal a nearly two-fold widened minor groove in the central region of the DNA as compared to all other currently available NF-κB-DNA complex structures, which have a central A/T bp. Microsecond molecular dynamics (MD) simulations of free DNAs and p52 bound complexes reveal that free DNAs exhibit distinct preferred conformations, and p52:p52 homodimer induces the least amount of DNA conformational changes when bound to the more transcriptionally active natural G/C-centric PSel-κB, but adopts closed conformation when bound to the mutant A/T and swap DNAs due to their narrowed minor grooves. Our binding assays further demonstrate that the fast kinetics favored by entropy is correlated with higher transcriptional activity. Overall, our studies have revealed a novel conformation for κB DNA in complex with NF-κB and pinpoint the importance of binding kinetics, dictated by DNA conformational and dynamic states, in controlling transcriptional activation for NF-κB.

An intrinsically disordered transcription activation domain increases the DNA binding affinity and reduces the specificity of NFκB p50/RelA

Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with coactivators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. Whether these results can be generalized to more TADs is not clear. Here, we biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the TAD’s influence on NFκB–DNA interactions. In solution, we show the RelA TAD is disordered but compact, with helical tendency in two regions that interact with coactivators. We determined that the presence of the TAD increased the stoichiometry of NFκB–DNA complexes containing promoter DNA sequences with tandem κB recognition motifs by promoting the binding of NFκB dimers in excess of the number of κB sites. In addition, we measured the binding affinity of p50/RelA for DNA containing tandem κB sites and single κB sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all κB sequences tested, it also increased the affinity for nonspecific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB DNA sequences. In contrast, previous studies have generally reported that TADs decrease DNA-binding affinity and increase sequence specificity. Our results reveal a novel function of the RelA TAD in promoting binding to nonconsensus DNA, which sheds light on previous observations of extensive nonconsensus DNA binding by NFκB in vivo in response to strong inflammatory signals.

Structural and dynamic studies of DNA recognition by NF-κB p50 RHR homodimer using methyl NMR spectroscopy.

Protein dynamics involving higher-energy sparsely populated conformational substates are frequently critical for protein function. This study describes the dynamics of the homodimer (p50)2 of the p50 Rel homology region (RHR) of the transcription factor NF-κB, using 13C relaxation dispersion experiments with specifically (13C, 1H)-labeled methyl groups of Ile (δ), Leu and Val. Free (p50)2 is highly dynamic in solution, showing μs-ms relaxation dispersion consistent with exchange between the ground state and higher energy substates. These fluctuations propagate from the DNA-binding loops through the core of the domain. The motions are damped in the presence of κB DNA, but the NMR spectra of the DNA complexes reveal multiple local conformations of the p50 RHR homodimer bound to certain κB DNA sequences. Varying the length and sequence of κB DNA revealed two factors that promote a single bound conformation for the complex: the length of the κB site in the duplex and a symmetrical sequence of guanine nucleotides at both ends of the recognition motif. The dynamic nature of the DNA-binding loops, together with the multiple bound conformations of p50 RHR with certain κB sites, is consistent with variations in the transcriptional activity of the p50 homodimer with different κB sequences.

RNA-binding protein hnRNP UL1 binds κB sites to attenuate NF-κB-mediated inflammation

Heterogeneous nuclear ribonucleoproteins (hnRNPs), a family of RNA-binding proteins, play important roles in various biological processes. However, the roles of hnRNPs members in immunity and inflammation remain to be fully understood. By a functional screening for hnRNPs members in LPS-stimulated macrophage inflammatory response, we identified hnRNP UL1 as a negative regulator of NF-κB-mediated inflammation. hnRNP UL1 constrains NF-κB-triggered transcriptional expression of pro-inflammatory cytokines in response to innate stimuli. Perturbation of hnRNP UL1 enhanced pro-inflammatory cytokine production in macrophages. In vivo deficiency of hnRNP UL1 increased the pro-inflammatory cytokine production once challenged with LPS. Accordingly, the expression of hnRNP UL1 decreased in peripheral blood mononuclear cells of rheumatoid arthritis patients. Mechanistically, hnRNP UL1 competes with NF-κB to bind κB sites to constrain the magnitude and duration of inflammatory response. Meanwhile, the broadly and dynamically binding of hnRNP UL1 on the target genes’ promoter during inflammatory response is unraveled. Our study adds new insight into the functions of hnRNPs in NF-κB-mediated inflammation, proposing a potential therapeutic strategy for controlling inflammatory autoimmune diseases.

Two NF-κB subunits are associated with antimicrobial immunity in Hyriopsis cumingii

The NF-κB pathway activated by bacteria and viruses produces a series of antimicrobial peptides that participate in the innate immune response. In this study, two NF-κB subunits were cloned and identified from Hyriopsis cumingii (named Hcp65 and Hcp105) using RT-PCR and RACE. The predicted Hcp65 protein possessed a N-terminal Rel homology domain (RHD) and an Ig-like/plexins/transcription factors domain (IPT); the Hcp105 contained an RHD, an IPT domain, 6 ankyrin (ANK) domain and a death domain. Quantitative reverse transcription PCR (qRT-PCR) showed that Hcp65 and Hcp105 were constitutively expressed in the detected tissues, and were significantly up-regulated in hemocytes, hepatopancreas and gill of mussels challenged with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly I: C). The dsRNA-mediated silencing of Hcp65 and Hcp105 caused significant reduction of immune genes such as lysozyme (HcLyso), theromacin (Hcther), whey acid protein (HcWAP), LPS-binding protein/bactericidal permeability protein (HcLBP/BPI) 1 and 2. In addition, subcellular localization experiments showed that Hcp65 and Hcp105 proteins were expressed in both the nucleus and cytoplasm of HEK-293T cells, and Hcp50 proteins (mature peptide of Hcp105) were mainly localized in the nucleus. The recombinant Hcp65 and Hcp50 protein could form homodimer and heterodimer and bind κB site in vitro. These results provide useful information for understanding the role of NF-κB in mollusks.

HIVEP1 Is a Negative Regulator of NF-κB That Inhibits Systemic Inflammation in Sepsis.

Our previous work identified human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1) as a putative driver of LPS-induced NF-κB signaling in humans in vivo. While HIVEP1 is known to interact with NF-ĸB binding DNA motifs, its function in mammalian cells is unknown. We report increased HIVEP1 mRNA expression in monocytes from patients with sepsis and monocytes stimulated by Toll-like receptor agonists and bacteria. In complementary overexpression and gene deletion experiments HIVEP1 was shown to inhibit NF-ĸB activity and induction of NF-ĸB responsive genes. RNA sequencing demonstrated profound transcriptomic changes in HIVEP1 deficient monocytic cells and transcription factor binding site analysis showed enrichment for κB site regions. HIVEP1 bound to the promoter regions of NF-ĸB responsive genes. Inhibition of cytokine production by HIVEP1 was confirmed in LPS-stimulated murine Hivep1-/- macrophages and HIVEP1 knockdown zebrafish exposed to the common sepsis pathogen Streptococcus pneumoniae. These results identify HIVEP1 as a negative regulator of NF-κB in monocytes/macrophages that inhibits proinflammatory reactions in response to bacterial agonists in vitro and in vivo.

Nanoparticle-based fluorescence probe for detection of NF-κB transcription factor in single cell via steric hindrance.

A novel nanoparticle-based fluorescence probe was developed for NF-κB transcription factor detection and in situ imaging via steric hindrance. The probe contains gold nanoparticles (AuNPs) to quench fluorescence, and nucleic acids immobilized on the surface of AuNPs to output fluorescence. In the basal state, Cy5 labeled DNA1 folds its long chain into a hairpin structure and quenches fluorescence by forcing the Cy5 fluorophore close to the surface of AuNPs. After the probe enters the cell, the NF-κB transcription factor can bind to the κB site in the DNA duplex of the nucleic acids. The steric hindrance caused by NF-κB leads to the extension of the long chain of DNA1 and the removal of the Cy5 fluorophore from the surface of AuNPs, thereby restoring the fluorescence of the probe. By measuring NF-κB in cell lysis in vitro, the probe obtains a detection limit of 0.38nM and the linear range from 0.5 to 16nM. Repeated measurements showed the recovery in the cell nuclear extract was between 93.38 and 109.32%, with relative standard deviation less than 5%. By monitoring the sub-localization of the Cy5 fluorophore in single cell, the probe system can effectively distinguish active NF-κB (nucleus) and inactive NF-κB (cytoplasm) through in situ imaging. The well-designed probe will make up for the shortcomings of the existing technology, and reveal the regulatory role of transcription factors in many disease processes.

Oscillating lncRNA Platr4 regulates NLRP3 inflammasome to ameliorate nonalcoholic steatohepatitis in mice

Background: Understanding the molecular events and mechanisms underlying development and progression of nonalcoholic steatohepatitis is essential in an attempt to formulating a specific treatment. Here, we uncover Platr4 as an oscillating and NF-κB driven lncRNA that is critical to the pathological conditions in experimental steatohepatitisMethods: RNA-sequencing of liver samples was used to identify differentially expressed lncRNAs. RNA levels were analyzed by qPCR and FISH assays. Proteins were detected by immunoblotting and ELISA. Luciferase reporter, ChIP-sequencing and ChIP assays were used to investigate transcriptional gene regulation. Protein interactions were evaluated by Co-IP experiments. The protein-RNA interactions were studied using FISH, RNA pull-down and RNA immunoprecipitation analysesResults: Cyclic expression of Platr4 is generated by the core clock component Rev-erbα via two RevRE elements (i.e., -1354/-1345 and -462/-453 bp). NF-κB transcriptionally drives Platr4 through direct binding to two κB sites (i.e., -1066/-1056 and -526/-516 bp), potentially accounting for up-regulation of Platr4 in experimental steatohepatitis. Intriguingly, Platr4 serves as a circadian repressor of Nlrp3 inflammasome pathway by inhibiting NF-κB-dependent transcription of the inflammasome components Nlrp3 and Asc. Loss of Platr4 down-regulates Nlrp3 inflammasome activity in the liver, blunts its diurnal rhythm, and sensitizes mice to experimental steatohepatitis, whereas overexpression of Platr4 ameliorates the pathological conditions in an Nlrp3-dependent manner. Mechanistically, Platr4 prevents binding of the NF-κB/Rxrα complex to the κB sites via a physical interaction, thereby inhibiting the transactivation of Nlrp3 and Asc by NF-κB.Conclusions: Platr4 functions to inactivate Nlrp3 inflammasome via intercepting NF-κB signaling. This lncRNA might be an attractive target that can be modulated to ameliorate the pathological conditions of steatohepatitis.

Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness.

Transcriptional memory allows certain genes to respond to previously experienced signals more robustly. However, whether and how the key proinflammatory cytokine TNF-α mediates transcriptional memory are poorly understood. Using HEK293F cells as a model system, we report that sustained TNF-α stimulation induces transcriptional memory dependent on TET enzymes. The hypomethylated status of transcriptional regulatory regions can be inherited, facilitating NF-κB binding and more robust subsequent activation. A high initial methylation level and CpG density around κB sites are correlated with the functional potential of transcriptional memory modules. Interestingly, the CALCB gene, encoding the proven migraine therapeutic target CGRP, exhibits the best transcriptional memory. A neighboring primate-specific endogenous retrovirus stimulates more rapid, more strong, and at least 100-fold more sensitive CALCB induction in subsequent TNF-α stimulation. Our study reveals that TNF-α-mediated transcriptional memory is governed by active DNA demethylation and greatly sensitizes memory genes to much lower doses of inflammatory cues.

Genetic Characterization of Full-Length HIV-2 Long Terminal Repeat Sequences: Identification of Rare Promoter Variants.

In this study, we sequenced the full-length HIV type 2 (HIV-2) long terminal repeat region from the proviral DNA of 23 HIV-2-infected individuals from the southern parts of India. We identified two different promoter variant strains circulating in this region along with the globally circulating common promoter variant. Seven sequences had an additional nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) binding motif and the sequence from another subject showed one NF-κB and one RBE-III binding site. Phylogenetic and subtyping analyses revealed that the circulating strains comprised HIV-2 subtype A. The occurrence of two NF-κB binding sites in ∼30% of the sequences analyzed in our study prompts us to hypothesize that as in the case of HIV-1 subtype C viruses that possess additional κB sites, the two NF-κB HIV-2 variants might possess superior replication fitness because of the increased magnitude of transcription, thus leading to the expansion of these variants in the country.

Nuclear Factor Kappa B Signaling Complexes in Acute Inflammation.

Significance: Nuclear factor kappa B (NF-κB) is a master regulator of the inflammatory response and represents a key regulatory node in the complex inflammatory signaling network. In addition, selective NF-κB transcriptional activity on specific target genes occurs through the control of redox-sensitive NF-κB interactions. Recent Advances: The selective NF-κB response is mediated by redox-modulated NF-κB complexes with ribosomal protein S3 (RPS3), Pirin (PIR). cAMP response element-binding (CREB)-binding protein (CBP)/p300, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), activator protein-1 (AP-1), signal transducer and activator of transcription 3 (STAT3), early growth response protein 1 (EGR-1), and SP-1. NF-κB is cooperatively coactivated with AP-1, STAT3, EGR-1, and SP-1 during the inflammatory process, whereas NF-κB complexes with CBP/p300 and PGC-1α regulate the expression of antioxidant genes. PGC-1α may act as selective repressor of phospho-p65 toward interleukin-6 (IL-6) in acute inflammation. p65 and nuclear factor erythroid 2-related factor 2 (NRF2) compete for binding to coactivator CBP/p300 playing opposite roles in the regulation of inflammatory genes. S-nitrosylation or tyrosine nitration favors the recruitment of specific NF-κB subunits to κB sites. Critical Issues: NF-κB is a redox-sensitive transcription factor that forms specific signaling complexes to regulate selectively the expression of target genes in acute inflammation. Protein-protein interactions with coregulatory proteins, other transcription factors, and chromatin-remodeling proteins provide transcriptional specificity to NF-κB. Furthermore, different NF-κB subunits may form distinct redox-sensitive homo- and heterodimers with distinct affinities for κB sites. Future Directions: Further research is required to elucidate the whole NF-κB interactome to fully characterize the complex NF-κB signaling network in redox signaling, inflammation, and cancer.

NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells.

The dimeric transcription factor NF-κB regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-κB is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκBα). Activation of NF-κB requires the degradation of IκBα, which then exposes a nuclear localization signal on NF-κB and promotes its trafficking to the nucleus. Once in the nucleus, NF-κB binds to the promotor region of NF-κB target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression. The activation of NF-κB occurs independently of transcription or translation. Therefore, the activation state of NF-κB must be measured either by quantifying NF-κB specifically in the nucleus, or by quantifying expression of NF-κB target genes. In this protocol, cells stably transfected with an NF-κB::luciferase reporter construct are assayed for NF-κB activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-κB, which traffics to the nucleus and binds to κB sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-κB activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

The regulation of sequence specific NF-κB DNA binding and transcription by IKKβ phosphorylation of NF-κB p50 at serine 80.

Phosphorylation of the NF-κB transcription factor is an important regulatory mechanism for the control of transcription. Here we identify serine 80 (S80) as a phosphorylation site on the p50 subunit of NF-κB, and IKKβ as a p50 kinase. Transcriptomic analysis of cells expressing a p50 S80A mutant reveals a critical role for S80 in selectively regulating the TNFα inducible expression of a subset of NF-κB target genes including pro-inflammatory cytokines and chemokines. S80 phosphorylation regulates the binding of p50 to NF-κB binding (κB) sites in a sequence specific manner. Specifically, phosphorylation of S80 reduces the binding of p50 at κB sites with an adenine at the −1 position. Our analyses demonstrate that p50 S80 phosphorylation predominantly regulates transcription through the p50:p65 heterodimer, where S80 phosphorylation acts in trans to limit the NF-κB mediated transcription of pro-inflammatory genes. The regulation of a functional class of pro-inflammatory genes by the interaction of S80 phosphorylated p50 with a specific κB sequence describes a novel mechanism for the control of cytokine-induced transcriptional responses.

Genome reading by the NF-κB transcription factors.

The NF-κB family of dimeric transcription factors regulates transcription by selectively binding to DNA response elements present within promoters or enhancers of target genes. The DNA response elements, collectively known as κB sites or κB DNA, share the consensus 5'-GGGRNNNYCC-3' (where R, Y and N are purine, pyrimidine and any nucleotide base, respectively). In addition, several DNA sequences that deviate significantly from the consensus have been shown to accommodate binding by NF-κB dimers. X-ray crystal structures of NF-κB in complex with diverse κB DNA have helped elucidate the chemical principles that underlie target selection in vitro. However, NF-κB dimers encounter additional impediments to selective DNA binding in vivo. Work carried out during the past decades has identified some of the barriers to sequence selective DNA target binding within the context of chromatin and suggests possible mechanisms by which NF-κB might overcome these obstacles. In this review, we first highlight structural features of NF-κB:DNA complexes and how distinctive features of NF-κB proteins and DNA sequences contribute to specific complex formation. We then discuss how native NF-κB dimers identify DNA binding targets in the nucleus with support from additional factors and how post-translational modifications enable NF-κB to selectively bind κB sites in vivo.

Alternate NF-κB-Independent Signaling Reactivation of Latent HIV-1 Provirus.

Current combination antiretroviral therapies (cART) are unable to eradicate HIV-1 from infected individuals because of the establishment of proviral latency in long-lived cellular reservoirs. The shock-and-kill approach aims to reactivate viral replication from the latent state (shock) using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells (kill) by specific therapeutics. The NF-κB RelA/p50 heterodimer has been characterized as an essential component of reactivation of the latent HIV-1 long terminal repeat (LTR). Nevertheless, prolonged NF-κB activation contributes to the development of various autoimmune, inflammatory, and malignant disorders. In the present study, we established a cellular model of HIV-1 latency in J-Lat CD4+ T cells that stably expressed the NF-κB superrepressor IκB-α 2NΔ4 and demonstrate that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivated HIV-1 from latency, even under conditions where NF-κB activation was repressed. Using specific calcineurin phosphatase, p38, and MEK1/MEK2 kinase inhibitors or specific short hairpin RNAs, c-Jun was identified to be an essential factor binding to the LTR enhancer κB sites and mediating the combined synergistic reactivation effect. Furthermore, acetylsalicylic acid (ASA), a potent inhibitor of the NF-κB activator kinase IκB kinase β (IKK-β), did not significantly diminish reactivation in a primary CD4+ T central memory (TCM) cell latency model. The present work demonstrates that the shock phase of the shock-and-kill approach to reverse HIV-1 latency may be achieved in the absence of NF-κB, with the potential to avoid unwanted autoimmune- and or inflammation-related side effects associated with latency-reversing strategies.IMPORTANCE The shock-and-kill approach consists of the reactivation of HIV-1 replication from latency using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells. The cellular transcription factor NF-κB is considered a master mediator of HIV-1 escape from latency induced by LRAs. Nevertheless, a systemic activation of NF-κB in HIV-1-infected patients resulting from the combined administration of different LRAs could represent a potential risk, especially in the case of a prolonged treatment. We demonstrate here that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivate HIV-1 from latency, even under conditions where NF-κB activation is repressed. Our study provides a molecular proof of concept for the use of anti-inflammatory drugs, like aspirin, capable of inhibiting NF-κB in patients under combination antiretroviral therapy during the shock-and-kill approach, to avoid potential autoimmune and inflammatory disorders that can be elicited by combinations of LRAs.