κB Sites Research Articles

Regulation of c-Fos Gene Expression by NF-κB: A p65 Homodimer Binding Site in Mouse Embryonic Fibroblasts but Not Human HEK293 Cells

The immediate early gene c-Fos is reported to be regulated by Elk-1 and cAMP response element-binding protein (CREB), but whether nuclear factor (NF)-κB is also required for controlling c-Fos expression is unclear. In this study, we determined how NF-κB’s coordination with Elk/serum response factor (SRF) regulates c-fos transcription. We report that PMA strongly induced c-Fos expression, but tumor necrosis factor (TNF)-α did not. In mouse embryonic fibroblasts, the PMA induction of c-Fos was suppressed by a deficiency in IKKα, IKKβ, IKKγ, or p65. By contrast, in human embryonic kidney 293 cells, PMA induced c-Fos independently of p65. In accordance with these results, we identified an NF-κB binding site in the mouse but not human c-fos promoter. Under PMA stimulation, IKKα/β mediated p65 phosphorylation and the binding of the p65 homodimer to the NF-κB site in the mouse c-fos promoter. Furthermore, our studies demonstrated independent but coordinated functions of the IKKα/β-p65 and extracellular signal-regulated kinase (ERK)-Elk-1 pathways in the PMA induction of c-Fos. Collectively, these results reveal the distinct requirement of NF-κB for mouse and human c-fos regulation. Binding of the p65 homodimer to the κB site was indispensable for mouse c-fos expression, whereas the κB binding site was not present in the human c-fos promoter. Because of an inability to evoke sufficient ERK activation and Elk-1 phosphorylation, TNF-α induces c-Fos more weakly than PMA does in both mouse and human cells.

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription

Diabetes mellitus results from immune cell invasion into pancreatic islets of Langerhans, eventually leading to selective destruction of the insulin-producing β-cells. How this process is initiated is not well understood. In this study, we investigated the regulation of the CXCL1 and CXCL2 genes, which encode proteins that promote migration of CXCR2(+) cells, such as neutrophils, toward secreting tissue. Herein, we found that IL-1β markedly enhanced the expression of the CXCL1 and CXCL2 genes in rat islets and β-cell lines, which resulted in increased secretion of each of these proteins. CXCL1 and CXCL2 also stimulated the expression of specific integrin proteins on the surface of human neutrophils. Mutation of a consensus NF-κB genomic sequence present in both gene promoters reduced the ability of IL-1β to promote transcription. In addition, IL-1β induced binding of the p65 and p50 subunits of NF-κB to these consensus κB regulatory elements as well as to additional κB sites located near the core promoter regions of each gene. Additionally, serine-phosphorylated STAT1 bound to the promoters of the CXCL1 and CXCL2 genes. We further found that IL-1β induced specific posttranslational modifications to histone H3 in a time frame congruent with transcription factor binding and transcript accumulation. We conclude that IL-1β-mediated regulation of the CXCL1 and CXCL2 genes in pancreatic β-cells requires stimulus-induced changes in histone chemical modifications, recruitment of the NF-κB and STAT1 transcription factors to genomic regulatory sequences within the proximal gene promoters, and increases in phosphorylated forms of RNA polymerase II.

IκBα beyond the NF-kB dogma

The NF-κB pathway participates in a myriad of processes including cell differentiation, proliferation, survival, and regulation of the immune response. In mammals there are five NF-κB proteins: p65/RelA, p50, RelB, cRel and p52. All these proteins can form dimers to generate more than a dozen of possible combinations. Different NF-κB dimers bind the DNA at specific κB sites, acting as activators or repressors of gene transcription. However, this is only an example of how complex the NF-κB pathway is, since it also involves several post-translational modifications of upstream and downstream elements and multiple negative and positive regulatory loops. IκB proteins, including IκBα, IκBβ and IκBe constitute the canonical inhibitors of the NF-κB pathway. However p105 and p100 proteins, the precursors of p50 and p52, contain a C-terminal end that is functionally comparable to IκB (this is the reason they are also called IκBγ and IκBδ, respectively). In unstimulated cells, NF-κB dimers reside in the cytoplasm bound to IκB proteins. Upon stimulation, IκBs become phosphorylated, subsequently polyubiquitinated and degraded by the proteasome. Degradation of IκB leads to the release of the NF-κB factors that then translocate to the nucleus to activate transcription of specific genes including IκBα itself. Termination of the transcriptional response primarily depends on IκBα resynthesis that is required not only for retaining NF-κB in the cytoplasm but also for removing active NF-κB dimers from the DNA [1]. Because this mechanism implies that IκBα continuously shuttles from the cytoplasm to the nucleus to maintain the cells in a silent NF-κB state [2], it is not so surprising that IκBα has been found associated with other proteins that are primarily nuclear such as histone deacetylases (HDACs) or nuclear corepressors [3, 4]. Now, we have demonstrated that IκBα is predominantly nuclear in primary keratinocytes where it binds the chromatin at specific genes promoters associated with their transcriptional repression [5]. Mechanistically, we found that IκBα facilitates the association of the Polycomb Repressive Complex 2 (PRC2) to specific promoters and supply PRC2 with the capacity to respond to TNFα stimulation, thus establishing an unexpected link between inflammatory signals and skin homeostasis. This result is particularly relevant since to date it is still not clear how PRC2 is recruited to the chromatin in mammals, unlike in flies where specific PRC2 recognition DNA sequences have been identified. The physiologic relevance of IκB and Polycomb (Pc) association has been validated in Drosophila using compose mutants of Cactus, the homolog of IκBα in flies, and Pc, which in fact indicates the elevated conservation of this function during evolution. The identification of IκBα as a chromatin interacting protein apparently contradicts the notion that IκBα cannot bind the DNA. However, it is not just IκBα but a SUMOylated and phosphorylated form of the protein that binds the chromatin, suggestive of structural changes that affect the biochemistry of the protein including its capacity to bind NF-κB or the chromatin in a mutually exclusive manner. Another member of the family, IκBβ, also binds the chromatin but only when it is hypophosphorylated [6], suggesting that post-translational modifications of the NF-κB elements link this pathway with specific cellular functions. This is also the case of IKKα and NEMO that exert additional NF-κB-related but independent functions such as cell cycle control and DNA-damage repair, respectively [7, 8]. However, which are the modifications that regulate alternative IKKα and NEMO functions is not completely understood. Detection of cytoplasmic IκBα has never been considered as pathologic in any tissue, however this seems to be the case in the skin (Fig. ​(Fig.1).1). In Mulero et al we found that nuclear exclusion and cytoplasmic accumulation of IκBα strongly associated with progression of Squamous Cell Carcinoma (SCC) in patient samples. Although this is not addressed in the study, it is plausible that this is not cytoplasmic accumulation of conventional IκBα but of SUMOylated IκBα what exerts pro-tumorigenic functions, likely by relieving the nucleus from transcriptional regulators. Further studies should be performed to validate this idea. Why this is observed in the skin but not in other tissues can be basically a matter of levels of either phosphatases and/or desumoylases, which will be deciphered in the near future. However, this does not exclude that low amounts of SUMOylated IκBα exert comparable functions in cells other than the keratinocytes, which will be also investigated. Figure 1 Cytoplasmic localization of IκBα in the skin is associated with tumorigenesis In conclusion, the identification of SUMOylated and phosphorylated IκBα as a spinoff of IκBα that plays unexpected but important physiologic function opens a whole field of research and the possibility to re-interpret some old unexpected results with a novel and renewed perspective.

Analysis of the RelA:CBP/p300 Interaction Reveals Its Involvement in NF-κB-Driven Transcription

Author SummaryThe NF-κB family of transcription factors regulate the expression of numerous genes involved in the immune response, cell survival, differentiation, and proliferation. The interaction of the RelA subunit of NF-κB with the general co-activator protein CBP/p300 is vital for RelA-dependent gene transcription. Although the recruitment of RelA to its cognate genomic κB sites for target gene activation is well-established, the involvement of CBP/p300 in this process remains unclear. Through our structure/function-based approach we provide the molecular and functional details of the RelA:CBP/p300 interaction and its contribution to the regulation of distinct subsets of target genes. We also show that disruption of this interaction deregulates the NF-κB pathway by interfering with its negative feedback loop. Furthermore, our study indicates a possible reciprocal role for CBP/p300 in the recruitment of RelA to its target gene promoters.

Role of lysine methylation of NF-κB in differential gene regulation

Lysine methylation of the p65 subunit of nuclear factor κB (NF-κB) on K218 and K221 together or K37 alone strongly enhances gene expression in response to cytokines. We analyzed the effects of K-to-Q mutations in the REL homology domain of p65 on the response to IL-1β in 293 cells with low levels of p65. The K218/221Q mutation greatly reduced the expression of 39 of 82 genes, whereas the K37Q mutation reduced the expression of 23 different genes. Enhanced expression of the lysine demethylase FBXL11, which catalyzes the demethylation of K218 and K221 specifically, inhibited the expression of most of the genes that were inhibited by the DKQ mutation. CHIP-Seq analysis showed that the K218/221Q mutation greatly reduces the affinity of p65 for many promoters and that the K37Q mutation does not. Structural modeling showed that the newly introduced methyl groups of K218 and K221 interact directly with DNA to increase the affinity of p65 for specific κB sites. Thus, the K218/221Q and K37Q mutations have dramatically different effects because methylations of these residues affect different genes by distinct mechanisms.

Failure in activation of the canonical NF-κB pathway by human T-cell leukemia virus type 1 Tax in non-hematopoietic cell lines

Human T-cell leukemia virus type 1 (HTLV-1) Tax (Tax1) plays crucial roles in leukemogenesis in part through activation of NF-κB. In this study, we demonstrated that Tax1 activated an NF-κB binding (gpκB) site of the gp34/OX40 ligand gene in a cell type-dependent manner. Our examination showed that the gpκΒ site and authentic NF-κB (IgκB) site were activated by Tax1 in hematopoietic cell lines. Non-hematopoietic cell lines including hepatoma and fibroblast cell lines were not permissive to Tax1-mediated activation of the gpκB site, while the IgκB site was activated in those cells in association with binding of RelB. However RelA binding was not observed in the gpκB and IgκB sites. Our results suggest that HTLV-1 Tax1 fails to activate the canonical pathway of NF-κB in non-hematopoietic cell lines. Cell type-dependent activation of NF-κB by Tax1 could be associated with pathogenesis by HTLV-1 infection.

Characterization of genome-wide binding of NF-κB in TNFα-stimulated HeLa cells

This study characterized the genome-wide binding of NF-κB RelA with ChIP-Seq and explored its effects on the gene transcription with DNA microarray. It was found that NF-κB showed several significant binding characteristics, including the inter- and intra-chromosomal differential high-fold enrichment binding, the dominant intronic binding to vast majority of target genes through multiple ChIP-seq peaks and κB sites, extensively binding to large number of genes in the human genome, and binding its target genes more broadly through noncanonical κB sites than canonical κB sites. These in vivo genome-wide binding characteristics exerted effects on the transcription of its direct target genes in genome, reflecting some important traits of this protein which acts as a stimulatory transcription factor involving in many biological processes and responding to various internal and external stimuli.

The eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NF-κB and is aberrantly regulated in acute myeloid leukemia

The eukaryotic translation initiation factor eIF4E is a potent oncogene elevated in many cancers, including the M4 and M5 subtypes of acute myeloid leukemia (AML). Although eIF4E RNA levels are elevated 3- to 10-fold in M4/M5 AML, the molecular underpinnings of this dysregulation were unknown. Here, we demonstrate that EIF4E is a direct transcriptional target of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) that is dysregulated preferentially in M4 and M5 AML. In primary hematopoietic cells and in cell lines, eIF4E levels are induced by NF-κB activating stimuli. Pharmacological or genetic inhibition of NF-κB represses this activation. The endogenous human EIF4E promoter recruits p65 and cRel to evolutionarily conserved κB sites in vitro and in vivo following NF-κB activation. Transcriptional activation is demonstrated by recruitment of p300 to the κB sites and phosphorylated Pol II to the coding region. In primary AML specimens, generally we observe that substantially more NF-κB complexes associate with eIF4E promoter elements in M4 and M5 AML specimens examined than in other subtypes or unstimulated normal primary hematopoietic cells. Consistently, genetic inhibition of NF-κB abrogates eIF4E RNA levels in this same population. These findings provide novel insights into the transcriptional control of eIF4E and a novel molecular basis for its dysregulation in at least a subset of M4/M5 AML specimens.

Identification of nuclear factor-κB sites in the Slc2a4 gene promoter

Glucose transporter GLUT4 protein, codified by Slc2a4 gene plays a key role in glycemic homeostasis. Insulin resistance, as in obesity, has been associated to inflammatory state, in which decreased GLUT4 is a feature. Inflammatory NF-κB transcriptional factor has been proposed as a repressor of Slc2a4; although, the binding site(s) in Slc2a4 promoter and the direct repressor effect have never been reported yet. A motif-based sequence analysis of mouse Slc2a4 promoter revealed two putative κB sites located inside −83/−62 and −134/−113bp. Eletrophoretic mobility assay showed that p50 and p65 NF-κB subunits bind to both putative κB sites. Chromatin immunoprecipitation assay using genomic DNA from adipocytes confirmed p50- and p65-binding to Slc2a4 promoter. Moreover, transfection experiments revealed that NF-κB binds to the −134/−113bp region of the mouse Slc2a4 gene promoter, inhibiting the Slc2a4 gene transcription. The current findings demonstrate the existence of two κB sites in Slc2a4 gene promote, and that NF-κB has a direct repressor effect upon the Slc2a4 gene, providing an important link between insulin resistance and inflammation.

P65 Chromatin Immunoprecipitation Protocol

Chromatin Immunoprecipitation (ChIP) is an important procedure that allows you to verify if a certain protein is physically located at a regulatory region. This information, taken together with other procedures such as luciferase assays and EMSAs, will give definitive proof that the query protein is involved in the transcription of a protein. This procedure for p65 ChIP can be adapted to investigate other proteins; just a change of the antibody will suffice. The transcription factor known as NF-κB is a homo- or hetero-dimer consisting of members of the Rel/NFKB family. The most abundant NF-κB complexes are made of two different proteins, p65 (Rel-A) and p50 (NFKB1). The NF-κB complex is initially inhibited by IκB by direct binding, thus trapping NF-κB in the cytoplasm. After a stimulatory signal, IκB kinase (IKK) phosphorylates IκB, allowing IκB to undergo proteasome-mediated degradation. The degradation of IκB and phosphorylation of p65 by multiple kinases activates NF-κB, allowing it to transport to the nucleus and cause the transcriptional activation of many of its target genes containing κB sites (consensus sequence: gggRNNYYcc, R = purine Y = pyrimidine), such as PUMA, IL-6, and TNF.

Nuclear Factor-κB (NF-κB) Regulates the Expression of Human Testis-Enriched Leucine-Rich Repeats and WD Repeat Domain Containing 1 (LRWD1) Gene

The human Leucine-rich Repeats and WD repeat Domain containing 1 (LRWD1) gene was originally identified by cDNA microarray as one of the genes down-regulated in the testicular tissues of patients with severe spermatogenic defects. Human LRWD1 is a testicular-enriched protein that is present predominantly in the cytoplasm of spermatocytes and spermatids and colocalizes with the centrosome at the base of sperm tail. Reporter assay, Chromatin immunoprecipitation (ChIP) analysis, and gel electrophoretic mobility shift assay (EMSA) were used to identify the core promoter region of LRWD1. A 198 bp segment upstream of the LRWD1 transcription initiation site exhibited promoter activity. The LRWD1 core promoter lacked a TATA box but contained a NF-κB binding site. Chromatin immunoprecipitation (ChIP) analysis and gel electrophoretic mobility shift assay (EMSA) showed basal binding of the NF-κB subunit to the LRWD1 promoter. LRWD1 promoter activity was positively regulated by NF-κB, and this regulation was dependent on the presence of the conserved κB site in the LRWD1 promoter region. Our data suggest that NF-κB is an important regulator for the expression of LRWD1. This is the first study showing that the expression of the testis-enriched LRWD1 gene is regulated by NF-κB.

Subset of genes targeted by transcription factor NF-κB in TNFα-stimulated human HeLa cells

Nuclear factor κB (NF-κB) is a ubiquitous transcription factor that plays a pivotal role in controlling important cellular processes, ranging from normal cell growth and differentiation to apoptosis and cancer. In recent years, many new target genes of NF-κB have been identified in several cell lines that were treated with various stimuli using chromatin immunoprecipitation (ChIP)-based high-throughput techniques. However, the target genes from various cell lines and stimuli are not identical, and many of them are cell or stimulus specific. This suggests that it is necessary to investigate different cell lines and stimuli for identifying all target genes of this transcription factor. In this study, the direct target genes (DTGs) of NF-κB in the TNFα-stimulated HeLa cells were identify by using ChIP-Seq, RNAi, and gene expression profiling techniques. As a result, 584 DTGs were identified, in which 266 were activated and 318 were repressed. The κB motif searching revealed that 50% of these genes contained canonical κB sites in their ChIP peaks and 90% contained non-canonical κB sites in their ChIP peaks. In comparison with target genes identified in LPS-treated U937 and THP-1, only limited numbers (10∼23) of target genes were shared by each of two cell lines, and only two gene (NFKB2 and STAT5A) were commonly shared by three cell lines.

IL-36γ/IL-1F9, an Innate T-bet Target in Myeloid Cells

By concerted action in dendritic (DC) and T cells, T-box expressed in T cells (T-bet, Tbx21) is pivotal for initiation and perpetuation of Th1 immunity. Identification of novel T-bet-regulated genes is crucial for further understanding the biology of this transcription factor. By combining siRNA technology with genome-wide mRNA expression analysis, we sought to identify new T-bet-regulated genes in predendritic KG1 cells activated by IL-18. One gene robustly dependent on T-bet was IL-36γ, a recently described novel IL-1 family member. Promoter analysis revealed a T-bet binding site that, along with a κB site, enables efficient IL-36γ induction. Using knock-out animals, IL-36γ reliance on T-bet was extended to murine DC. IL-36γ expression by human myeloid cells was confirmed using monocyte-derived DC and M1 macrophages. The latter model was employed to substantiate dependence of IL-36γ on endogenous T-bet in human primary cells. Ectopic expression of T-bet likewise mediated IL-36γ production in HaCaT keratinocytes that otherwise lack this transcription factor. Additional experiments furthermore revealed that mature IL-36γ has the capability to establish an inflammatory gene expression profile in human primary keratinocytes that displays enhanced mRNA levels for TNFα, CCL20, S100A7, inducible NOS, and IL-36γ itself. Data presented herein shed further light on involvement of T-bet in innate immunity and suggest that IL-36γ, besides IFNγ, may contribute to functions of this transcription factor in immunopathology.

Multiple NF-κB Sites in HIV-1 Subtype C Long Terminal Repeat Confer Superior Magnitude of Transcription and Thereby the Enhanced Viral Predominance

We demonstrate that at least three different promoter variant strains of HIV-1 subtype C have been gradually expanding and replacing the standard subtype C viruses in India, and possibly in South Africa and other global regions, over the past decade. The new viral strains contain an additional NF-κB, NF-κB-like, or RBEIII site in the viral promoter. Although the acquisition of an additional RBEIII site is a property shared by all the HIV-1 subtypes, acquiring an additional NF-κB site remains an exclusive property of subtype C. The acquired κB site is genetically distinct, binds the p50-p65 heterodimer, and strengthens the viral promoter at the levels of transcription initiation and elongation. The 4-κB viruses dominate the 3-κB "isogenic" viral strains in pairwise competition assays in T-cell lines, primary cells, and the ecotropic human immunodeficiency virus mouse model. The dominance of the 4-κB viral strains is also evident in the natural context when the subjects are coinfected with κB-variant viral strains. The mean plasma viral loads, but not CD4 counts, are significantly different in 4-κB infection suggesting that these newly emerging strains are probably more infectious. It is possible that higher plasma viral loads underlie selective transmission of the 4-κB viral strains. Several publications previously reported duplication or deletion of diverse transcription factor-binding sites in the viral promoter. Unlike previous reports, our study provides experimental evidence that the new viral strains gained a potential selective advantage as a consequence of the acquired transcription factor-binding sites and importantly that these strains have been expanding at the population level.

Forkhead Transcription Factor FOXP3 Upregulates CD25 Expression through Cooperation with RelA/NF-κB

Considerable evidence supports the prediction that CD25 is directly regulated by the forkhead transcription factor FOXP3. However, given that CD25 is normally upregulated in activated T cells, regardless of whether they express FOXP3, this issue has still to be definitively demonstrated. Here we describe that FOXP3, induced by CD28 signals in human CD4+CD25− T lymphocytes, synergizes with RelA on a regulatory region of Cd25 promoter to mediate the transcriptional activation of Cd25 gene. We found that a striking feature of this regulatory region is the presence of a κB site and of two tandem copies of a non-consensus FOXP3 binding site separated at 5′ ends by 19 nucleotides that allow FOXP3 and RelA binding to DNA and their physical interaction. The occupancy of the two FOXP3 binding sites in conjunction with RelA binding site occupancy allows FOXP3 to function as a positive activator of Cd25 gene. Indeed mutations of both FOXP3 binding sites such as mutation of κB site on Cd25 promoter abolished FOXP3 activatory functions. Moreover, FOXP3 mutation ΔE251, that compromises FOXP3 homotypic interactions, failed to trans activate Cd25 promoter, suggesting that both FOXP3 DNA binding and dimerization are required to trans activate Cd25 promoter. These findings identify a novel mechanism by which RelA and FOXP3 cooperate to mediate transcriptional regulation of target genes and characterize a region on Cd25 promoter where FOXP3 dimer could bridge intramolecularly two DNA sites and trans activate Cd25 gene.

The Transcriptional Specificity of NF-κB Dimers Is Coded within the κB DNA Response Elements

SUMMARYNuclear factor κB (NF-κB) regulates gene expression by binding to specific DNA elements, known collectively as κB sites, that are contained within the promoters/enhancers of target genes. We found that the identity of the central base pair (bp) of κB sites profoundly affects the transcriptional activity of NF-κB dimers. RelA dimers prefer an A/T bp at this position for optimal transcriptional activation (A/T-centric) and discriminate against G/C-centric κB sites. The p52 homodimer, in contrast, activates transcription from G/C-centric κB sites in complex with Bcl3 but represses transcription from the A/T-centric sites. The p52:Bcl3 complex binds to these two classes of κB sites in distinct modes, permitting the recruitment of coactivator, corepressor, or both coactivator and corepressor complexes in promoters that contain G/C-, A/T-, or both G/C- and A/T-centric sites. Therefore, through sensing of bp differences within κB sites, NF-κB dimers modulate biological programs by activating, repressing, and altering the expression of effector genes.

Oct-1 acts as a transcriptional repressor on the C-reactive protein promoter

C-reactive protein (CRP), a plasma protein of the innate immune system, is produced by hepatocytes. A critical regulatory region (−42 to −57) on the CRP promoter contains binding site for the IL-6-activated transcription factor C/EBPβ. The IL-1β-activated transcription factor NF-κB binds to a κB site located nearby (−63 to −74). The κB site overlaps an octamer motif (−59 to −66) which is the binding site for the constitutively active transcription factor Oct-1. Oct-1 is known to function both as a transcriptional repressor and as an activator depending upon the promoter context. Also, Oct-1 can regulate gene expression either by binding directly to the promoter or by interacting with other transcription factors bound to the promoter. The aim of this study was to investigate the functions of Oct-1 in regulating CRP expression. In luciferase transactivation assays, overexpressed Oct-1 inhibited (IL-6+IL-1β)-induced CRP expression in Hep3B cells. Deletion of the Oct-1 site from the promoter drastically reduced the cytokine response because the κB site was altered as a consequence of deleting the Oct-1 site. Surprisingly, overexpressed Oct-1 inhibited the residual (IL-6+IL-1β)-induced CRP expression through the promoter lacking the Oct-1 site. Similarly, deletion of the Oct-1 site reduced the induction of CRP expression in response to overexpressed C/EBPβ, and overexpressed Oct-1 inhibited C/EBPβ-induced CRP expression through the promoter lacking the Oct-1 site. We conclude that Oct-1 acts as a transcriptional repressor of CRP expression and it does so by occupying its cognate site on the promoter and also via other transcription factors by an as yet undefined mechanism.

Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-κB-Dependent Transcription of Specific Genes after Genotoxic Stress

The NF-κB pathway is involved in immune and inflammation responses, proliferation, differentiation and cell death or survival. It is activated by many external stimuli including genotoxic stress. DNA double-strand breaks activate NF-κB in an ATM-dependent manner. In this manuscript, a direct interaction between p65(RelA) and the N-terminal extremity of ATM is reported. We also report that only one of the five potential ATM-(S/T)Q target sites present in p65, namely Ser547, is specifically phosphorylated by ATM in vitro. A comparative transcriptomic analysis performed in HEK-293 cells expressing either wild-type HA-p65 or a non-phosphorylatable mutant HA-p65S547A identified several differentially transcribed genes after an etoposide treatment (e.g. IL8, A20, SELE). The transcription of these genes is increased in cells expressing the mutant. Substitution of Ser547 to alanine does not affect p65 binding abilities on the κB site of the IL8 promoter but reduces p65 interaction with HDAC1. Cells expressing p65S547A have a higher level of histone H3 acetylated on Lys9 at the IL8 promoter, which is in agreement with the higher gene induction observed. These results indicate that ATM regulates a sub-set of NF-κB dependent genes after a genotoxic stress by direct phosphorylation of p65.

OCT-1 acts as a repressor of C-reactive protein gene expression (172.13)

Abstract C-reactive protein (CRP), an ancient host-defense plasma protein, is produced by hepatocytes. In Hep3B cells, CRP expression is induced by IL-6; IL-1β synergistically enhances the IL-6 effect. A critical regulatory segment on the CRP promoter contains binding sites for transcription factors C/EBPβ (-48 to -57) and NF-κB (-63 to -74). The κB site overlaps an octamer motif (-59 to -66) which is the binding site for the constitutively expressed transcription factor OCT-1. OCT-1 is known to function both as a transcriptional repressor and activator depending upon the promoter context. The aim of this study was to investigate the function of OCT-1 on the CRP promoter. In luciferase transactivation assays, overexpressed OCT-1 inhibited cytokine-induced CRP expression. EMSA, mutational analysis of the κB site, and the OCT-1 dose-response assays suggested that OCT-1 and NF-κB compete for binding to their overlapping cognate sites. Deletion of the OCT-1 site drastically reduced the cytokine response because the κB site was altered as a consequence of deleting the OCT-1 site. Surprisingly, overexpressed OCT-1 inhibited the residual cytokine-induced CRP expression through the promoter lacking the OCT-1 site. OCT-1 also inhibited overexpressed C/EBPβ-induced CRP expression. We conclude that OCT-1 acts as a repressor of CRP gene expression and it does so by occupying its cognate site on the promoter and also via other transcription factors by an as yet undefined mechanism.

Sequence Insertions in the HIV Type 1 Subtype C Viral Promoter Predominantly Generate an Additional NF-κB Binding Site

After screening a large number of clinical samples of HIV-1 subtype C in India, a subset of viral strains containing sequence insertions upstream of the viral enhancer has been identified. The sequence insertions contained binding sites for at least two different transcription factors NF-κB and RBEIII, importantly, in a mutually exclusive fashion. Furthermore, while some of the viral strains contained insertions of κB-like sites, a few others contained dual insertions of the RBEIII and κB sites together but only one of the two was intact. NF-κB acquisition appears to be the most common phenotype unique for subtype C with nearly half of the variant strains containing such insertions. Given that subtype C already contains three functional NF-κB sites in the viral enhancer, acquisition of a fourth NF-κB motif in some variant viral strains is intriguing. Further investigation is warranted to examine the significance of the sequence insertions for the replicative fitness of the variant viral strains.