Regulation of the human fas promoter by YB-1, Puralpha and AP-1 transcription factors.

Fas (CD95/Apo-1) gene expression is dysregulated in a number of diseased states. Towards understanding the regulation of fas gene expression, we previously identified activator and repressor elements within the human fas promoter. Using a combination of expression screening and reporter gene assays, we have identified transcription factors which bind to these elements and thereby regulate transcription of the fas promoter. These are three single-stranded DNA binding proteins, YB-1, Puralpha and Purbeta and two components of the AP-1 complex, c-Fos and c-Jun. c-Jun is a potent transcriptional activator of fas and stimulated expression levels up to 184-fold in reporter gene assays. Co-expression with c-Fos abrogated c-Jun-mediated activation. YB-1 and Puralpha are transcriptional repressors of fas and decreased basal transcription by 60-fold in reporter gene assays. Purbeta was predominantly an antagonist of YB-1/Puralpha-mediated repression. Overexpression of YB-1 and Puralpha in Jurkat cells was shown to reduce the level of cell surface Fas staining, providing further evidence that these proteins regulate the fas promoter. It has been suggested that YB-1 plays a role in cell proliferation as an activator of growth-associated gene expression. We have shown that YB-1 is a repressor of a cell death-associated gene fas. These results suggest that YB-1 may play an important role in controlling cell survival by co-ordinately regulating the expression of cell growth-associated and death-associated genes.

Functional genomics with protein-protein interactions.

Knowing the sequence of a gene does not mean knowing its function. Although, information stored at the DNA level can be used to predict biological processes, proteins are the final executors of the various response programs of a cell. Transient information, like posttranslational modifications or interactions among proteins, cannot be deduced from DNA sequences. The rapid accumulation of large amounts of DNA sequence data in genomics projects has led to an increasing demand for powerful tools to analyze proteins and their behaviour at a large scale. This review aims to compare different technologies used for identification of interacting proteins and discusses recent developments in the field of high-throughput protein-protein interaction mapping.

Genomics and proteomics tools for the clinic.

The sequencing of the human genome was only made possible by the massively parallel use of automated high-throughput technologies. These technologies have required the development and interfacing of new hardware and software in a way which would have been hardly conceivable only ten years ago. As a consequence, an unbroken trend to more 'industrialized science' is apparent. The wealth of information generated by intensive sequencing efforts is now being further exploited by using complex tools to comprehensively analyze complex systems at the DNA, RNA and protein level. A landmark innovation was the introduction of the biochip principle, best exemplified with the development of the DNA chip, mainly used for RNA expression profiling. The chip principle, together with miniaturization, has now become the dominating theme for a number of new genomics and proteomics technologies, culminating in the lab-on-a-chip concept which, in the next five to ten years, could advance at a comparable rate to that of computers over the last 50 years. Some of the new technologies are already used for comprehensive analysis of clinical samples in an attempt to describe disease and disease risk at the molecular level. However, all of these technologies are far from routine in clinical use and it is also too early to decide whether molecular fingerprints or signature profiles will have the diagnostic and prognostic power currently predicted.

Selection of a peptide ligand to the p75 neurotrophin receptor death domain and determination of its binding sites by NMR.

The p75 neurotrophin receptor (p75(NTR)) contains a conserved death domain module similar to that of the cytotoxic receptors Fas and TNFR-1. Here, we describe the selection of peptide ligands from a combinatorial library using a variation of the selectively-infective phage (SIP) method directed to the death domain of p75(NTR). The binding sites on the death domain of p75(NTR) were identified for a 15 amino acid residue peptide by nuclear magnetic resonance (NMR) spectroscopy. The selected peptides may be useful for probing the function of the p75(NTR) death domain and aid in defining its downstream signalling mechanism.

A phage-based system to select multiple protein-protein interactions simultaneously from combinatorial libraries.

Selectively infective phage (SIP) can be used to identify protein-protein interactions. SIP was modified to facilitate the simultaneous selection of interacting protein pairs from large combinatorial libraries. An interference-resistant phage was constructed which non-covalently, but stably links the genetic information of an interacting pair, encoded separately on phage and phagemid vectors, by co-packaging into heteropolyphages. In a model system, the interaction between a SIP-selected peptide and the intracellular domain of the p75 neurotrophin receptor was detected in the presence of a 10(4)-fold excess of a non-interacting control pair (jun leucine zipper and p75 intracellular domain) via SIP hetero-polyphage transductants. To minimize the redundancy of transductants and to minimize possible ligand exchange generated in a solution-based SIP screening, a filter-based in situ infectivity screening was developed. The combination of the above techniques may provide a powerful system for rapid screening of very large sequence spaces.

Silencer and enhancer regions in the human CD95 (Fas/APO-1) gene with sequence similarity to the granulocyte-macrophage colony-stimulating factor promoter: binding of single strand-specific silencer factors and AP-1 and NF-AT-like enhancer factors.

The CD95 (Fas/APO-1) apoptosis receptor is expressed in a variety of tissues and transiently upregulated in lymphocytes during activation-induced cell death. A silencer (S1; -1035 to -1008) and an adjacent enhancer (E1; -1007 to -964) region have been mapped in the CD95 gene. The S1 region shows similarity to binding sites for the transcriptional repressor NF-GMb, which prefers binding to single-stranded DNA. The E1 contains an everted repeat of two CATTA/T elements spaced by 2 bp (ER2). Such motifs are directly repeated in the CLE0 region of the human granulocyte-macrophage colony-stimulating factor (huGM-CSF) promoter. A motif (TGATGTCA) which matches a CREB site and is similar to an AP-1 site is embedded within ER2. Sequence-specific binding of nuclear factors to single-stranded S1 probes involved, to some extent, a central heptamer motif (ATCCAAA) also present in E1. Competition binding studies suggested that AP-1 or AP-1 components, as well as factors related, but not identical, to NF-AT bound to E1 probes. S1-binding-proteins/complexes of 47, 77, and 100 kDa were detected by Southwestern analysis and ultraviolet crosslinking. Complexes of 70 and 80 kDa were formed with a double-stranded E1 probe in UV-crosslinking, whereas Southwestern analysis with this probe revealed single binding species of 59 and 113 kDa. ER2 autonomously enhanced transcription from the heterologous HSV tk promoter in a cell type-specific manner only in the absence of the S1 region. This analysis has identified a small region in the CD95 gene containing adjacent opposing regulatory elements which are likely to be involved in the cell type- and activation state-specific gene expression under physiologic conditions.

Apoptosis through CD95 (Fas/APO-1), but not a CD40/CD95 chimeric receptor, is inhibited by phorbol-12-myristate-13-acetate.

CD95 (Fas/APO-1)-mediated apoptosis appears to be regulated by positive and negative signaling molecules. A human CD40/CD95 chimeric receptor was stably transfected into CD95-expressing human Jurkat T cells, and signaling through native and chimeric CD95 was compared in the same cell type to assess contributions of the CD95 extracellular and intracellular domains. Apoptosis was induced in these transfectants by soluble CD40 ligand and also by the anti-CD40 monoclonal antibodies (mAb) M2 and M3. The M2 mAb was more effective than M3 in these transfectants. In contrast to apoptosis mediated through native CD95, CD40/CD95-mediated apoptosis was not inhibited by phorbol-12-myristate-13-acetate (PMA). The apoptotic response to the anti-CD40 mAb M3, but not M2, was enhanced by PMA and dibutyryl cyclic adenosine monophosphate (db-cAMP), which also increased mRNA levels and surface expression of CD40/CD95. The enhancing effects of PMA, but not those of db-cAMP, were sensitive to cycloheximide. The M2 and M3 mAbs appeared to have virtually identical binding affinities but, when added to cells together, M3 inhibited M2-induced apoptosis. These mAbs may bind neighboring epitopes, but M2 induces a more effective signaling-competent conformation upon the chimeric receptor. These data suggest that dimerization, however only in a signaling-competent conformation, was sufficient to induce apoptosis. When expressed as a chimera with the CD40 extracellular domain, the CD95 intracellular domain was not inhibited by protein kinase C (PKC)-dependent pathways, suggesting that the CD95 extracellular domain is required for association with a molecule that inhibits the apoptotic signal.

CD95 (Fas/Apo-1)-induced apoptosis results in loss of glucose transporter function.

Treatment of activated human T cells with CD95 (Fas/Apo-1) ligand or Abs against CD95 results in apoptotic cell death. Although cellular responses to CD95 ligation have been described in some detail, the early molecular events that result in T cell death are only now beginning to be elucidated. Using Jurkat cells as a model of activated human T cells, we have investigated the effects of CD95 ligation on glucose transport and on glucose transporter function. We show that within minutes of CD95 activation, the ability to transport glucose across the plasma membrane is compromised and that transient exposure to Abs against CD95 for as little as 3 min results in reduced glucose transport and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) responses measured at 16 h. The effects of CD95 ligation on glucose transport are shown to be associated with loss of affinity of glucose transporters for glucose without altered maximum velocity and without changes in the cell surface expression of Glut 1, the predominant glucose transporter isotype on Jurkat cells. These results support a model of CD95 induced cell death that, at least in its early stages, does not depend on signaling to the nucleus or on macromolecular synthesis. Acute regulation of glucose transport is proposed to be an early effector mechanism in CD95-induced apoptotic cell death.

pLEF, a novel vector for expression of glutathione S-transferase fusion proteins in mammalian cells.

An expression vector, pLEF, has been used to produce the intracellular domain (IC) of the human CD95 (Fas/APO-1) apoptosis receptor as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts. GST::CD95IC was affinity-purified in a single step using glutathione-Sepharose. Purification of GST::CD95IC from 32P-labelled L929 cells and cleavage with thrombin revealed that CD95IC was phosphorylated in vivo when produced as a GST fusion protein. Therefore, pLEF may facilitate the mapping of in vivo-modified sites of eukaryotic proteins.

Identification of a silencer, enhancer, and basal promoter region in the human CD95 (Fas/APO-1) gene.

Genomic clones for the human CD95 (Fas/APO-1) and CD40 genes have been isolated and 2.3 kb of the CD95 and 0.8 kb of the CD40 gene 5'-flanking regions sequenced. Comparisons of the human CD95 gene with the human CD40 and the murine CD40 and TNFR-II genes showed a low degree of sequence similarity. However, dot matrix analyses revealed conservation of two stretches between human CD95 (-387 to -362 and -288 to 261 in CD95) and murine TNFR-II genes. Additionally, TCCTCC motifs are present within 400 bp up-stream of the ATG of all genes examined. Repeated interferon-beta (IFN-beta) silencer B motifs and a lysozyme silencer 1 motif have been found in the CD95 gene at approximately -1,600 and -1,100, respectively. Sequence comparison of the 5'-flanking regions of the murine and human CD40 genes revealed the presence of a conserved AP-4 site and two SP-1 sites. CD95, CD40, and TNFR-II genes all lack classical TATA and CAAT boxes. However, a strongly increased frequency of CpG dinucleotides was found. Primer extension analysis revealed multiple transcriptional start sites in the CD95 gene, where the usage of individual start sites appeared to be cell type-specific. Functional analysis, using reporter constructs and transient transfections, identified a silencer activity residing between nucleotide positions -1,781 and -1007 and a strong enhancer region between -1,007 and -425 in the human CD95 gene. The region between -425 and -1 retained a basal promoter activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcripts from opposite strands of gamma satellite DNA are differentially expressed during mouse development.

Using in vitro immuno-selected retinoic acid response elements, we have isolated mouse genomic clones containing major (gamma) satellite DNA repeats that are considered as typical of chromosome centromeres. Several cDNA clones were then isolated from a F9 cell cDNA library and were found to harbor variants of the 234-base pair consensus gamma satellite monomer. In Northern analysis, these satellite DNA sequences hybridized predominantly to an approximately 1.8-kb RNA species in polyadenylated RNA from P19 cells. These transcripts were strongly repressed by retinoic acid, and nuclear run-on assays revealed that this repression was, at least in part, mediated at the transcriptional level. Satellite transcripts were also detected in HeLa cells, where they were similarly down-regulated by retinoids. Heterogeneously sized satellite transcripts were detected in RNA from specific mouse tissues, such as fetuses (but not placenta), adult liver, and testis. In situ hybridization analysis revealed that satellite transcripts are generated from opposite DNA strands and are differentially expressed in cells of the developing central nervous system as well as in adult liver and testis. These data may have implications on retinoic acid-mediated transcriptional regulation and centromere function.

Apoptosis in L929 cells expressing a CD40/Fas chimeric receptor: dissociation of stimulatory from inhibitory death signalling functions.

A chimeric transmembrane receptor was constructed by fusing the extracellular domain of human CD40 to the transmembrane/intracellular domain of human Fas. When stably overexpressed in L929, the chimera retained the ligand binding specificity of CD40 and the basic signalling properties of Fas since an apoptotic response could be induced in a dose-dependent manner upon administration of recombinant, soluble CD40 ligand trimer or immobilized antiCD40 antibodies. However, this apoptotic response was independent of actinomycin D which is in contrast to results previously reported for L929 cells stably expressing wildtype human Fas. A labile protein factor was postulated to be responsible for inhibition of Fas-induced apoptosis in these cells. The apoptotic Fas signal tranduced by the chimeric CD40/Fas receptor is not regulated by this putative inhibitor. Our data suggest that the extracellular domains of CD40 and Fas form functionally similar mono- and multimeric structures and that the extracellular domain of Fas participates in the regulation of Fas-specific signal transduction.

Cloning a pseudogene and cDNA encoding a 17-kDa ribosomal protein from mouse: structure and regulation of expression.

An rp lambda 5 cDNA encoding a ribosomal protein (r-protein) and a pseudogenic form of the corresponding gene (rp lambda 7) have been cloned from mouse. This cDNA codes for a highly basic protein of 160 amino acids (aa) with a deduced M(r) of 17,601, and most likely represents the species homolog of a recently cloned rat cDNA, which has been proposed to encode a homolog of the yeast r-protein, YL43. The entire rp lambda 5 gene encompasses less than 1.5 kb of genomic DNA and apparently is composed of only two exons, as deduced from sequence comparison with its very similar pseudogenic variant, rp lambda 7. Southern analysis, using the rp lambda 5 cDNA as a probe, indicates the existence of a great number of highly related sequences in the mouse genome. The mRNA for rp lambda 5 is approximately 800 nucleotides (nt) long and is found to be ubiquitously expressed at high levels in embryonic and adult mouse tissues, as shown by Northern and in situ analyses. Retinoic acid (RA) seems to have a moderate down-regulatory effect on this mRNA in differentiating P19 embryonal carcinoma cells. Several degenerate/nondegenerate RA-response element (RARE) motifs are found within 560 bp upstream from the degenerate start codon in rp lambda 7. However, it is unknown whether this RA effect is exerted at the transcriptional and/or posttranscriptional levels.

Retinoic acid-response elements with a highly repetitive structure isolated by immuno-selection from genomic DNA.

In vitro binding sites of retinoic acid receptors (RARs) were isolated from mouse genomic DNA by immunoprecipitation of receptor/DNA complexes. PuG(G/T)TCA half-site motifs, which constitute RA-responsive elements (RAREs), were identified in the immuno-selected fragments (ISFs), some of which contained highly repetitive arrangements of this motif. Genomic Southern analysis of a number of ISFs showed them to be of a single or low copy number. Several, but not all, ISFs acted as ligand-dependent RAREs in transient transfection assays. Two ISFs with repetitive RARE motifs responded preferentially to 9-cis retinoic acid-liganded retinoid X receptor in the presence or absence of co-transfected RAR, while little activation was seen with RAR alone in the presence of either all-trans or 9-cis retinoic acid. Another ISF, containing consensus TATA and CAAT box motifs, was shown to have RA-inducible promoter activity. The results suggest a high degree of promiscuity in response element recognition by retinoid receptors.

Ubiquitous nuclear factors bind specifically to a 5'-region conserved in carcinoembryonic antigen-related genes.

We recently cloned members of the murine carcinoembryonic antigen (CEA) gene family, some of which are differentially expressed during placental development. By intra- and interspecies sequence comparisons, we identified an element in the putative promoter and/or 5'-nontranslated region which is conserved within all human and rodent CEA-related genes analyzed so far. Using gel retardation analysis and DNaseI hypersensitive site mapping, we now show that ubiquitously expressed nuclear factors specifically bind to the conserved region derived from the mouse gene Cea-2 in vitro and probably also in vivo. Another DNaseI hypersensitive site lies within or close to a simple sequence motif [(GGA)n] located in the first intron of Cea-2. Such sequences have been reported to play a role in the regulation of certain genes. Therefore, this analysis has identified putative regulatory regions for Cea-2 and possibly CEA-related genes in general.

Characterization of murine carcinoembryonic antigen gene family members.

The carcinoembryonic antigen (CEA) is a human tumor marker whose gene belongs to a family with more than 20 members. This gene family codes for a group of proteins with in vitro cell adhesion properties and for a group of abundantly expressed pregnancy-specific glycoproteins (PSG) with unknown functions. As a basis for in vivo functional studies, we have started to analyze the murine CEA gene family and have identified five new members (Cea-2 to Cea-6). cDNA clones were isolated for Cea-2, Cea-3, and Cea-6. The deduced amino acid sequences of Cea-2 and Cea-6 indicate three IgV-like (N), followed by one IgC-like (A) domain (N1-N2-N3-A). We have also partially characterized the Cea-2 gene and two additional ones, Cea-4 and Cea-5. Cea-2 and Cea-4 are separated by only 16 kb, suggesting a close linkage of murine CEA-related genes, as found for the human CEA gene family. Cea-5 was located to the proximal region of mouse Chromosome (Chr) 7, which is syntenic to part of human Chr 19, containing the human CEA gene family cluster. Cea-2, Cea-3, and a Cea-4-like gene are differentially transcribed in the placenta during pregnancy, but not in other organs tested. This expression pattern strongly suggests that they represent counterparts of the human PSG subgroup members, despite the presence of multiple IgV-like domains, a feature not found for human PSGs. The more distantly related Cea-5 seems to be ubiquitously expressed. The putative promoter region of Cea-2 lacks typical TATA- or CAAT-boxes, but contains other conserved motifs that could play a role in the initiation of transcription.

Intra- and interspecies analyses of the carcinoembryonic antigen (CEA) gene family reveal independent evolution in primates and rodents.

Various rodent and primate DNAs exhibit a stronger intra- than interspecies cross-hybridization with probes derived from the N-terminal domain exons of human and rat carcinoembryonic antigen (CEA)-like genes. Southern analyses also reveal that the human and rat CEA gene families are of similar complexity. We counted at least 10 different genes per human haploid genome. In the rat, approximately seven to nine different N-terminal domain exons that presumably represent different genes appear to be present. We were able to assign the corresponding genomic restriction endonuclease fragments to already isolated CEA gene family members of both human and rat. Highly similar subgroups, as found within the human CEA gene family, seem to be absent from the rat genome. Hybridization with an intron probe from the human nonspecific cross-reacting antigen (NCA) gene and analysis of DNA sequence data indicate the conservation of noncoding regions among CEA-like genes within primates, implicating that whole gene units may have been duplicated. With the help of a computer program and by calculating the rate of synonymous substitutions, evolutionary trees have been derived. From this, we propose that an independent parallel evolution, leading to different CEA gene families, must have taken place in, at least, the primate and rodent orders.

Chromosomal localization of the carcinoembryonic antigen gene family and differential expression in various tumors.

Carcinoembryonic antigen (CEA) is a glycoprotein which is important as a tumor marker for a number of human cancers. It is a member of a gene family comprising about 10 closely related genes. In order to characterize mRNAs transcribed from individual genes we have identified by DNA and RNA hybridization experiments, gene-specific sequences from the 3' noncoding regions of CEA, and of nonspecific cross-reacting antigen (NCA) mRNAs, which have been recently cloned. With these probes, CEA mRNAs with lengths of 3.5 and 3.0 kilobases and an NCA mRNA species of 2.5 kilobases were identified in various human tumors. A 2.2-kilobase mRNA species, however, could only be detected in leukocytes of patients with chronic myeloid leukemia by hybridization with a probe from the immunoglobulin-like repeat domain of CEA. This region is known to be very similar among the various members of the CEA gene family, and indeed the probe hybridizes with all four mRNA species. In situ hybridization with a cross-hybridizing probe from the NCA gene localized the members of the CEA gene family to the short and to the long arm of chromosome 19. In addition, a CEA cDNA probe was found to hybridize to the long arm of chromosome 19 only.