Identification by in vivo genomic footprinting of a transcriptional switch containing NF-kappaB and Sp1 that regulates the IkappaBalpha promoter.

In unstimulated cells, NF-kappaB transcription factors are retained in the cytoplasm by inhibitory IkappaB proteins. Upon stimulation by multiple inducers including cytokines or viruses, IkappaBalpha is rapidly phosphorylated and degraded, resulting in the release of NF-kappaB and the subsequent increase in NF-kappaB-regulated gene expression. IkappaBalpha gene expression is also regulated by an NF-kappaB autoregulatory mechanism, via NF-kappaB binding sites in the IkappaBalpha promoter. In previous studies, tetracycline-inducible expression of transdominant repressors of IkappaBalpha (TD-IkappaBalpha) progressively decreased endogenous IkappaBalpha protein levels. In the present study, we demonstrate that expression of TD-IkappaBalpha blocked phorbol myristate acetate-phytohemagglutinin or tumor necrosis factor alpha-induced IkappaBalpha gene transcription and abolished NF-kappaB DNA binding activity, due to the continued cytoplasmic sequestration of RelA(p65) by TD-IkappaBalpha. In vivo genomic footprinting revealed stimulus-responsive protein-DNA binding not only to the -63 to -53 kappaB1 site but also to the adjacent -44 to -36 Sp1 site of the IkappaBalpha promoter. In vivo protection of both sites was inhibited by tetracycline-inducible TD-IkappaBalpha expression. Prolonged NF-kappaB binding and a temporal switch in the composition of NF-kappaB complexes bound to the -63 to -53 kappaB1 site of the IkappaBalpha promoter were also observed; with time after induction, decreased levels of transcriptionally active p50-p65 and increased p50-c-Rel heterodimers were detected at the kappaB1 site. Mutation of either the kappaB1 site or the Sp1 site abolished transcription factor binding to the respective sites and the inducibility of the IkappaBalpha promoter in transient transfection studies. These observations provide the first in vivo characterization of a promoter proximal transcriptional switch involving NF-kappaB and Sp1 that is essential for autoregulation of the IkappaBalpha promoter.

Coordination of cell signaling, chromatin remodeling, histone modifications, and regulator recruitment in human matrix metalloproteinase 9 gene transcription.

Transcriptional activation of eukaryotic genes depends on the precise and ordered recruitment of activators, chromatin modifiers/remodelers, coactivators, and general transcription factors to the promoters of target genes. Using the human matrix metalloproteinase 9 (MMP-9) gene as a model system, we investigated the sequential assembly and dynamic formation of transcription complexes on a human promoter under the influence of mitogen signaling. We find that, coincident with activation of the MMP-9 gene, activators, chromatin remodeling complexes, and coactivators are recruited to the preassembled MMP-9 promoter in a stepwise and coordinated order, which is dependent on activation of MEK-1/extracellular signal-regulated kinase and NF-kappa B signaling pathways. Conversely, corepressor complexes are released from the MMP-9 promoter after transcriptional activation. Histone modifications shift from repressive to permissive modifications concurrent with activation of the MMP-9 gene. Chromatin remodeling induced by Brg-1 is required for MMP-9 gene transcription, which is concomitant with initiation of transcription. Therefore, coordination of cell signaling, chromatin remodeling, histone modifications, and stepwise recruitment of transcription regulators is critical to precisely regulate MMP-9 gene transcription in a temporally and spatially dependent manner. Given the important role of MMP-9 in both normal development and pathological conditions, understanding MMP-9 gene regulation is of great relevance.

Constitutive expression of the Id-1 promoter in human metastatic breast cancer cells is linked with the loss of NF-1/Rb/HDAC-1 transcription repressor complex.

The helix-loop-helix protein Id-1 is a dominant negative regulator of basic helix-loop-helix transcription factors, and plays a key role in the control of breast epithelial cell growth, invasion and differentiation. Previous investigations in our laboratory have shown that Id-1 mRNA was constitutively expressed in highly aggressive and invasive human breast cancer cells in comparison to non-transformed or non-aggressive cancerous cells, and that this loss of regulation is mediated by a 2.2-kb region of the human Id-1 promoter. Here we show that a 31 bp sequence within this 2.2-kb promoter, located 200 bp upstream of the initiation of transcription, is responsible for the constitutive expression of Id-1 in metastatic human breast cancer cells. Using gel shift experiments, we identified a high molecular weight complex present only in non-aggressive breast cancer cells cultured in serum-free medium and which appear to be necessary for proper Id-1 repression. In contrast, nuclear extracts from highly aggressive and metastatic cell lines do not contain this large molecular weight complex. Using DNA affinity precipitation assays (DAPA), we show that this complex contains SP-1, NF-1, Rb and HDAC-1 proteins. On the basis of these findings, we propose a mechanism for the loss of regulation of Id-1 promoter in invasive and metastatic human breast cancer cells.

c-fos is a positive regulator of carcinogen enhancement of adenovirus transformation.

The early gene expression changes mediating carcinogen enhancement of viral transformation (CET) remain to be elucidated. A model cell culture system has been developed that is now permitting a molecular analysis of CET. Pretreatment of cloned rat embryo fibroblast (CREF) cells with methyl methanesulfonate (MMS) prior to infection with the cold-sensitive host-range type 5 adenovirus mutant, H5hr1, results in a dose-dependent increase in viral transformation. The present study investigates the role of immediate-early response genes, specifically c-fos, in the CET process. MMS pretreatment, alone or in combination with infection with H5hr1 temporally and differentially increases c-fos, c-jun, jun-B, jun-D and c-myc steady-state mRNA levels. Maximum induction occurs with c-fos and c-jun 8 to 12 h posttreatment and the magnitude of response is generally greatest in CREF cells pretreated with MMS and then infected with H5hr1. Enhancement in RNA levels is observed in the presence of cycloheximide indicating that ongoing protein synthesis is not required for induction of c-fos, c-jun, jun-B or c-myc expression. Nuclear run-on analysis indicates an enhancement in transcriptional rates for c-fos, c-jun, jun-B and c-myc in CREF cells treated with MMS or MMS plus infection with H5hr1. A requirement for elevated c-fos in the early stages of CET is indicated by the ability of c-fos antisense oligonucleotides to prevent the CET process. Direct evidence implicating early increases in c-fos as a mediator of the CET process is demonstrated by stably expressing mouse mammary tumor virus promoter-regulated human sense and antisense c-fos genes in CREF cells. Induction of c-fos sense expression by dexamethasone (DEX) in the absence of MMS treatment results in enhanced c-fos mRNA, Fos protein, AP-1 DNA-binding activity and H5hr1-induced transformation and CET. Induction of c-fos expression by DEX in stable c-fos-sense CREF constructs also results in elevated levels of c-jun, jun-B and c-myc mRNA and protein. Conversely, induction of c-fos antisense expression prevents the increase in c-fos mRNA, Fos protein and AP-1 DNA-binding activity and eliminates CET. In the antisense-c-fos constructs, increases in c-jun, jun-B and c-myc mRNA and protein normally induced by MMS also are not apparent. Thus, induction or inhibition in c-fos expression affects the level of expression of additional immediate-early response genes, including c-jun, jun-B and c-myc.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of ZiRF1 and basal SP1 transcription factor MRE-binding activity by transition metals.

The metal-dependent activation of metallothionein (MT) genes requires the interaction of positive trans-activators (MRFs) with metal-regulatory (MRE) regions of MT promoters. In this report, we examined the role of transition metals in modulating the MRE-binding activities of two different MRE-binding proteins: the metal-regulated factor ZiRF1 and the basal factor SP1. We showed the ability of both proteins to interact with a similar sequence specificity with the cognate target site (MRE-S) of another known MRE-binding protein, mMTF1. We next evaluated the role of metal ions in modulating the MRE-binding activity of recombinant ZiRF1 and basal SP1 proteins by measuring the effect of different metal chelators on DNA interaction. We observed a dose-dependent inhibition of the GST-ZiRF1/MRE-binding activity using three different metal chelators: EDTA, 1,10 PHE and TPEN. Interestingly, EDTA treatment failed to inhibit the recombinant SP1 MRE-binding activity while the effect of 1,10 PHE was comparable to that obtained analyzing 1,10 PHE-treated GST-ZiRF1. The MRE-binding complexes detected in cell extracts showed a response to metal chelator treatment very similar to that displayed by the recombinant ZiRF1 and SP1 proteins. The hypothesis of mutual interactions of both basal and metal-regulated transcription factors with the same metal-regulatory regions is discussed.

Differential modulation by estradiol of P-glycoprotein drug resistance protein expression in cultured MCF7 and T47D breast cancer cells.

BACKGROUND: Multidrug resistance (MDR) is associated with over expression of the P-glycoprotein (P-gp) drug transporter, which is encoded by the MDR1 gene. Estradiol (E2) is thought to regulate P-gp expression in breast cancer and the aim of this study was to determine the role of estrogen receptor subtypes (ERalpha and ERbeta) in modulating drug resistance and P-gp expression in cultured breast carcinoma cells. MATERIALS AND METHODS: The cytotoxic effects of doxorubicin and P-gp concentrations were determined in E2-treated and untreated T47D and MCF7 breast carcinoma cells. Western blot and mobility shift/super shift analyses were used to determine estrogen receptor subtype interaction with AP1 and Sp1 transcription factors. RESULTS: ERalpha-positive MCF7 cells were resistant to doxorubicin cytotoxicity, while ERbeta-expressing T47D cells were sensitive to doxorubicin treatment. E2 increased the cytoplasmic concentration of P-gp in MCF7 cells but not in T47D cells. ERalpha binds both AP1 and Sp1 transcription factors in extracts from MCF7 cells, while ERbeta binds AP1 in extracts from T47D cells. CONCLUSION: These interactions of the ER subtypes with transcription factors correlates with their functional effects on the MDR1 promoter and the observed effects of E2 on drug resistance.

Genomic structure of human cystatin A.

Cystatin A is a cysteine proteinase inhibitor with a molecular mass of 11 kDa, and is located mainly in the keratohyaline granules of the stratum granulosum and the cornified envelope of the stratum corneum in the epidermis. In this study, we demonstrated the genomic structure of this proteinase inhibitor in which there were three exons of 111 bp, 102 bp and 226 bp in length, while the lengths of the 1st and 2nd intron were approximately 14 Kbp and 4 Kbp, respectively. The conserved sequence of QVVAG was encoded in the 2nd exon and was not inserted by any introns. There were binding sites for SP-1 and AP-2 in the promoter region and an AP-1 binding site in the 1st intron. The successful amplification of each exon of cystatin A may possibly contribute to the detection of the genomic abnormality of some skin disorders e.g. keratinization disorder, chronic bacterial infection or photophobia.

Podophyllum toxicity: a report of a fatal case and a review of the literature.

A 59-yr-old male ingested 10 g of podophyllum in a fatal suicide attempt. Previously unreported nuclear and cytoplasmic changes were observed in circulating leukocytes. Symptoms did not occur until 10 h after ingestion with loss of reflexes, coma, and a marked lactic acidosis. Despite hemoperfusion, the patient expired 39 h after ingestion. The literature on podophyllum toxicity is reviewed.

Identification of a novel variant of the human NR2B gene promoter region and its possible association with schizophrenia.

N-methyl-D-aspartate (NMDA) receptor dysfunction is involved in the pathogenesis of schizophrenia. We determined the nucleotide sequence of the 5'-upstream region of the human NMDA receptor 2B (NR2B) subunit gene and identified a novel T-200G variant located in one of the Sp1 binding sites. To investigate the effect of this variant on the transcriptional activity of the hNR2B gene, we performed gene reporter assays using PC12 pheochromocytoma cells transiently transfected with luciferase reporter plasmids. In the absence of nerve growth factor (NGF), luciferase activities did not significantly differ between the two alleles and the control plasmid. However, luciferase reporter activity of the T allele was significantly up-regulated compared to that of the G allele in the presence of NGF (P = 0.0013), indicating that this polymorphic site is a critical region for NR2B gene regulation through NGF-induced Sp1-binding. A case control study showed that the frequency of the G allele (P = 0.0164) was significantly higher in 100 schizophrenics than in 100 controls. These findings suggest that the T-200G variant causes dysfunction of NMDA receptors consisting of the NR2B subunit and may be involved in the development of schizophrenia. Replication studies of independent samples and family-based association studies are necessary to further evaluate the significance of our findings.

Characterization of 5'-flanking region of human MRP3.

We previously cloned MRP3, which is responsible for the cellular extrusion of organic anions, as an inducible transporter in the liver under cholestatic conditions. In the present study, we investigated the mechanism for the expression of human MRP3. The cap site hunting method revealed that the transcription starts at -25 and -27 nt upstream of the initiation codon. Luciferase assay with a series of truncated 5'-flanking regions indicated that the region from -127 to -23 nt is important for MRP3 expression. Moreover, carrying out a gel shift assay indicated that Sp1 binds to the sequence between -92 and -58 nt. Collectively, it was demonstrated that human MRP3 is under the control of TATA-less promoter and Sp1 binding sites may be involved in the transcription.

Expression of the human cytochrome c1 gene is controlled through multiple Sp1-binding sites and an initiator region.

It is widely accepted that nuclear genes that encode proteins of the oxidative-phosphorylation system are regulated by nuclear factors believed to be specific for such genes. In the present study we show that the promoter for the human cytochrome c1 gene is an exception, in that it involves only conserved Sp1 core elements and an initiator region. Maximal promoter activity within a 1.4-kb 5' flanking region of the cytochrome c1 gene is contained in a fragment (-72 to +18) that lacks TATA and CCAAT elements. The transcriptional start site was mapped to an initiator region by RNase protection of mRNA from human HepG2 cells, and by primer extension of in vitro-generated transcripts, to a sequence that is highly similar to the dihydrofolate reductase family of initiators. Deletion of this region (+1 to +18) severely impairs transcription initiation. Sp1 core elements centered at nucleotides -21 and -39 define the activation domain of the proximal promoter. Only the -39 element is protected from DNase I in the presence of crude nuclear extracts. However, transfection, gel-mobility-shift, supershift and in vitro-transcription experiments show that the -21 element binds Sp1 protein and contributes to transcription activation. No other functional oxidative-phosphorylation-specific response elements have been identified. These data implicate Sp1 as a single activating factor for an oxidative-phosphorylation gene.

Human Sug1/p45 is involved in the proteasome-dependent degradation of Sp1.

The transcription factor Sp1 was previously shown to undergo proteasome-dependent degradation when cells were glucose-starved and stimulated with the adenylate cyclase inducer, forskolin. However, the control of the Sp1 degradation process is largely unknown. Using in vitro and in vivo interaction studies, we show in the present study that Sp1 interacts with human Sug1 [hSug1, also known as p45 or thyroid-hormone-receptor interacting protein ('TRIP1')], an ATPase subunit of the 26 S proteasome and a putative transcriptional modulator. This interaction with Sp1 occurs through the C-terminus of hSug1, the region that contains the conserved ATPase domain in this protein. Both in vitro studies, in reconstituted degradation assays, and in vivo experiments, in which hSug1 is overexpressed in normal rat kidney cells, show that full-length hSug1 is able to stimulate the proteasome-dependent degradation of Sp1. However, hSug1 truncations that lack either the N- or C-terminal domain of hSug1 act as dominant negatives, inhibiting Sp1 degradation in vitro. Also, an ATPase mutant of hSug1, while still able to bind Sp1, acts as a dominant negative, blocking Sp1 degradation both in vitro and in vivo. These results demonstrate that hSug1 is involved in the degradation of Sp1 and that ATP hydrolysis by hSug1 is necessary for this process. Our findings indicate that hSug1 is an exchangeable proteasomal component that plays a critical regulatory role in the proteasome-dependent degradation of Sp1. However, hSug1 is not the factor limiting Sp1 degradation in the cells treated with glucosamine. This and other considerations suggest that hSug1 co-operation with other molecules is necessary to target Sp1 for proteasome degradation.

Biosynthesis of yatein in Anthriscus sylvestris.

Little is known about the biosynthesis of yatein, in spite of its importance as a typical heartwood lignan and a key biosynthetic intermediate of the antitumor lignan podophyllotoxin. The present study, based on individual administration of [13C]phenylalanine and deuterium labelled lignans and simultaneous administration of two distinct lignans labelled with deuterium atoms to Anthriscus sylvestris, established the two independent branch pathways from matairesinol, one to afford yatein via thujaplicatin, 5-methylthujaplicatin, and 4,5-dimethylthujaplicatin and the other to bursehernin via pluviatolide. The latter pathway did not lead to yatein, eliminating the presence of a metabolic grid from matairesinol to yatein.

Identification of PU.1 and Sp1 as essential transcriptional factors for the promoter activity of mouse tec gene.

Tec is a cytoplasmic protein-tyrosine kinase abundantly expressed in hematopoietic precursor cells. To investigate the mechanism regulating the expression of Tec molecule, we cloned and analysed 5' flanking region of mouse tec gene up to -2kb from the transcriptional initiation site. Luciferase assays using successive deletion mutants demonstrated that regions from -364 to -323 and from -122 to -63, which contain the consensus binding sequences for PU.1 (GGAA) and Sp1 (GGGCGG), respectively, are important for the transcriptional activity. Gel-shift and supershift assays revealed that PU.1 and Sp1 bind to the these regions through their consensus binding motifs. In addition, introduction of mutations into these motifs resulted in marked decrease in the promoter activity. These results indicate that PU.1 and Sp1 are essential for the transcriptional activity of the tec promoter and suggest that the cooperation of PU.1 and Sp1 plays a substantial role in the preferential expression of the Tec molecule in the hematopoietic lineages.

Suppression of stellate cell type I collagen gene expression involves AP-2 transmodulation of nuclear factor-1-dependent gene transcription.

The regulation of collagen gene expression was studied in culture-activated rat hepatic stellate cells, the fibrogenic effector cell involved in hepatic fibrogenesis. Treatment of cells with a 5-lipoxygenase-specific inhibitor caused a reduction in alphaI(I) collagen mRNA transcript abundance, which suggested that leukotriene production was involved in maintaining the activated cell's high level of collagen mRNA production. The underlying mechanism involved a decrease in collagen gene transcription. Suppression of gene transcription was localized to an nuclear factor-1 (NF-1) binding domain in the proximal promoter and an AP-2 binding domain adjacent to it. Gel retardation assays demonstrated that an increase in AP-2 binding adjacent to the NF-1 site was likely to be the transmodulator responsible for the suppression of the NF-1-dependent gene expression. The data suggest that post-translational alterations in AP-2 activity are responsible for this unappreciated mechanism of regulating the collagen gene.

Efficient transcription and replication of simian immunodeficiency virus in the absence of NF-kappaB and Sp1 binding elements.

Ten mutants of the simian immunodeficiency virus (SIV) SIVmac239 bearing deletions (delta) or substitutions (subst) in the NF-kappaB and/or Sp1 binding elements were created, and the replicative capacities of the mutants were analyzed. All mutants, including one extensively mutagenized strain entirely missing the NF-kappaB and four Spl binding elements, replicated with wild-type kinetics and to a wild-type level in peripheral blood mononuclear cell cultures in 50 to 100% of the experiments. One group of mutants replicated very similarly to SIVmac239 in kinetics and yield in CEMxl74 cells (2xNFKappaB > or = SlVmac239 approximately deltaNFkappaB approximately deltaSpl234 approximately substNFkappaB approximately substSpl2 approximately substSp23), while a second group replicated with delayed or slightly delayed kinetics in CEMxl74 cells (SIVmac239 > substSp34 > deltaNFkappaBdeltaSpl234 approximately deltaNFkappaBdeltaSp1 > substSpl234). Reversions or additional mutations were not detected in the U3 and R regions of proviral DNA from CEMxl74 cells infected with the SIVmac239 mutants. Similar results were obtained when mutants of SIVmacMER (a macrophage-competent derivative of SIVmac239) were tested in peripheral blood mononuclear cell and CEMx174 cultures. However, the growth of most mutated viruses was suppressed in primary rhesus monkey alveolar macrophages (SIVmacMER approximately 2xNFkappaB approximately substNFkappaB > deltaNFkappaB > deltaNFkappaBdeltaSpl234 approximately deltaNFkappaBdeltaSpl > deltaSpl234 approximately substSpl2 > substSp23 approximately substSp34 approximately substSpl234 > or = SIVmac239). Thus, changes in the Sp1 binding sites had the most dramatic effects on SIVmac replication in primary macrophage cultures. Analysis of long terminal repeat-driven secreted alkaline phosphatase activity in transient assays showed that, unlike human immunodeficiency virus type 1, the SIV long terminal repeat possesses an enhancer region just upstream of the NF-kappaB element which maintains significant levels of basal transcription in the absence of NF-kappaB and Sp1 sites. This region is responsive to transactivation by Tat. In addition, the SIV TATA box was shown to be stronger than that of human immunodeficiency virus type 1. Therefore, the surprisingly high replicative capacity of NF-kappaB and Sp1 binding site mutants of SIVmac is due to unique features or the enhancer/promoter region.

Silencer activity of NFATc2 in the interleukin-12 receptor beta 2 proximal promoter in human T helper cells.

Interleukin 12 (IL-12) is a potent enhancer of interferon gamma production by activated T cells. The high-affinity IL-12 receptor (IL-12R) is a heterodimer of a beta1 and a beta2 subunit. Expression of the signaling IL-12Rbeta2 chain is usually low, as compared with the more abundant beta1 chain, and may be rate-limiting for IL-12 sensitivity. Little is known about the mechanisms controlling IL-12Rbeta2 gene expression. Reporter gene assays in IL-12Rbeta2-expressing Jurkat cells showed that truncation of the region from -151 to -61 abrogated promoter activity. The proximal promoter region does not contain a typical TATA box, suggesting a role for SP-1. Indeed, mutagenesis of the -63 SP-1 consensus site decreased transcription by 50%. Electrophoretic mobility shift experiments confirmed the binding of SP-1 and SP-3 at this site. In contrast, truncation of -252 to -192 increased promoter activity. Likewise, mutagenesis of the consensus nuclear factor of activated T cells site at -206 increased promoter activity by 70%, suggesting silencer activity of this element. Electrophoretic mobility shift experiments with primary Th (T helper) cells showed the formation of a specific, T-cell receptor-inducible complex at this site that is sensitive to cyclosporin A and supershifted with anti-NFATc2 in both Th1 and Th2 cells. Accordingly, cyclosporin A dose-dependently increased IL-12Rbeta2 mRNA expression. These first data on IL-12Rbeta2 gene regulation indicate a TATA-less promoter, depending on SP-1/SP-3 transcription factors, and a negative regulatory NFAT element at -206. This element may contribute to the overall low level of IL-12Rbeta2 expression on Th cells.