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Sequence annotation is fundamental for studying the evolution of protein families, particularly when working with nonmodel species. Given the rapid, ever-increasing number of species receiving high-quality genome sequencing, accurate domain modeling that is representative of species diversity is crucial for understanding protein family sequence evolution and their inferred function(s). Here, we describe a bioinformatic tool called Taxon-Informed Adjustment of Markov Model Attributes (TIAMMAt) which revises domain profile hidden Markov models (HMMs) by incorporating homologous domain sequences from underrepresented and nonmodel species. Using innate immunity pathways as a case study, we show that revising profile HMM parameters to directly account for variation in homologs among underrepresented species provides valuable insight into the evolution of protein families. Following adjustment by TIAMMAt, domain profile HMMs exhibit changes in their per-site amino acid state emission probabilities and insertion/deletion probabilities while maintaining the overall structure of the consensus sequence. Our results show that domain revision can heavily impact evolutionary interpretations for some families (i.e., NLR’s NACHT domain), whereas impact on other domains (e.g., rel homology domain and interferon regulatory factor domains) is minimal due to high levels of sequence conservation across the sampled phylogenetic depth (i.e., Metazoa). Importantly, TIAMMAt revises target domain models to reflect homologous sequence variation using the taxonomic distribution under consideration by the user. TIAMMAt’s flexibility to revise any subset of the Pfam database using a user-defined taxonomic pool will make it a valuable tool for future protein evolution studies, particularly when incorporating (or focusing) on nonmodel species.

The heterogeneity of traumatic brain injury (TBI)-induced secondary injury has greatly hampered the development of effective treatments for TBI patients. Targeting common processes across species may be an innovative strategy to combat debilitating TBI. In the present study, a cross-species transcriptome comparison was performed for the first time to determine the fundamental processes of secondary brain injury in Sprague-Dawley rat and C57/BL6 mouse models of TBI, caused by acute controlled cortical impact. The RNA sequencing data from the mouse model of TBI were downloaded from the Gene Expression Omnibus (ID: GSE79441) at the National Center for Biotechnology Information. For the rat data, peri-injury cerebral cortex samples were collected for transcriptomic analysis 24 hours after TBI. Differentially expressed gene-based functional analysis revealed that common features between the two species were mainly involved in the regulation and activation of the innate immune response, including complement cascades as well as Toll-like and nucleotide oligomerization domain-like receptor pathways. These findings were further corroborated by gene set enrichment analysis. Moreover, transcription factor analysis revealed that the families of signal transducers and activators of transcription (STAT), basic leucine zipper (BZIP), Rel homology domain (RHD), and interferon regulatory factor (IRF) transcription factors play vital regulatory roles in the pathophysiological processes of TBI, and are also largely associated with inflammation. These findings suggest that targeting the common innate immune response might be a promising therapeutic approach for TBI. The animal experimental procedures were approved by the Beijing Neurosurgical Institute Animal Care and Use Committee (approval No. 201802001) on June 6, 2018.

Rel/nuclear factor (NF)-κB family of transcription factors paly vital roles in innate immunity response to bacterial and viral infection. Here, we cloned and identified a dorsal homologue (named as MnDorsal) from Macrobrachium nipponense. The full-length cDNA of MnDorsal is 2573 bp with a 1986 bp open reading frame that encodes 661 amino acids. Predicted MnDorsal protein contained a RHD (Rel homology domain), an IPT (Iglike, plexins, and transcriptions factors) domain, and two low complexity regions. Phylogenetic analysis showed that MnDorsal has a closer genetic distance with dorsal homologues from invertebrates. MnDorsal was widely expressed in a variety of tissues, including hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Expression patterns analysis showed that the transcriptional level of MnDorsal in the gills was evidently up-regulated after Staphylococcus aureus, Vibrio parahaemolyticus, white spot syndrome virus, or polyinosinic-polycytidylic acid challenge, suggesting that MnDorsal participates in the immune defenses against pathogens and stimulant challenges. Additionally, the dsRNA-mediated RNA interference analysis showed that knockdown of MnDorsal can significantly inhibit the expression of anti-lipopolysaccharide factor (ALF) and crustin. Further studies revealed that the up-regulated expression of ALFs (MnALF2, MnALF3, and MnALF4) and crustins (MnCrustin3 and MnCrustin4) caused by S. aureus infection were obviously decreased after silencing MnDorsal. These findings suggest that MnDorsal positively regulate the expression of antibacterial peptides (AMPs) during S. aureus infection. Our study will promote to better understand the role of Toll-Dorsal-AMPs pathway in innate immunity response to gram-positive bacterial infection in crustacean.

The Rel/NF-κB transcription factor family has myriad roles in immunity, development, and differentiation in animals, and was considered a key innovation for animal multicellularity. Rel homology domain-containing proteins were previously hypothesized to have originated in a last common ancestor of animals and some of their closest unicellular relatives. However, key taxa were missing from previous analyses, necessitating a systematic investigation into the distribution and evolution of these proteins. Here, we address this knowledge gap by surveying taxonomically broad data from eukaryotes, with a special emphasis on lineages closely related to animals. We report an earlier origin for Rel/NF-κB proteins than previously described, in the last common ancestor of animals and fungi, and show that even in the sister group to fungi, these proteins contain elements that in animals are necessary for the subcellular regulation of Rel/NF-κB., This work was supported by a European Research Council Consolidator Grant (ERC-2012-CO-616960) grant and a grant (BFU2017-90114-P) from Ministerio de Economía y Competitividad (MINECO), Agencia Estatal de Investigación (AEI), and Fondo Europeo de Desarrollo Regional (FEDER) to I.R.-T. M.M.L. was supported by a Marie Skłodowska-Curie Individual Fellowship under the EU Framework Programme for Research and Innovation Horizon 2020 (Project ID 747789) and an Ayuda Juan de la Cierva-Incorporación postdoctoral fellowship (IJC2018-036657-I) from the Spanish Ministry of Sciece and Innovation. N.R.-R. was supported by a “Formación del Profesorado Universitario (FPU13/01840)” PhD scholarship from the Spanish Ministerio de Educación, Cultura y Deporte (MECD).

Background It is well-established that activation of nuclear factor-kappa B (NF-κB) signaling plays important roles in cancer development and progression. However, the underlying mechanism by which the NF-κB pathway is constitutively activated in cancer remains largely unclear. The present study aimed to investigate the effect of PICALM interacting mitotic regulator (PIMREG) on sustaining NF-κB activation in breast cancer. Methods The underlying mechanisms in which PIMREG-mediated NF-κB constitutive activation were determined via immunoprecipitation, EMSA and luciferase reporter assays. The expression of PIMREG was examined by quantitative PCR and western blotting analyses and immunohistochemical assay. The effect of PIMREG on aggressiveness of breast cancer cell was measured using MTT, soft agar clonogenic assay, wound healing and transwell matrix penetration assays in vitro and a Xenografted tumor model in vivo. Findings PIMREG competitively interacted with the REL homology domain (RHD) of NF-κB with IκBα, and sustained NF-κB activation by promotion of nuclear accumulation and transcriptional activity of NF-κB via disrupting the NF-κB/IκBα negative feedback loop. PIMREG overexpression significantly enhanced NF-κB transactivity and promoted the breast cancer aggressiveness. The expression of PIMREG was markedly upregulated in breast cancer and positively correlated with clinical characteristics of patients with breast cancer, including the clinical stage, tumor-node-metastasis classification and poorer survival. Interpretation PIMREG promotes breast cancer aggressiveness via disrupting the NF-κB/IκBα negative feedback loop, which suggests that PIMREG might be a valuable prognostic factor and potential target for diagnosis and therapy of metastatic breast cancer. Fund The science foundation of China, Guangdong Province, Guangzhou Education System, and the Science and Technology Program of Guangzhou.

The activity and close regulation of nuclear factor kappaB (NF-κB) transcription factors is critical for a variety of cellular processes including inflammation, immunity, differentiation and cell survival. Thus, dysregulation of the NF-κB system could lead to serious diseases, e.g. uncoordinated growth of the normal tissue during the development of cancer. Transcriptional activity of the NF-κB factor RelA is regulated by a number of mechanisms which comprise ubiquitinylation by a multimeric ubiquitin ligase containing Elongins B and C, cullin-2 (Cul2) and suppressor of cytokine signaling 1 (SOCS1), but also USP48-dependent deubiquitinylation. Further, USP48 promotes cell survival and antagonizes also other E3 ligase functions which are involved in genome stability and DNA repair. The regulation of RelA by USP48 has been investigated in detail, but the domains of USP48 and RelA for direct interaction are not known. In this study we report that USP48 interacts physically with RelA in the nucleus. Further, we show by overexpression of truncated proteins that the catalytic USP domain of USP48 interacts with the N-terminal region of the Rel homology domain (RHD) of RelA. This study provides first evidence that the USP domain of USP48 is important for the physical association with substrate proteins, and a suitable target for small molecule inhibitors for therapeutic intervention strategies.

In this report, we investigate the evolution of transcription factor NF-κB by examining its structure, activity, and regulation in two protists using phylogenetic, cellular, and biochemical techniques. In Capsaspora owczarzaki (Co), we find that full-length NF-κB has an N-terminal DNA-binding domain and a C-terminal Ankyrin (ANK) repeat inhibitory domain, and its DNA-binding activity is more similar to metazoan NF-κB rather than Rel proteins. As with mammalian NF-κB proteins, removal of the ANK repeats is required for Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by co-expression of IKK in human cells. Exogenously expressed Co-NF-κB localizes to the nucleus in Co cells. NF-κB mRNA and DNA-binding levels differ across three life stages of Capsaspora, suggesting distinct roles for NF-κB in these life stages. RNA-seq and GO analyses identify possible gene targets and biological functions of Co-NF-κB. We also show that three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) all consist of primarily the N-terminal conserved Rel Homology domain sequences of NF-κB, and lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of human and Co cells, but differ in their DNA-binding and transcriptional activation activities. Furthermore, all three As-NF-κB proteins can form heterodimers, indicating that NF-κB diversified into multi-subunit families at least two times during evolution. Overall, these results present the first functional characterization of NF-κB in a taxonomic kingdom other than Animalia and provide information about the evolution and diversification of this biologically important transcription factor.SignificanceThese results represent the first functional characterization of the biologically important transcription factor NF-κB in a taxonomic kingdom other than Animalia. As such, they provide information on the evolutionary origins and basal diversification of NF-κB outside of metazoans. These results suggest that NF-κB plays life stage-specific roles in Capsaspora, the closest unicellular ancestor to all metazoans. Finally, the analysis of three NF-κB proteins in a single choanoflagellate indicates that choanoflagellates have subclasses of NF-κBs, which can form heterodimers, suggesting that NF-κB subunit expansion and diversification has occurred at least twice in evolution.

R-loops are three-stranded, RNA–DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

Recurrent RELA and YAP1 fusions are intimately associated with tumorigenesis in supratentorial ependymomas. Chromothripsis and focal copy number alterations involving 11q are hallmarks of these tumors. However, it is unknown whether the chromosomal alterations are a direct causal event resulting in fusion transcripts. In addition, the biological significance of the RELA fusion variants and YAP1 fusions is not yet fully characterized. In this study, we generated gene rearrangements on 11q with the CRISPR/Cas9 system and investigated the formation of oncogenic ependymoma fusion genes. Further, we examined the oncogenic potential of RELA fusion variants and YAP1 fusions in a lentiviral gene transfer model. We observed that endogenous RELA fusion events were successfully induced by CRISPR/Cas9-mediated genome rearrangement in cultured cells. In vivo genome editing in mouse brain resulted in the development of ependymoma-like brain tumors that harbored the Rela fusion gene. All RELA fusion variants tested, except a variant lacking the Rel homology domain, were able to induce tumor formation, albeit with different efficacy. Furthermore, expression of YAP1-FAM118B and YAP1-MAMLD1 fusions induced the formation of spindle-cell-like tumors at varying efficacy. Our results indicate that chromosomal rearrangements involving the Rela locus are the causal event for the formation of Rela fusion-driven ependymomas in mice. Furthermore, the type of RELA. fusion might affect the aggressiveness of tumors and that the Rel homology domain is essential for the oncogenic functions of RELA. fusions. The YAP1 fusion genes are also oncogenic when expressed in mice.

The nuclear factor-κB (NF-κB) signaling pathway plays a crucial role in regulating many physiological processes such as development, inflammation, apoptosis, cell proliferation, differentiation and immune responses. And the NF-κB/Rel family members were considered as the most important transcription factors in the NF-κB signaling pathway. In this study, we cloned a Rel homolog gene (named as CgRel2) from the Pacific oyster, Crassostrea gigas. The 2115-bp open reading frame (ORF) encodes 704 amino acids and CgRel2 possesses a conserved Rel Homology Domain (RHD) at the N-terminus. Phylogenetic analysis revealed that CgRel2 is most closely related to Pinctada fucata dorsal protein. CgRel2 transcripts are widely expressed in all tested tissues, with the highest expression observed in the labial palp and the gill. Moreover, the expression of CgRel2 is significantly upregulated after lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)] challenge. CgRel2 transfection into human cell lines activated NF-κB, TNFα and oyster IL-17 (CgIL-17) reporter genes in a dose-dependent manner, while CgRel2 overexpression cannot induce ISRE (Interferon stimulation response element) reporter gene's transcriptional activity. Additionally, the results of co-immunoprecipitation showed that CgRel2 or CgRel1 could interact with oyster IκB1, IκB2 and IκB3 proteins strongly, which may be critical for the immune signaling transduction and the regulation of its immune functions. Together, these results suggest that CgRel2 could respond to pathogenic infection, participate in the immune signal transduction and activate NF-κB, TNFα and CgIL-17 reporter genes. Thus, CgRel2 could play an important role in the oyster immune system.

Background Human immunodeficiency virus type 1 (HIV-1) Tat protein plays an essential role in HIV-1 gene transcription. Tat transactivates HIV-1 long terminal repeat (LTR)-directed gene expression through direct interactions with the transactivation-responsive region (TAR) element and other cis elements in the LTR. The TAR-independent Tat-mediated LTR transactivation is modulated by several host factors, but the mechanism is not fully understood. Results Here, we report that Tat interacts with the Rel homology domain of RelB through its core region. Furthermore, RelB significantly increases Tat-mediated transcription of the HIV-1 LTR and viral gene expression, which is independent of the TAR. Both Tat and RelB are recruited to the HIV-1 promoter, of which RelB facilitates the recruitment of Tat to the viral LTR. The NF-κB elements are key to the accumulation of Tat and RelB on the LTR. Knockout of RelB reduces the accumulation of RNA polymerase II on the LTR, and decreases HIV-1 gene transcription. Together, our data suggest that RelB contributes to HIV-1 transactivation. Conclusions Our results demonstrate that RelB interacts with Tat and enhances TAR-independent activation of HIV-1 LTR promoter, which adds new insights into the multi-layered mechanisms of Tat in regulating the gene expression of HIV-1.

Dorsal, a member of the nuclear factor-kappa B (NF-κB) family of transcription factors, is a critical downstream component of the Toll pathway that regulates the expression of antimicrobial peptides (AMPs) against pathogen invasion. In this study, the full-length ORF of Dorsal was identified from the RNA-seq database of the mealworm beetle Tenebrio molitor (TmDorX2). The ORF of TmDorX2 was 1,482 bp in length, encoding a polypeptide of 493 amino acid residues. TmDorX2 contains a conserved Rel homology domain (RHD) and an immunoglobulin-like, plexins, and transcription factors (IPT) domain. TmDorX2 mRNA was detected in all developmental stages, with the highest levels observed in 3-day-old adults. TmDorX2 transcripts were highly expressed in the adult Malpighian tubules (MT) and the larval fat body and MT tissues. After challenging the larvae with Staphylococcus aureus and Escherichia coli, the TmDorX2 mRNA levels were upregulated 6 and 9 h post infection in the whole body, fat body, and hemocytes. Upon Candida albicans challenge, the TmDorX2 mRNA expression were found highest at 9 h post-infection in the fat body. In addition, TmDorX2-knockdown larvae exposed to E. coli, S. aureus, or C. albicans challenge showed a significantly increased mortality rate. Furthermore, the expression of 11 AMP genes was downregulated in the gut and fat body of dsTmDorX2-injected larvae upon E. coli challenge. After C. albicans and S. aureus challenge of dsTmDorX2-injected larvae, the expression of 11 and 10 AMPs was downregulated in the gut and fat body, respectively. Intriguingly, the expression of antifungal transcripts TmTenecin-3 and TmThaumatin-like protein-1 and -2 was greatly decreased in TmDorX2-silenced larvae in response to C. albicans challenge, suggesting that TmDorX2 regulates antifungal AMPs in the gut in response to C. albicans infection. The AMP expression profiles in the fat body, hemocytes, gut, and MTs suggest that TmDorX2 might have an important role in promoting the survival of T. molitor larvae against all mentioned pathogens.

The nuclear factor-kappa B (NF-κB) is a pleiotropic transcription factor regulating the expression of genes involved in various biological processes including the immune response and inflammation. Lamprey is regarded as a key species to provide meaningful clues for understanding the evolution of immune system; nevertheless, no information about lamprey NF-κB is reported. Thus, we have characterized a NF-κB homolog in lamprey (lj-NF-κB) for the deeper understanding of the role it played in lamprey immune system. The sequence and 3D structure analyses demonstrate that lj-NF-κB contained a Rel homology domain (RHD) and seven ankyrin repeats domains (ANKs), which would exhibit functional similarities to NF-κB superfamily proteins. This hypothesis was further proved by experiments. We found that the RHD of lj-NF-κB could interact with a mammalian κB response element, translocate to the nucleus to modulate gene (IL-6, IL-1β and TNF-α) expression, and the nuclear localization signals (NLS) was essential for the nuclear translocation. Furthermore, the ANKs of lj-NF-κB are the inhibition signal for the RHD of lj-NF-κB. The present results allow us to surmise that the lj-NF-κB should play a key role in immune response of lamprey, and the function of NF-κB has been maintained during evolution.

We deduced the amino acid (aa) sequence of the nuclear factor kappa B (NFκB) protein from genomic data for the American alligator (Alligator mississippiensis), the estuarine crocodile (Crocodylus porosus), and the Indian gharial (Gavialis gangeticus). A 105kDa protein, NFκB1 exhibits complex post-translational processing, multiple mechanisms of activation, and acts as precursor for a p50, a Rel homology transcription factor which influences the expression of key genes for developmental processes, apoptosis, and immune function. The aa sequences of the crocodylian proteins share very high identity with each other (97.2±0.7%), birds (81.0±1.1%, n=6), mammals (75.3±1.6%, n=4), reptiles (80.3±5.1%, n=2), and less identity with fish (55.5±5.5%, n=4) and one amphibian (66.1±0.8%). The crocodylian protein has a well-conserved Rel homology domain, a nuclear localization signal, and a glycine-rich region which facilitates proteasome-mediated generation of p50. The Rel homology domain contains sequences responsible for dimerization, DNA-binding, and nuclear translocation. In addition, seven ankyrin repeats were located, which putatively allow for inhibition of transcriptional regulation by mediating interaction with Inhibitor kappa B. Other features include a death domain, and conserved serine residues, near the C-terminal end, which act as potential phosphorylation sites for activation of the proteolytic generation of p50. Western blot analysis showed both the 105kDa precursor and the 50kDa mature NFκB were expressed in the alligator liver. Nuclear factor κB exhibited diffuse cytoplasmic distribution in alligator hepatocytes, and almost no cytoplasmic localization in infected animals. In addition, nuclear NFκB exhibited specific binding to the consensus NFκB promoter element.

Dorsal is a Rel/NF-κB transcription factor, which forms a key part of the Toll pathway. Lysozyme is a ubiquitous enzyme that degrades bacterial cell walls. In this study, a Dorsal homolog was cloned and characterized from triangle sail mussel Hyriopsis cumingii, namely, HcDorsal. Dorsal consisted of 3041 bp, including a 1938 bp open reading frame encoding a 645 amino acid protein. The deduced HcDorsal protein contained a Rel homology domain and an Ig-like, plexin, transcription factor domain. Analysis of expression patterns showed that HcDorsal was highly expressed in the hepatopancreas of H. cumingii. The expression level of HcDorsal continuously increased after Vibrio parahaemolyticus stimulation. When HcDorsal was knocked down by siRNA interference, two phage lysozyme genes (HcLyso1 and HcLyso2) obtained by horizontal gene transfer were significantly downregulated in hemocytes of mussels. Furthermore, knockdown of HcLyso1 and HcLyso2 could weaken V. parahaemolyticus clearance ability. Recombinant HcLyso1 and HcLyso2 proteins accelerated the bacterial clearance in vivo in mussels and evidently inhibited the growth of V. parahaemolyticus. These results suggested that HcDorsal could be activated after V. parahaemolyticus stimulation and then modulate the immune response through the transcriptional regulation of HcLyso1 and HcLyso2, thereby playing a protective role in mussels.

Although some novel antimicrobial peptides (AMP) have been successfully isolated from Bactrocera dorsalis Hendel, the mechanisms underlying the induction of these peptides are still elusive. The homolog of NF-κB transcription factor Relish, designated as BdRelish, was cloned from B. dorsalis. The full length cDNA of BdRelish is 3954 bp with an open reading frame that encodes 1013 amino acids. Similar to Drosophila Relish and the mammalian p100, it is a compound protein containing a conserved Rel homology domain, an IPT (Ig-like, plexins, transcription factors) domain and an IκB-like domain (four ankyrin repeats), the nuclear localization signal RKRRR is also detected at the residues 449–453, suggesting that it has homology to Relish and it is a member of the Rel family of transcription activator proteins. Reverse transcription quantitative polymerase chain reaction analysis reveals that BdRelish mRNAs are detected in different quantities from various tissues and the highest transcription level of BdRelish is determined in fat body. The injection challenge of Escherichia coli and Staphylococcus aureas significantly upregulated the expression of BdRelish. The injection of BdRelish dsRNA markedly reduced the expression of BdRelish and decreased the transcription magnitude of antimicrobial peptides. Individuals injected BdRelish dsRNA died at a significantly faster rate compared with the control groups. Therefore, BdRelish is vital for the transcription of AMPs to attack the invading bacteria.

Nuclear factor κB (NF-κB) plays a key role in the innate immunity of invertebrates. Relish belongs to the NF-κB family. In insects, alternative splicing induces the sequence diversity of the Relish gene. However, information on the roles of various relish isoforms in crustacean innate immune response is limited. Here, two alternatively spliced Relish isoforms (designated as SPcRelish and LPcRelish) were identified from freshwater crayfish (Procambarus clarkii), and functional analysis was performed. The Relish gene has 25 exons and 24 introns. The long isoform LPcRelish is fully spliced, whereas the short isoform SPcRelish is alternatively spliced and contains exon 1-9 and a retention of intron 9. LPcRelish contains the Rel homology domain (RHD), the ig-like, plexins, transcription factors (IPT), and ankyrin-repeat (ANK) inhibitory domain. However, SPcRelish contains only the RHD and IPT domain, and does not have an ANK domain. The transcripts of SPcRelish and LPcRelish can be regulated by Vibrio parahaemolyticus. The intestinal immunological barrier and bacterial balance in the intestine play crucial roles in host health. In this study, we analyzed the connection between Relish isoforms and the transcripts of antimicrobial peptides (AMPs) in intestine. The transcripts of all the tested AMPs, except ALF-41125, were upregulated by V. parahaemolyticus. The knock down of the SPcRelish gene resulted in a significant decrease in the expression levels of ALF-7032, ALF-13162, and Crustin-42012 during V. parahaemolyticus invasion. The expression levels of four AMP genes (ALF-41125, ALF-42430, Crustin-41354, and Crustin-42993) were obviously increased in V. parahaemolyticus-challenged SPcRelish-silenced crayfish. ALF-7032, ALF-9228, ALF-13162, ALF-42430, Crustin-41354, Crustin-42012, and Crustin-42993 were evidently downregulated in V. parahaemolyticus-infected LPcRelish-silenced crayfish. Overall, generating the two Relish isoforms by alternative splicing may be an important mechanism of the host immune system to promote molecular diversity, which results in the functional diversity of the relish transcription factor.

Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus, can regulate the antiviral response of NF-κB signaling, which is critical for cell survival, growth transformation, and virus latency. Here, we showed that tegument protein BGLF2 could inhibit TNF-α-induced NF-κB activity. BGLF2 was shown to interplay with the NF-κB subunits p65 and p50, and the Rel homology domain of p65 was the pivotal region to interact with BGLF2. Nonetheless, BGLF2 did not influence the development of p65-p50 dimerization. Yet, overexpression of BGLF2 inhibited the phosphorylation of p65 Ser536 (but not Ser276) and blocked the nuclear translocation of p65. In addition, knockdown of BGLF2 during EBV lytic replication elevated NF-κB activity and the phosphorylation of p65 Ser536. Taken together, these results suggest that the inhibition of NF-κB activation may serve as a strategy to escape the host's antiviral innate immunity to EBV during its lytic infection.-Chen, T., Wang, Y., Xu, Z., Zou, X., Wang, P., Ou, X., Li, Y., Peng, T., Chen, D., Li, M., Cai, M. Epstein-Barr virus tegument protein BGLF2 inhibits NF-κB activity by preventing p65 Ser536 phosphorylation.

Nuclear factor-κB (NF-κB) proteins are transcription factors that play key roles in regulating most immune responses and cell death. Constitutively active NF-κB has been shown to exhibit chemoresistance by inducing anti-apoptosis in tumor cells. Multiple myeloma is known as a constitutive NF-κB activating disease, and the proteasome inhibitor bortezomib is used to treat multiple myeloma and mantle cell lymphoma. We demonstrate here that DANFIN (N,N'-bis-(2,4-dimethyl-phenyl)-ethane-1,2-diamine) functions as an inhibitor of the p65 family proteins and induces chemosensitization to bortezomib in multiple myeloma. DANFIN was found to be an inhibitor of interactions between p65 and IκBα without the inhibition of the DNA binding activity of the p65 protein. In addition, DANFIN affected the IκBα binding region in Rel Homology Domain (RHD) and suppressed the nuclear translocalization of the p65 protein in cells. Furthermore, in multiple myeloma cells, DANFIN suppressed the expression level of NF-κB target genes and induced apoptosis. The combination therapy of DANFIN with bortezomib dramatically enhanced the apoptosis of multiple myeloma cells and indicated a remarkable anti-tumor effect in a multiple-myeloma xenograft mouse model.

Relish is an NF-kB transcription factor involved in immune-deficiency (IMD) signal pathway. In this study, a Relish gene (MrRelish) was identified from Macrobrachium rosenbergii. The full length of MrRelish comprises 5072 bp, including a 3510 bp open reading frame encoding a 1169 bp amino acid protein. MrRelish contains a Rel homology domain (RHD), a nucleus localization signal, an IκB-like domain (6 ankyrin repeats), and a death domain. Phylogenetic analysis showed that MrRelish and other Relish from crustaceans belong to one group. MrRelish was expressed in all detected tissues, with the highest expression level in hemocytes and intestines. MrRelish was also upregulated in hepatopancreas at 6 h after Vibrio anguillarum challenge. The over-expression of MrRelish could induce the expression of antimicrobial peptides (AMPs), such as Drosophila Metchnikowin (Mtk), Attacin (Atta), Drosomycin (Drs), and Cecropin (CecA) and shrimp Penaeidin (Pen4). The RNAi of MrRelish in gills showed that the expression of crustin (cru) 2, Cru5, Cru8, lysozyme (Lyso) 1, and Lyso2 was inhibited. However, the expression of anti-lipopolysaccharide factor (ALF) 1 and ALF3 did not change when MrRelish was knocked down. These results indicate that MrRelish may play an important role in innate immune defense against V. anguillarum in M. rosenbergii.

Summary Upon infection of epithelial cells, enteropathogenic Escherichia coli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180, within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector.

The Werner syndrome helicase (WRN) plays a role in maintaining genomic stability. The lack of WRN results in Werner syndrome, a rare autosomal recessive genetic disorder, which causes premature aging accompanied by many complications such as rare forms of cancer and type 2 diabetes. However, the underlying mechanisms of these complications, arising due to the loss of WRN, are poorly understood. In this study, we demonstrated the function of WRN in transcriptional regulation of NF-κB targets. WRN physically interacts via its RecQ C-terminal (RQC) domain with the Rel homology domain of both the RelA (p65) and the p50 subunits of NF-κB. In the steady state, WRN is recruited to HIV-1 long terminal repeat (LTR), a typical NF-κB-responsive promoter, as well as the p50/p50 homodimer, in an NF-κB site-dependent manner. The amount of WRN on LTR increased along with the transactivating RelA/p50 heterodimer in response to TNF-α stimulation. Further, a knockdown of WRN reduced the transactivation of LTR in exogenous RelA/p50-introduced or TNF-α-stimulated cells. Additionally, knockdown of WRN reduced TNF-α stimulation-induced activation of the endogenous promoter of IL-8, an NF-κB-responsive gene, and WRN increased its association with the IL-8 promoter region together with RelA/p50 after TNF-α stimulation. In conjunction with studies that have shown NF-κB to be a key regulator of aging and inflammation, our results indicate a novel role of WRN in transcriptional regulation. Along with NF-κB, the loss of WRN is expected to result in incorrect regulation of downstream targets and leads to immune abnormalities and homeostatic disruption.

Rel/NF-κB transcription factors play critical roles in induction and regulation of innate immune response in organisms. In this study, the full length of a Relish homolog cDNA from Exopalaemon carinicauda named EcRelish was 2141 bp encoding a 660 amino-acid polypeptide. EcRelish cDNA contained a conserved Rel homology domain and two nucleus localization signals. Sequence analysis indicated that the deduced amino acid sequence of the EcRelish showed high similarities to that of other crustaceans. Real time RT-PCR analysis showed that EcRelish mRNA expressed with different levels in tested tissues, and the highest expression was observed in the hemocytes. With longer infection time, the cumulative mortality rates increased gradually followed by the proliferation of Vibrio anguillarum and WSSV. The expression profiles of EcRelish gene were analyzed after V. a nguillarum, white spot syndrome virus (WSSV) challenge, and ammonia-N stress. The results showed that the expression levels of EcRelish mRNA in the hemocytes were up-regulated at 1–24 h after V. a nguillarum challenge. Meanwhile, the expression levels of EcRelish mRNA were up-regulated at 3 h after WSSV challenge. The expression of EcRelish in hemocytes was down-regulated significantly under ammonia-N stress during the experimental time. The results indicated that EcRelish might be involved in immune defense against pathogens and ammonia-N stress in E. c arinicauda.

Nuclear factor κB (NF-κB) is a family of critical transcription factors that play a critical role in innate immune responses and inflammation, yet the molecular mechanisms responsible for its tight regulation is not fully understood. In this study, we identified LRRC25, a member of leucine-rich repeat (LRR)-containing protein family, as a negative regulator in the NF-κB signaling pathway. Ectopic expression of LRRC25 impaired NF-κB activation, whereas knockout of LRRC25 potentiated NF-κB activation and enhanced the production of inflammatory cytokines. Further study demonstrated that the LRR domain of LRRC25 interacted with the Rel Homology domain (RHD) of p65/RelA and promotes the degradation of p65/RelA. Furthermore, LRRC25 enhanced the interaction between p65/RelA and cargo receptor p62, thus facilitating the degradation of p65/RelA through autophagy pathway. Our study has not only identified LRRC25 as a novel inhibitor of NF-κB signaling pathway, but also uncovers a new mechanism of crosstalk between NF-κB signaling and autophagy pathways.

We recently discovered that IκBα enhances the rate of release of nuclear factor kappa B (NFκB) from DNA target sites in a process we have termed molecular stripping. Coarse-grained molecular dynamics simulations of the stripping pathway revealed two mechanisms for the enhanced release rate: the negatively charged PEST region of IκBα electrostatically repels the DNA, and the binding of IκBα appears to twist the NFκB heterodimer so that the DNA can no longer bind. Here, we report amide hydrogen/deuterium exchange data that reveal long-range allosteric changes in the NFκB (RelA-p50) heterodimer induced by DNA or IκBα binding. The data suggest that the two Ig-like subdomains of each Rel-homology region, which are connected by a flexible linker in the heterodimer, communicate in such a way that when DNA binds to the N-terminal DNA-binding domains, the nuclear localization signal becomes more highly exchanging. Conversely, when IκBα binds to the dimerization domains, amide exchange throughout the DNA-binding domains is decreased as if the entire domain is becoming globally stabilized. The results help understand how the subtle mechanism of molecular stripping actually occurs.

The inflammation regulating transcription factor NFκB and the tumor-suppressing transcription factor p53 can act as functional antagonists. Chronic inflammation (NFκB activity) may contribute to the development of cancer through the inhibition of p53 function, while, conversely, p53 activity may dampen inflammation. Here we report that the E3 ubiquitin ligase MDM2, whose gene is transcriptionally activated by both NFκB and p53, can bind and inhibit the p65RelA subunit of NFκB. The interaction is mediated through the N-terminal and the acidic/zinc finger domains of MDM2 on the one hand and through the N-terminal Rel homology domain of p65RelA on the other hand. Co-expression of MDM2 and p65RelA caused ubiquitination of the latter in the nucleus, and this modification was dependent of a functional MDM2 RING domain. Conversely, inhibition of endogenous MDM2 by small-molecule inhibitors or siRNA significantly reduced the ubiquitination of ectopic and endogenous p65RelA. MDM2 was able to equip p65RelA with mutated ubiquitin moieties capable of multiple monoubiquitination but incapable of polyubiquitination; moreover, MDM2 failed to destabilize p65RelA detectably, suggesting that the ubiquitin modification of p65RelA by MDM2 was mostly regulatory rather than stability-determining. MDM2 inhibited the NFκB-mediated transactivation of a reporter gene and the binding of NFκB to its DNA binding motif in vitro. Finally, knockdown of endogenous MDM2 increased the activity of endogenous NFκB as a transactivator. Thus, MDM2 can act as a direct negative regulator of NFκB by binding and inhibiting p65RelA.

The Rel/NF-κB transcription factors can function as key regulators to modulate the expression of immune-related genes in response to immune challenge or environmental stress. In the present study, a gene coding Rel/NF-κB homologue was identified from scallop Chlamys farreri (designated CfRel). Its deduced protein comprised 359 amino acids, and contained a conserved N-terminal Rel homology domain (RHD) and an IPT domain. There was an NF-κB/Rel/dorsal domain signature sequence in the RHD domain. The mRNA transcripts of CfRel could be detected in all the tested tissues including adductor muscle, mantle, gill, gonad, haemocytes, kidney and hepatopancreas, with the highest expression level in hepatopancreas. After LPS stimulation, there were two peaks of CfRel mRNA expression level in haemocytes at 6 h (25.25-fold, P < 0.05) and 24 h (59.66-fold, P < 0.05) respectively, while the mRNA expression of CfRel was only up-regulated at 3 h after PGN stimulation (2.35-fold, P < 0.05). By Western blotting technique, CfRel protein was observed in the cytoplasm and nucleus of scallop haemocytes, and its concentration in the haemocyte nucleus increased significantly at 3 h and 12 h after LPS stimulation. The noticeable NF-κB transcription activity of CfRel protein was determined by NF-κB luciferase reporter assays (122.43%, P < 0.05), and it decreased significantly (17.61%, P < 0.05) after the coexpression of scallop IκB protein. These results collectively suggested that CfRel mRNA transcripts and protein were induced by immune stimulation, and CfRel protein could extricate itself from IκB protein and transfer into the haemocyte nucleus to modulate the immune response in scallop.

Background: Dietary phytochemicals consist of a wide variety of biologically active compounds that are ubiquitous in plants, many of which have been reported to have anti-tumor as well as anti-inflammatory properties. Objective: In the present study, we aimed to validate these findings by using docking protocols and explicate the possible mechanism of action for a dataset of nine phytochemicals namely boswellic acid, 1-caffeoylquinic acid, ellagic acid, emodin, genistein, guggulsterone, quercetin, resveratrol, and sylibinin from different plants against the nuclear factor- kappaB (NF-κB) precursor protein p105, an important transcription factor reported to be overexpressed in breast cancer. Materials and Methods: 2-D structures of all phytochemicals were retrieved from PubChem Compound database and their subsequent conversion into 3-D structures was performed by using online software system CORINA. The X-ray crystallographic structure of the NF-κB precursor p105 was extracted from Brookhaven Protein Data Bank. Molecular docking simulation study was carried out by using AutoDock Tools 4.0. Results: Our results showed significant binding affinity of different phytochemicals with the Rel homology domain of the NF-κB precursor protein p105. Quercetin and 1-caffeoylquinic acid were found to be very effective inhibitors against target molecule as they showed binding energy of −12.11 and −11.50 Kcal/mol, respectively. The order of affinity of other ligands with p105 was found as follows: guggulsterone > sylibinin > emodin > resveratrol > genistein > boswellic acid > ellagic acid. Conclusion: Our in silico study has explored the possible chemopreventive mechanism of these phytochemicals against the NF-κB precursor protein p105 and deciphered that quercetin, 1-caffeoylquinic acid and guggulsterone were the potent inhibitors against target molecule. In addition, large scale preclinical and clinical trials are needed to explore the role of these chemotherapeutic molecules against the NF-κB precursor protein p105 in cure and prevention of breast cancer.

Many citrullinated proteins are known autoantigens in rheumatoid arthritis, a disease mediated by inflammatory cytokines, such as tumor necrosis factor-α (TNFα). Citrullinated proteins are generated by converting peptidylarginine to peptidylcitrulline, a process catalyzed by the peptidylarginine deiminases (PADs), including PAD1 to PAD4 and PAD6. Several major risk factors for rheumatoid arthritis are associated with heightened citrullination. However, the physiological role of citrullination in immune cells is poorly understood. We report that suppression of PAD activity attenuates Toll-like receptor-induced expression of interleukin-1β (IL-1β) and TNFα by neutrophils in vivo and in vitro but not their global transcription activity. Mechanistically, PAD4 directly citrullinates nuclear factor κB (NF-κB) p65 and enhances the interaction of p65 with importin α3, which brings p65 into the nucleus. The citrullination-enhanced interaction of p65 with importin α3 and its nuclear translocation and transcriptional activity can be attributed to citrullination of four arginine residues located in the Rel homology domain of p65. Furthermore, a rheumatoid arthritis-prone variant of PAD4, carrying three missense mutations, is more efficient in interacting with p65 and enhancing NF-κB activity. Together, these data not only demonstrate a critical role of citrullination in an NF-κB-dependent expression of IL-1β and TNFα but also provide a molecular mechanism by which heightened citrullination propagates inflammation in rheumatoid arthritis. Accordingly, attenuating p65-mediated production of IL-1β and TNFα by blocking the citrullination of p65 has great therapeutic potential in rheumatoid arthritis.

Nuclear factor κB (NF-κB) plays an essential role in regulation of innate immunity. In mammals, NF-κB factors can form homodimers and heterodimers to activate gene expression. In insects, three NF-κB factors, Dorsal, Dif and Relish, have been identified to activate antimicrobial peptide (AMP) gene expression. However, it is not clear whether Dorsal (or Dif) and Relish can form heterodimers. Here we report the identification and functional analysis of a Dorsal homologue (MsDorsal) and two Relish short isoforms (MsRel2A and MsRel2B) from the tobacco hornworm, Manduca sexta. Both MsRel2A and MsRel2B contain only a Rel homology domain (RHD) and lack the ankyrin-repeat inhibitory domain. Overexpression of the RHD domains of MsDorsal and MsRel2 in Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells can activate AMP gene promoters from M. sexta and D. melanogaster. We for the first time confirmed the interaction between MsDorsal-RHD and MsRel2-RHD, and suggesting that Dorsal and Rel2 may form heterodimers. More importantly, co-expression of MsDorsal-RHD with MsRel2-RHD suppressed activation of several M. sexta AMP gene promoters. Our results suggest that the short MsRel2 isoforms may form heterodimers with MsDorsal as a novel mechanism to prevent over-activation of antimicrobial peptides.

Rationale: Both nuclear factors of activated T cells (NFAT) and nuclear factor-κB (NFκB) are Rel homology domain (RHD)-containing transcription factors whose independent activities are critically involved in regulating cardiac hypertrophy and failure. Objective: To determine the potential functional interaction between NFAT and NFκB signaling pathways in cardiomyocytes and its role in cardiac hypertrophy and remodeling. Methods and Results: We identified a novel transcriptional regulatory mechanism whereby NFκB and NFAT directly interact and synergistically promote transcriptional activation in cardiomyocytes. We show that the p65 subunit of NFκB coimmunoprecipitates with NFAT in cardiomyocytes, and this interaction maps to the RHD within p65. Overexpression of the p65-RHD disrupts the association between endogenous p65 and NFATc1, leading to reduced transcriptional activity. Overexpression of IκB kinase β (IKKβ) or p65-RHD causes nuclear translocation of NFATc1, and expression of a constitutively nuclear NFATc1-SA mutant similarly facilitated p65 nuclear translocation. Combined overexpression of p65 and NFATc1 promotes synergistic activation of NFAT transcriptional activity in cardiomyocytes, whereas inhibition of NFκB with IκBαM or dominant negative IKKβ reduces NFAT activity. Importantly, agonist-induced NFAT activation is reduced in p65 null mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs. In vivo, cardiac-specific deletion of p65 using a Cre-loxP system causes a ≈50% reduction in NFAT activity in luciferase reporter mice. Moreover, ablation of p65 in the mouse heart decreases the hypertrophic response after pressure overload stimulation, reduces the degree of pathological remodeling, and preserves contractile function. Conclusions: Our results suggest a direct interaction between NFAT and NFκB that effectively integrates 2 disparate signaling pathways in promoting cardiac hypertrophy and ventricular remodeling.

Full-length IL-33 is a member of the IL-1 family of cytokines, which can act in an autocrine or paracrine manner by binding to the IL-33R on several different target cell types. In addition, IL-33 can act in an intracrine fashion by translocating to the nucleus, where it binds to the chromatin and modulates gene expression. In this article, we report that full-length IL-33, but not mature IL-33, interacts with the transcription factor NF-κB. This interaction occurs between the N-terminal part of IL-33 from aa 66–109 and the N-terminal Rel homology domain of NF-κB p65. Coimmunoprecipitation experiments in cells overexpressing IL-33 or endogenously expressing IL-33 revealed rhIL-1β–stimulated association between IL-33 and p65, whereas binding to the p50 subunit was constitutive. The biological consequence of IL-33/NF-κB complex formation was reduction in NF-κB p65 binding to its cognate DNA and impairment of p65-triggered transactivation. Overexpression of IL-33 resulted in a reduction and delay in the rhIL-1β–stimulated expression of endogenous NF-κB target genes such as IκBα, TNF-α, and C-REL. We suggest that nuclear IL-33 sequesters nuclear NF-κB and reduces NF-κB–triggered gene expression to dampen proinflammatory signaling.

Nuclear factor κB (NF-κB) transcription factors are involved in controlling numerous cellular processes, including inflammation, innate and adaptive immunity, and cell survival. Here we show that the immunosuppressive measles virus (MV; Morbillivirus genus, Paramyxoviridae ) has evolved multiple functions to interfere with canonical NF-κB signaling in epithelial cells. The MV P, V, and C proteins, also involved in preventing host cell interferon responses, were found to individually suppress NF-κB-dependent reporter gene expression in response to activation of the tumor necrosis factor (TNF) receptor, RIG-I-like receptors, or Toll-like receptors. NF-κB activity was most efficiently suppressed in the presence of V, while expression of P or C resulted in moderate inhibition. As indicated by reporter gene assays involving overexpression of the IκB kinase (IKK) complex, which phosphorylates the inhibitor of κB to liberate NF-κB, V protein targets a downstream step in the signaling cascade. Coimmunoprecipitation experiments revealed that V specifically binds to the Rel homology domain of the NF-κB subunit p65 but not of p50. Notably, the short C-terminal domain of the V protein, which is also involved in binding STAT2, IRF7, and MDA5, was sufficient for the interaction and for preventing reporter gene activity. As observed by confocal microscopy, the presence of V abolished nuclear translocation of p65 upon TNF-α stimulation. Thus, MV V appears to prevent NF-κB-dependent gene expression by retaining p65 in the cytoplasm. These findings reveal NF-κB as a key target of MV and stress the importance of the V protein as the major viral immune-modulatory factor.

Under many circumstances, the NF-κB transcription factor signaling pathway is activated to enable animals to respond to environmental stresses, such as pathogens, chemicals, and UV light (14). Once activated, NF-κB transcription factors alter the expression of target genes to counteract these stresses. Examples of genes regulated by NF-κB include those encoding innate immune response factors (e.g., cytokines and antimicrobial peptides), antioxidizing enzymes (e.g., superoxide dismutase and nitric oxide synthase), and antiapoptosis molecules (e.g., Bcl-2, tumor necrosis factor receptor-associated factors [TRAFs], and inhibitor of apoptosis proteins [IAPs]) (14). NF-κB family members have additional roles in development. For example, the Dorsal protein controls the establishment of dorsal-ventral polarity in the Drosophila melanogaster embryo, and mammalian NF-κB proteins control various aspects of immune and liver cell growth and survival (23, 36). In Drosophila, there are three NF-κB proteins, while in humans and other mammals, there are five NF-κB proteins. All NF-κB family transcription factors have a conserved DNA-binding/dimerization domain called the Rel homology domain (RHD), and all NF-κB family proteins bind specific DNA sequences (κB sites) as homodimers or heterodimers. NF-κB proteins can be divided into two classes based on sequences C-terminal to the RHD. One class (the “NF-κB proteins”) includes Drosophila Relish and the mammalian p50/p105 and p52/p100 proteins. Proteins of this class contain C-terminal ankyrin repeat sequences, which restrict the protein to the cytosol and inhibit the DNA-binding activity of the RHD. The C-terminal regions of the Relish, p105, and p100 NF-κB proteins must be proteolytically cleaved for the proteins to become active. In contrast, members of the “Rel class” of proteins (Dorsal and Dif in Drosophila and c-Rel, RelA, and RelB in mammals) contain C-terminal transactivation domains that are not removed. The overall regulation of NF-κB activity in mammals has been extensively characterized (14). In its inactive state, NF-κB is bound to its inhibitor, IκB, in the cytoplasm. Upon activation of the pathway by an upstream signal, IκB is phosphorylated by an IκB kinase (IKK), which targets IκB for ubiquitination and degradation. Free NF-κB then translocates to the nucleus and binds to κB sites in the promoters of specific target genes to alter transcription. One well-characterized transcriptional target of NF-κB is the IκBα gene (15). Thus, NF-κB regulates its own activity by promoting the transcription of its primary inhibitor in a negative feedback loop. In mammals, there are several IκB proteins that have overlapping but nevertheless distinct functions (these proteins are IκBα, IκBβ, IκBɛ, and Bcl-3). All IκB proteins contain multiple copies of ankyrin repeat domain sequences, which mediate binding to the RHD sequences. The C-terminal regions of the mammalian NF-κB proteins also contain ankyrin repeat domains (Fig. ​(Fig.1A)1A) and consequently function as IκBs to inhibit the nuclear translocation of NF-κB. These C-terminal regions are phosphorylated and degraded in response to certain NF-κB-activating signals. FIG. 1. Cloning and expression of Nv-NF-κB, Nv-IκB, Nv-Bcl-3, and Nv-IKK proteins. (A) Structures of human (Homo sapiens [Hs]) and Nematostella vectensis (Nv) orthologs. Conserved domains are shaded in gray or black. Numbers indicate amino acid ... Nematostella vectensis is a small, burrowing sea anemone native to estuaries on the Atlantic coast of North America (29). Nematostella is a member of the phylum Cnidaria, which includes sea anemones, hydras, jellyfish, and corals. Like all anemones, it has two tissue layers, the endoderm and the ectoderm, and lacks the mesodermal layer found in triploblastic metazoans such as nematodes, insects, and vertebrates. The most recent common ancestor of Nematostella and triploblasts is thought to have lived approximately 600 million years ago (26, 27). Nematostella is emerging as the leading model system for cnidarians, in part because its complete genome sequence is known (28). Mining of genomic and expressed sequence tag (EST) databases revealed a single NF-κB gene and two IκB-like genes in Nematostella (33). The RHD of the Nematostella vectensis NF-κB protein (Nv-NF-κB) is ∼50% identical to the RHDs of human p50 and p52 but is only ∼35% identical to the Rel proteins (RelA, c-Rel, and RelB). Moreover, the RHD is immediately followed by a glycine-rich region, which is also present in mammalian p50 and p52. Surprisingly, the Nv-NF-κB protein lacks the C-terminal ankyrin repeat domain found in all other NF-κB proteins (Fig. ​(Fig.1A).1A). The ankyrin repeats of the Nv-IκB proteins, Nv-IκB and Nv-Bcl-3, are 42% and 48% identical to the ankyrin repeat domains of human p105 and Bcl-3, respectively. Additionally, Nematostella has two naturally occurring Nv-NF-κB alleles whose encoded proteins differ at 10 amino acid residues and have distinct DNA-binding and transactivating abilities (35). NF-κB homologs have been identified in a few other basal marine organisms. The coral Acropora millepora, an anthozoan cnidarian like Nematostella, has ESTs corresponding to NF-κB (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EZ002200.1","term_id":"222546010","term_text":"EZ002200.1"}}EZ002200.1) (21) and IκB (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"DY582971","term_id":"89137426","term_text":"DY582971"}}DY582971) (22). However, the genome of the cnidarian Hydra magnipapillata apparently lacks an NF-κB homolog (6). A single NF-κB homolog exists in the demisponge Amphimedon queenslandica of the phylum Porifera (13). This sponge NF-κB protein contains both the RHD and the C-terminal ankyrin repeat domain, suggesting that the independent NF-κB and IκB genes in Nematostella are the result of a gene splitting event. The biological function of the NF-κB signaling pathway in these simple marine organisms is not known; however, NF-κB has been proposed to play a role in mediating host-symbiont interactions in Cnidaria (37). In an effort to understand the evolutionary origins of NF-κB signaling, we have cloned cDNAs for Nv-NF-κB, Nv-IκB, Nv-Bcl-3, and Nv-IKK and expressed these proteins in a variety of cell-based assays. We demonstrated that the activities of these Nematostella proteins are conserved with respect to their vertebrate homologs. In addition, we showed that Nv-NF-κB is expressed primarily in a subset of ectodermal cells in both juvenile and adult Nematostella anemones.

Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor κB (NF-κB) pathway through the action of its transactivator BTas to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question, we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced long terminal repeat [LTR] transactivation) and that this process requires both the localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. The knockdown of the cellular endogenous RelB protein using small interfering RNA (siRNA) significantly attenuated BTas-induced LTR transcription. The results of chromatin immunoprecipitation (ChIP) analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages the RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection upregulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive-feedback circuit in which BFV utilizes the host's NF-κB pathway through the RelB protein for efficient viral transcription.

NF-κB is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-κB-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor κB (IκB)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-κBs and phylogenetic analysis suggested that EsRelish was a member of the NF-κB family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.

Rel/NF-kappaB transcription factors play central roles in induction and regulation of innate immune responses. Here, identification and functional analysis of LvDorsal, a Dorsal homologue from the Pacific white shrimp Litopenaeus vannamei, were described. The full-length cDNA of LvDorsal is 2204bp with an open reading frame that encodes 400 amino acids. The deduced LvDorsal contains a conserved Rel homology domain (RHD), an IPT (Ig-like, plexins and transcription factors) domain and a nucleus localization signal, suggesting that it belongs to the class II NF-kappaB. RT-PCR analysis showed that LvDorsal mRNAs were expressed in all the tissues tested, including gill, epidermis, hemocytes, intestine, stomach, eyestalk, brain, hepatopancreas, muscle, heart and pyloric caecum. Immunofluorescence assay showed that recombinant LvDorsal was translocated into the nucleus of Drosophila S2 cells. Electrophoretic mobility shift assay illustrated that recombinant LvDorsal RHD from S2 cells bound specifically with D. melanogaster kappaB motifs. Additionally, the dual-luciferase reporter assays indicated that LvDorsal could transactivate the reporter gene controlled by the 5' flanking region of shrimp penaeidin-4 and Drosophila attacin genes, suggesting that LvDorsal can regulate the transcription of shrimp penaeidin-4 gene. Study of LvDorsal will help us to better understand shrimp immunity and may help to obtain more effective methods to prevent shrimp diseases.

A cDNA encoding an IkappaB family protein was identified and the full nucleotide sequence was determined in the silkworm Bombyx mori. The IkappaB gene, designated BmCactus, was constitutively expressed mainly in the fat body and hemocytes. Transfection experiments on a B. mori cell line, NIAS-Bm-aff3, with expression vectors containing BmCactus, BmRelA, BmRelB, or the active portion of BmRelish1 showed that activation of the CecB1 gene promoter by either BmRelA or BmRelB, but not the active portion of BmRelish1, was strongly inhibited by BmCactus. In addition, activation of CecB1 gene by autoclaved E. coli in the cultured cells was observed regardless of the presence or absence of BmCactus. A glutathione S-transferase pull-down assay and analysis using a yeast two-hybrid system demonstrated that BmCactus interacted with the BmRel Rel homology domain, but not with the BmRelish Rel homology domain. These results suggest that BmCactus is involved in the Toll signal transduction pathway in B. mori.

International audience; Analyses of recently sequenced sponge, cnidarian, placozoan, and choanoflagellate genomes have revealed that most transcription factor (TF) classes and families expressed during bilaterian development originated at the dawn of the animal kingdom, before the divergence of contemporary animal lineages. The ancestral metazoan genome included members of the bHLH, Mef2, Fox, Sox, T-box, ETS, nuclear receptor, Rel/NF-kappaB, bZIP, and Smad families, and a diversity of homeobox-containing classes, including ANTP, Prd-like, Pax, POU, LIM-HD, Six, and TALE. As many of these TF classes and families appear to be metazoan specific and not present in choanoflagellates, fungi and more distant eukaryotes, their genesis and expansion may have contributed to the evolution of animal multicellularity.

The interplay of transcription factors, histone modifiers, and DNA modification can alter chromatin structure that epigenetically controls gene transcription. During severe systemic inflammatory (SSI), the generation of facultative heterochromatin from euchromatin reversibly silences transcription of a set of acute proinflammatory genes. This gene-specific silencing is a salient feature of the endotoxin tolerant phenotype that is found in blood leukocytes of SSI patients and in a human THP-1 cell model of SSI. We previously reported that de novo induction of the NF-kappaB transcription factor RelB by endotoxin activation is necessary and sufficient for silencing transcription of acute proinflammatory genes in the endotoxin tolerant SSI phenotype. Here, we examined how RelB silences gene expression and found that RelB induces facultative heterochromatin formation by directly interacting with the histone H3 lysine 9 methyltransferase G9a. We found that heterochromatin protein 1 (HP1) and G9a formed a complex at the interleukin-1beta promoter that is dependent on the Rel homology domain (RHD) of RelB. RelB knockdown disassociated the complex and reversed transcription silencing. We also observed that whereas RelB chromatin binding was independent of G9a, RelB transcriptional silencing required G9a accumulation at the silenced promoter. Binding between RelB and G9a was confirmed by glutathione S-transferase pulldown in vitro and coimmunoprecipitation in vivo. These data provide novel insight into how RelB is required to initiate silencing in the phenotype associated with severe systemic inflammation in humans, a disease with major morbidity and mortality.