SNW1, a Novel Transcriptional Regulator of the NF-?B Pathway.
Ontology highlight
ABSTRACT:
SUBMITTER: Verma S
PROVIDER: S-EPMC6336138 | biostudies-literature | 2019 Feb
REPOSITORIES: biostudies-literature
Publications
SNW1, a Novel Transcriptional Regulator of the NF-κB Pathway.
Verma Suneer S De Jesus Paul P Chanda Sumit K SK Verma Inder M IM
Molecular and cellular biology 20190116 3
The nuclear factor kappa B (NF-κB) family of transcription factors plays a central role in coordinating the expression of genes that control inflammation, immune responses, cell proliferation, and a variety of other biological processes. In an attempt to identify novel regulators of this pathway, we performed whole-genome RNA interference (RNAi) screens in physiologically relevant human macrophages in response to lipopolysaccharide and tumor necrosis factor alpha (TNF-α). The top hit was SNW1, a ...[more]
Similar Datasets
Project description:MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix-loop-helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT-REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT-REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer.
| S-EPMC7794610 | biostudies-literature
Project description:BACKGROUND:Overexpression of FAT1 gene and its oncogenic effects have been reported in several cancers. Previously, we have documented upregulation of FAT1 gene in glioblastoma (GBM) tumors which was found to increase the expression of proinflammatory markers, HIF-1?, stemness genes and EMT markers in glioma cells. Here, we reveal NF?B (RelA)/RelA/p65 as the transcriptional regulator of FAT1 gene in GBM cells. METHODS:In-silico analysis of FAT1 gene promoter was performed using online bioinformatics tool Promo alggen (Transfac 8.3) to identify putative transcription factor(s) binding motifs. A 4.0?kb FAT1 promoter (-?3220?bp to +?848?bp w.r.t. TSS?+?1) was cloned into promoter less pGL3Basic reporter vector. Characterization of FAT1 promoter for transcriptional regulation was performed by in-vitro functional assays using promoter deletion constructs, site directed mutagenesis and ChIP in GBM cells. RESULTS:Expression levels of NF?B (RelA) and FAT1 were found to be increased and positively correlated in GBM tumors (n?=?16), REMBRANDT GBM-database (n?=?214) and TCGA GBM-database (n?=?153). In addition to glioma, positive correlation between NF?B (RelA) and FAT1 expression was also observed in other tumors like pancreatic, hepatocellular, lung and stomach cancers (data extracted from TCGA tumor data). A 4.0?kb FAT1-promoter-construct [-?3220?bp/+?848?bp, transcription start site (TSS)?+?1, having 17 NF?B (RelA) motifs] showed high FAT1 promoter luciferase-activity in GBM cells (U87MG/A172/U373MG). FAT1 promoter deletion-construct pGL3F1 [-?200?bp/+?848?bp, with 3-NF?B (RelA)-motifs] showed the highest promoter activity. Exposure of GBM cells to known NF?B (RelA)-activators [severe-hypoxia/TNF-?/ectopic-NF?B (RelA)?+?IKBK vectors] led to increased pGL3F1-promoter activity and increased endogenous-FAT1 expression. Conversely, siRNA-mediated NF?B (RelA) knockdown led to decreased pGL3F1-promoter activity and decreased endogenous-FAT1 expression. Deletion of NF?B (RelA)-motif at -?90?bp/-?80?bp [pGL3F1?1-construct] showed significant decrease in promoter activity. Site directed mutagenesis at -90?bp/-?80?bp and ChIP assay for endogenous-NF?B (RelA) confirmed the importance of this motif in FAT1 expression regulation. Significant reduction in the migration, invasion as well as colony forming capacity of the U87MG glioma cells was observed on siRNA-mediated knockdown of NF?B (RelA). CONCLUSION:Since FAT1 and NF?B (RelA) are independently known to promote pro-tumorigenic inflammation and upregulate the expression of HIF-1?/EMT/stemness in tumors, targeting the NF?B (RelA)-FAT1 axis may attenuate an important tumor-promoting pathway in GBM. This may also be applicable to other tumors.
| S-EPMC6988320 | biostudies-literature
Project description:Riboflavin (vitamin B2) is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide, which are essential coenzymes in all free-living organisms. Riboflavin biosynthesis in many Bacteria but not in Archaea is controlled by FMN-responsive riboswitches. We identified a novel bifunctional riboflavin kinase/regulator (RbkR), which controls riboflavin biosynthesis and transport genes in major lineages of Crenarchaeota, Euryarchaeota and Thaumarchaeota. RbkR proteins are composed of the riboflavin kinase domain and a DNA-binding winged helix-turn-helix-like domain. Using comparative genomics, we predicted RbkR operator sites and reconstructed RbkR regulons in 94 archaeal genomes. While the identified RbkR operators showed significant variability between archaeal lineages, the conserved core of RbkR regulons includes riboflavin biosynthesis genes, known/predicted vitamin uptake transporters and the rbkR gene. The DNA motifs and CTP-dependent riboflavin kinase activity of two RbkR proteins were experimentally validated in vitro. The DNA binding activity of RbkR was stimulated by CTP and suppressed by FMN, a product of riboflavin kinase. The crystallographic structure of RbkR from Thermoplasma acidophilum was determined in complex with CTP and its DNA operator revealing key residues for operator and ligand recognition. Overall, this study contributes to our understanding of metabolic and regulatory networks for vitamin homeostasis in Archaea.
| S-EPMC5397151 | biostudies-literature
Project description:PURPOSE:The forkhead transcription factor (FoxO1) is a master transcriptional regulator of fundamental cellular processes ranging from cell proliferation and differentiation to inflammation and metabolism. However, despite its relevance, the mechanism(s) underlying FoxO1 gene regulation are largely unknown. We have previously shown that the chromatin factor high-mobility group A1 (HMGA1) plays a key role in the transcriptional regulation of glucose-responsive genes, including some that are involved in FoxO1-mediated glucose metabolism. Here we investigated the impact of HMGA1 on FoxO1 gene expression. METHODS:FoxO1 protein and gene expression studies were performed by Western blot analysis combined with qRT-PCR of material from human cultured cells and EBV-transformed lymphoblasts, and from primary cultured hepatocytes from wild-type and Hmga1 -/- mice. Reporter gene assays and chromatin immunoprecipitation for binding of HMGA1 to the endogenous FoxoO1 locus were performed in cells overexpressing HMGA1 and in cells pretreated with siRNA targeting HMGA1. RESULTS:HMGA1 increased FoxO1 mRNA and protein expression in vitro, in cultured HepG2 and HEK-293 cells by binding FoxO1 gene promoter, thereby activating FoxO1 gene transcription. Forced expression of HMGA1 in primary cultured hepatocytes from Hmga1 -/- mice and in EBV-transformed lymphoblasts from subjects with reduced expression of endogenous HMGA1 increased FoxO1 mRNA and protein levels. CONCLUSION:These findings may contribute to the understanding of FoxO1 gene regulation and its role in metabolism.
| S-EPMC5845622 | biostudies-literature
Project description:Burkholderia cepacia complex is a set of closely related bacterial species that are notorious pathogens of cystic fibrosis patients, responsible for life-threatening lung infections. Expression of several virulence factors of Burkholderia cepacia complex is controlled by a mechanism known as quorum sensing (QS). QS is a means of bacterial communication used to coordinate gene expression in a cell-density-dependent manner. The system involves the production of diffusible signaling molecules (N-acyl-l-homoserine lactones, AHLs), that bind to cognate transcriptional regulators and influence their ability to regulate gene expression. One such system that is highly conserved in Burkholderia cepacia complex consists of CepI and CepR. CepI is AHL synthase, whereas CepR is an AHL-dependent transcription factor. In most members of the Burkholderia cepacia complex group, the cepI and cepR genes are divergently transcribed and separated by additional genes. One of them, bcam1869, encodes the BcRsaM protein, which was recently postulated to modulate the abundance or activity of CepI or CepR. Here, we show the crystal structure of BcRsaM from B. cenocepacia J2315. It is a single-domain protein with unique topology and presents a novel fold. The protein is a dimer in the crystal and in solution. This regulator has no known DNA-binding motifs and direct binding of BcRsaM to the cepI promoter could not be detected in in vitro assays. Therefore, we propose that the modulatory action of RsaM might result from interactions with other components of the QS machinery rather than from direct association with the DNA promoter.The atomic coordinates and structure factors have been deposited in the Protein Data Bank under entry 4O2H.BcRsaM and BcRsaM bind by x-ray crystallography (View interaction) BcRsaM and BcRsaM bind by molecular sieving (View interaction).
| S-EPMC4169324 | biostudies-literature
Project description:BackgroundAxin1 is a scaffold protein in the β-catenin destruction complex, which, if disrupted, contributes to pathogenesis of various human diseases, including colorectal carcinogenesis and inflammatory bowel diseases (IBD). We have previously demonstrated that Salmonella infection promotes the degradation and plasma sequestration of Axin1, leading to bacterial invasiveness and inflammatory responses. Vitamin D and the vitamin D receptor (VDR) appear to be important regulators of IBD and colon cancer. Although VDR and Axin1 are all involved in intestinal inflammation, it remains unclear whether these processes are related or function independently. In the current study, we hypothesize that VDR is an important regulator for the maintenance of physiological level of Axin1.MethodsUsing the intestinal epithelial conditional VDR knockout mouse model (VDRΔIEC) and cultured cell lines, influences of VDR status on the expression of Axin1 was evaluated by Western blots and real-time PCR. Loss- and gain-of-function assays were used to investigate the regulation of VDR on Axin1 at the transcriptional and translational levels. Cells were treated with cycloheximide or actinomycin for molecular mechanistic studies. Candidate genomic VDR binding sites for Axin1 were tested by chromatin immunoprecipitation (ChIP) assay. Physical interactions among VDR, Axin1, and β-catenin were tested by immunoprecipitation. Cellular localization of Axin1 with different VDR status was determined by fractionation and immunohistochemistry.ResultsWe found that VDR deletion led to lower protein and mRNA levels of Axin1, whereas knockdown of Axin1 did not change the expression level of VDR protein. Immunoprecipitation data did not support physical interaction between VDR and Axin1. The VDR regulation of Axin1 was through a VDR genomic binding site for Axin1 gene on the regulatory region. Fractionation data showed that cytosolic Axin1 was significantly reduced due to VDR deletion, leaving the nuclear fraction unchanged. In ileum, Axin1 was distributed in the cytosol of apical epithelium and crypts.ConclusionVDR is important for the maintenance of physiological level of Axin1. The discovery of Axin1 as a VDR target gene provides novel and fundamental insights into the interactions between the VDR and β-catenin signaling pathways.
| S-EPMC5180453 | biostudies-literature
Project description:BACKGROUND: Geobacillus stearothermophilus is able to utilize phenol as a sole carbon source. A DNA fragment encoding a phenol hydroxylase catalyzing the first step in the meta-pathway has been isolated previously. Based on these findings a PCR-based DNA walk was performed initially to isolate a catechol 2,3-dioxygenase for biosensoric applications but was continued to elucidate the organisation of the genes encoding the proteins for the metabolization of phenol. RESULTS: A 20.2 kb DNA fragment was isolated as a result of the DNA walk. Fifteen open reading frames residing on a low-copy megaplasmid were identified. Eleven genes are co-transcribed in one polycistronic mRNA as shown by reverse transcription-PCR. Ten genes encode proteins, that are directly linked with the meta-cleavage pathway. The deduced amino acid sequences display similarities to a two-component phenol hydroxylase, a catechol 2,3-dioxygenase, a 4-oxalocrotonate tautomerase, a 2-oxopent-4-dienoate hydratase, a 4-oxalocrotonate decarboxylase, a 4-hydroxy-2-oxovalerate aldolase, an acetaldehyde dehydrogenase, a plant-type ferredoxin involved in the reactivation of extradiol dioxygenases and a novel regulatory protein. The only enzymes missing for the complete mineralization of phenol are a 2-hydroxymuconic acid-6-semialdehyde hydrolase and/or 2-hydroxymuconic acid-6-semialdehyde dehydrogenase. CONCLUSION: Research on the bacterial degradation of aromatic compounds on a sub-cellular level has been more intensively studied in gram-negative organisms than in gram-positive bacteria. Especially regulatory mechanisms in gram-positive (thermophilic) prokaryotes remain mostly unknown. We isolated the first complete sequence of an operon from a thermophilic bacterium encoding the meta-pathway genes and analyzed the genetic organization. Moreover, the first transcriptional regulator of the phenol metabolism in gram-positive bacteria was identified. This is a first step to elucidate regulatory mechanisms that are likely to be distinct from modes described for gram-negative bacteria.
| S-EPMC2590616 | biostudies-literature
Project description:Lipocalin 2 (Lcn2) has been previously characterized as an adipokine/cytokine and implicated in obesity and inflammation. Herein, we investigated the role and potential mechanism of Lcn2 in the regulation of macrophage polarization in obesity-associated inflammation. We observed that Lcn2-/- mice displayed an up-regulation of expression of M1 macrophage marker Cd11c but a down-regulation of M2 marker arginase 1 in adipose tissue and liver of mice upon a high-fat diet feeding. Lcn2-deficient bone marrow-derived macrophages (BMDMs) were more sensitive to lipopolysaccharide (LPS) stimulation, leading to a more profound up-regulation of expression of pro-inflammatory markers than wild-type (WT) BMDMs. Accordingly, LPS stimulation elicited an increase in the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B), c-Jun, and STAT3 signaling pathways as well as an up-regualtion of expression of NF-?B and STAT3 target genes such as IL-1?, IL-6, iNOS, and MCP-1 in Lcn2-/- BMDMs compared with WT controls. Pre-treatment of recombinant Lcn2 attenuated LPS-stimulated degradation of I?B? and STAT3 phosphorylation as well as LPS-induced gene expression of IL-6 and iNOS in Lcn2-/- BMDMs. Moreover, the NF?B inhibitor markedly blocked LPS-stimulated STAT3 phosphorylation in Lcn2-/- BMDMs. These results together with the time course of Lcn2 secretion, NF?B and STAT3 phosphorylation in response to LPS stimulation, suggest that Lcn2 plays a role as an anti-inflammatory regulator in macrophage activation via modulating a feed-forward activation of NF?B-STAT3 loop.
| S-EPMC4179633 | biostudies-literature
Project description:Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and thus neural crest specification.
| S-EPMC3039673 | biostudies-literature
Project description:We found that ZBTB2, a POK family transcription factor, is a potent repressor of the ARF-HDM2-p53-p21 pathway important in cell cycle regulation. ZBTB2 repressed transcription of the ARF, p53, and p21 genes, but activated the HDM2 gene. In particular, ZBTB2 repressed transcription of the p21 gene by acting on the two distal p53 binding elements and the proximal Sp1 binding GC-box 5/6 elements. ZBTB2 directly interacted with Sp1 via its POZ domain and zinc fingers, which was important in the repression of transcription activation by Sp1. ZBTB2 and Sp1 competed with each other in binding to the GC-box 5/6 elements and the two p53 binding elements. ZBTB2 directly interacted with p53 via its zinc fingers, inhibiting p53 binding and repressing transcription activation by p53. The POZ domain, required for transcription repression, interacted with corepressors such as BCoR, NCoR, and SMRT. The interactions deacetylated histones Ac-H3 and -H4 at the proximal promoter. Although ectopic ZBTB2 stimulated cell proliferation, knock-down of ZBTB2 expression decreased cell proliferation and DNA synthesis. Overall, our data suggest that ZBTB2 is a potential proto-oncogenic master control gene of the p53 pathway and, in particular, is a potent transcription repressor of the cell cycle arrest gene p21 by inhibiting p53 and Sp1.
| S-EPMC2709380 | biostudies-literature