Fritz Rudert

Regulation of the human fas promoter by YB-1, Purα 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.

Transcripts from opposite strands of ? 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 (γ) 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 γ satellite monomer. In Northern analysis, these satellite DNA sequences hybridized predominantly to an ≈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.

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

SummaryVarious 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.

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.

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.

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.