Characterization of Sp1, AP-1, CBF and KRC binding sites and minisatellite DNA as functional elements of the metastasis-associated mts1/S100A4 gene intronic enhancer.

The mts1/S100A4 gene encodes a small acidic calcium-binding protein that is expressed in a cell-specific manner in development, tumorigenesis and certain tissues of adult mice. A composite enhancer that is active in murine mammary adenocarcinoma cells was previously identified in the first intron of the mts1/S100A4 gene. Here we present a detailed analysis of the structure and function of this enhancer in the Mts1/S100A4-expressing CSML100 and non-expressing CSML0 mouse adenocarcinoma cell lines. In CSML100 cells the enhancer activity is composed of at least six cis-elements interacting with Sp1 and AP-1 family members and CBF/AML/PEBP2 and KRC transcription factors. In addition, a minisatellite-like DNA sequence significantly contributes to the enhancer activity via interaction with abundant proteins, which likely have been described previously under the name minisatellite-binding proteins. Extensive mutational analysis of the mts1/S100A4 enhancer revealed a cooperative function of KRC and the factors binding minisatellite DNA. This is the first example of an enhancer where two nuclear factors earlier implicated in different recombination processes cooperate to activate transcription. In Mts1/S100A4-negative CSML0 cells the strength of the enhancer was 7- to 12.5-fold lower compared to that in CSML100 cells, when referred to the activities of three viral promoters. In CSML0 cells the enhancer could be activated by exogenous AP-1 and CBF transcription factors.

The dimerization interface of the metastasis-associated protein S100A4 (Mts1): in vivo and in vitro studies.

The S100 calcium-binding proteins are implicated in signal transduction, motility, and cytoskeletal dynamics. The three-dimensional structure of several S100 proteins revealed that the proteins form non-covalent dimers. However, the mechanism of the S100 dimerization is still obscure. In this study we characterized the dimerization of S100A4 (also named Mts1) in vitro and in vivo. Analytical ultracentrifugation revealed that apoS100A4 was present in solution as a mixture of monomers and dimers in a rapidly reversible equilibrium (K(d) = 4 +/- 2 microm). The binding of calcium promoted dimerization. Replacement of Tyr-75 by Phe resulted in the stabilization of the dimer. Helix IV is known to form the major part of the dimerization interface in homologous S100 proteins. By using the yeast two-hybrid system we showed that only a few residues of helix IV, namely Phe-72, Tyr-75, Phe-78, and Leu-79, are essential for dimerization in vivo. A homology model demonstrated that these residues form a hydrophobic cluster on helix IV. Their role is to stabilize the structure of individual subunits rather than provide specific interactions across the dimerization surface. Our mutation data showed that the specificity at the dimerization surface is not particularly stringent, which is consistent with recent data indicating that S100 proteins can form heterodimers.

The three-dimensional structure of human S100A12.

The crystal structure of human EF-hand calcium-binding protein S100A12 in its calcium-bound form has been determined to 1.95 A resolution by molecular replacement using the structure of the S100B protein. The S100 family members are homologous to calmodulin and other related EF-hand calcium-binding proteins. Like the majority of S100 proteins, S100A12 is a dimer, with the interface between the two subunits being composed mostly of hydrophobic residues. The fold of S100A12 is similar to the other known crystal and solution structures of S100 proteins, except for the linker region between the two EF-hand motifs. Sequence and structure comparison between members of the S100 family suggests that the target-binding region in S100A12 is formed by the linker region and C-terminal residues of one subunit and the N-terminal residues of another subunit of the dimer. The N-terminal region of the target-binding site includes two glutamates that are conserved in most of the S100 sequences. The comparison also provided a better understanding of the role of the residues important for intra- and inter-subunit hydrophobic interactions. The precise role of S100A12 in cell behaviour is yet undefined, as is the case for the whole family, although it has been shown that the interaction of S100A12 with the RAGE receptor is implicated in inflammatory response.

Crystallization and preliminary X-ray diffraction analysis of human calcium-binding protein S100A12.

S100A12, a member of the calgranulin family, isolated from human blood, has been crystallized by vapour diffusion in the presence of Ca(2+). Crystals belong to the space group R3 with unit-cell dimensions a = b = 99.6 c = 64.2 A. There are two monomers per asymmetric unit, with a solvent content of 57.9%. The crystals diffract to at least 2.2 A resolution and complete X-ray data have been collected to 2.5 A on a conventional laboratory source.

A minisatellite "core" element constitutes a novel, chromatin-specific activator of mts1 gene transcription.

Expression of the mts1 gene is often associated with malignant transformation of tumor cells. Transcription of the gene is controlled by a number of positive and negative regulatory elements, all of them being localized in the first intron (+38 to +1215) of the mts1 gene. Through analysis of the distribution of DNase I hypersensitive sites in the first intron of the gene we revealed a structurally conserved region that consisted of a non-canonical NFkB binding site and a minisatellite "core" element. Deletion of the minisatellite core DNA in the context of the first intron had no effect on its regulatory capacity when assayed in transient transfections, while a fivefold decrease was observed in a pool of stably transfected cells. The minisatellite core sequence CTGGGCAGGCAG is involved in DNA-protein interactions in vivo, and is similar to a binding site for the previously identified minisatellite DNA sequence binding protein (Msbp-1). The core DNA interacted in vitro with a protein that had an apparent molecular mass of 40 kDa. These data indicate that the minisatellite DNA represents the novel, chromatin-specific element in the mts1 complex enhancer.

Molecular cloning and characterization of the mouse tag7 gene encoding a novel cytokine.

Cloning of the mouse tag7 gene encoding a novel cytokine is described. The Tag7 protein consists of 182 amino acids. Genomic organization of the tag7 gene and its promoter region remind those of the genes of the tumor necrosis factor locus, although the tag7 gene is not linked to this locus. The gene is located on chromosome 7 at the area that corresponds to band 7A3, which has genetic linkage with lupus-like disease in mouse models. tag7 transcription is essential for lymphoid organs. It is also detected in certain areas of lungs, brain, and intestine and in some tumors. Tag7 protein is detectable in both cell-associated and soluble forms. The soluble form of Tag7 triggers apoptosis in mouse L929 cells in vitro and does not involve NF-kappaB activation. The relationship between Tag7 and tumor necrosis factor family of ligands is discussed.

Expression of the metastasis-associated mts1 gene during mouse development.

The mts1 gene, a member of the S100 family, is specifically expressed in different metastatic tumor cell lines. After transfection in some nonmetastatic cell lines Mtsl can induce a metastatic phenotype. Mts1 protein can interact with non-muscle myosin, indicating that Mts1 plays a role in cell motility. In order to understand the function of this gene, we studied the expression of the mts1 mRNA and protein in vivo during mouse development. Both mRNA and protein were present in high concentrations from 12.5 to 18.5 days post coitum (dpc) in a variety of developing embryonic tissue of mesodermal origin. We found by double immunostaining with a macrophage-specific antibody that Mts1 protein was highly expressed in fetal macrophages throughout the embryonic mesenchyme and in macrophages colonizing developing lymphatic and non-lymphatic organs. Moreover, we found mts1 expression during differentiation and morphogenesis of mesenchymal tissues such as the mesenchyme surrounding the tips of digits, the mesenchyme underlying the epithelium of the bladder, and the mesenchyme between the primordia of the nasal capsule and the skin as well as in the developing dermal papilla of hair and tooth follicle. In developing bone, Mts1 was expressed in invasive mesenchymal cells and in osteoclasts. The results presented here suggest that Mtsl plays an important role in mouse development during differentiation and function of macrophages and might be involved in different processes associated with mesenchymal morphogenesis including mesenchymal-epithelial interaction, tissue remodeling, and invasion.

Spectral studies on the calcium-binding properties of Mts1 protein and its interaction with target protein.

Two calcium-binding sites of the Mts1 protein, a member of S-100 protein family, were distinguished with the Fluo-3 fluorescent technique. The geometric mean of the apparent dissociation constant (Kd) for these two sites is 2.6 microM; the Hill coefficient (nH) is 0.98. In the presence of a novel target protein p37, isolated from the mouse adenocarcinoma cell line CSML-100, Mts1 binds Ca2+ ions with higher affinity and with strong positive cooperativity (Kd = 0.2 microM, nH = 1.91). Interaction of Mts1 with p37 is confirmed by the fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS). Reaction with TNS shows that p37 interacts with the hydrophobic site of Mts1 which is exposed due to the binding of Ca2+ ions.

Calcium-binding characteristics and conformational changes in metastasin, a member of S-100 protein family.

Investigation of Ca(2+)-binding characteristics of metastasin (Mts-1) by competition with Fluo-3 revealed two types of Ca(2+)-binding sites in Mts-1 with the geometric mean of their dissociation constant (Kd) value of 2.6 microM for the two EF-sites. The Hill coefficient (nH) is 0.98. A substantial increase in the affinity of Mts-1 for Ca2+ and strong cooperative character of binding (kd = 0.2 microM, nH = 1.91) is observed in the presence of the target protein p37 isolated from mouse adenocarcinoma cell lines CSML-100 and CSML-0. Two different hydrophobic sites of binding with the fluorescent probe 2-(p-toludino)naphthalene-6-sulfonate (TNS) per Mts-1 molecule have been determined. The exposure of the hydrophobic binding sites of the first type are shown to be Ca(2+)-dependent and the hydrophobic binding sites of the second type are exposed independently of Ca2+ concentration. A decrease in the number of Ca(2+)-dependent hydrophobic centers in the presence of p37 protein was detected by measurements of TNS fluorescence.

The differentiation antigen Ly-6E.1 is expressed in mouse metastatic tumor cell lines.

We report the cloning of the mouse surface GPI-anchored Ly-6E.1 protein from a highly metastatic mouse adenocarcinoma cell line CSML-100 by differential display. The expression is specific for the metastatic cell line as the closely related, non-metastatic mouse adenocarcinoma cell line CSML-0 does not express Ly-6E.1. Northern blot analysis reveals expression in a number of mouse tumour cell lines, exclusively metastatic ones. To date, active Ly-6A/E has only been described in lymphoid cells. The correlation between Ly-6E.1 expression, and the ability to metastasize, is discussed.

[Analysis of homologous DNA sequences within the first intron of the mouse and human mts1 genes: kB-like site and microsatellite DNA]. / Analiz gomologichnykh posledovatel'nostei DNK v pervom introne gena mts1 myshi i cheloveka: kB-podobnyi sait i mikrosatellitnaia DNK.

Homologous regions were revealed and analyzed within the first intron of the mouse and human mts1 genes. Maximal homology was detected between microsatellite DNA sequences from the first intron of the mouse gene (+804, +863) and the human gene (+600, +645). DNase I hypersensitive sites were revealed within these regions in both mouse and human cell lines tested, thus showing functional significance of the homology detected. In both the mouse and the human genes, the 5' end of homologous regions is flanked by a DNA sequence similar to a NF-kB/Rel protein-binding consensus sequence. Previously, this sequence was demonstrated to be involved in a complex regulatory element of the mouse mts1 gene. In comparison with the kB-like sequence, mouse, rat, and human sequences were found to involve one, two, and three nucleotide substitutions, respectively. Alterations in a spectrum of nuclear proteins bound to various kB-like sequences were analyzed. Possible effects of these alterations on mts1 transcription regulation were discussed. The possibility of the human kB-like sequence to function as positive regulator of transcription initiation was demonstrated in vivo.

[Study of calcium-binding properties of the Mts-1 protein and its recombinant analog using the fluorescent probe Fura-2]. / Izuchenie kal'tsiisviazyvaiushchikh svoistv belka Mts-1 i ego rekombinantnogo analoga s pomoshch'iu fluorestsentnogo zonda Fura-2.

A procedure based on the ability of Mts-1 to competitively inhibit the formation of Fura-2 complexes with Ca(2+)-ions is described. It has been shown that Mts-I reversibly inhibits the complex formation between Fura-2 and calcium. The efficiency of natural Mts-I isolated from cells of the CSML-100 line and of its recombinant analog on the formation of the Fura-2+Ca complex is different. The inhibition constant, Ki, for the native protein is equal to 2,4 microM, that for the recombinant form is 8.5 microM. The data obtained are suggestive of posttranslational modification of Mts-I in cells under in vivo conditions.

Novel AP-1 binding site created by DNA-methylation.

DNA-methylation is known to repress transcription either by inactivation of positive regulatory cis-elements containing CpG dinucleotides or via the sequence-nonspecific and methylation-specific binding of inhibiting methyl-CpG dinucleotides or via the sequence-nonspecific and methylation-specific binding of inhibiting methyl-CpG binding protein 1 (MeCP1). In the present work we describe the novel way DNA-methylation can influence gene expression: a binding site for transcription factors AP-1 might be created by DNA-methylation. Such a DNA-methylation-dependent AP-1 binding site was found in the first intron of the metastasis-associated mts1 gene. The expression level of this gene correlates with the hypomethylation of the mts1 first intron sequence in mouse adenocarcinoma cells. The DNA - methylation-dependent AP-1 binding site was found to be functionally active in the nucleotide context of the mts1 gene. When methylated, this site reproducibly repressed transcription of CAT-containing DNA that had been transiently transfected into mouse adenocarcinoma CSML100 cells.

Activation of mts1 transcription by insertion of a retrovirus-like IAP element.

The mechanism of activation of mestatasis-associated mts1 gene transcription in the mouse myelomonocytic leukaemia WEHI-3 cell line is described. Northern blot analysis showed that WEHi-3 cells expressed two types of mts1-specific mRNA: standard (550 nt) and additional (about 800 nt). The steady-state expression level of the 800-nt RNA was isolated and sequence analysis showed that it contained a 357-bp fragment represented by long terminal repeat (LTR) sequences and a 5' untranslated region of the gag gene of the intracisternal A-particle (IAP) element. The chimeric IAP::mts1 800-nt mRNA is initiated from the 5' LTR promoter. The rearranged and normal loci of mts1 were cloned and partially sequenced. The results suggested that the insertion of the IAP sequences into the first intron of mts1 brings the gene under control of the strong IAP promoter. The additional chimeric 800-nt IAP::mts1 RNA transcript was the result of a splicing event linking IAP sequences with the coding part of mts1. We suggest that the chimeric IAP::mts1 RNA is capable of producing a functional Mts1 protein.