The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade.

A set of growth arrest-specific genes (gas) whose expression is negatively regulated after serum induction has previously been described (C. Schneider, R. M. King, and L. Philipson, Cell 54:787-793, 1988). The detailed analysis of one of them, gas6, is reported here, gas6 mRNA (2.6 kb) is abundantly expressed in serum-starved (48 h in 0.5% fetal calf serum) NIH 3T3 cells but decreases dramatically after fetal calf serum or basic fibroblast growth factor stimulation. The human homolog of gas6 was also cloned and sequenced, revealing a high degree of homology and a similar pattern of expression in IMR90 human fibroblasts. Computer analysis of the protein encoded by murine and human gas6 cDNAs showed significant homology (43 and 44% amino acid identity, respectively) to human protein S, a negative coregulator in the blood coagulation pathway. By using an anti-human Gas6 monospecific affinity-purified antibody, we show that the biosynthetic level of human Gas6 fully reflects mRNA expression in IMR90 human fibroblasts. This finding thus defines a new member of vitamin K-dependent proteins that is expressed in many human and mouse tissues and may be involved in the regulation of a protease cascade relevant in growth regulation.

A growth arrest-specific (gas) gene codes for a membrane protein.

A set of growth arrest-specific (gas) genes whose expression is negatively regulated by serum has recently been identified. We report on the detailed analysis of one of these genes (gas3). The kinetics of regulation by the presence and absence of serum were investigated, and it was found that this gene is regulated at the post-transcriptional level. The encoded protein deduced from the nucleotide sequence showed some similarity to a mitochondrial oxyreductase, and in vitro translation established that the protein product is a transmembrane glycoprotein.

Inhibition of HMGI-C protein synthesis suppresses retrovirally induced neoplastic transformation of rat thyroid cells.

Elevated expression of the three high-mobility group I (HMGI) proteins (HMGI, HMGY, and HMGI-C) has previously been correlated with the presence of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells and in experimental thyroid, lung, mammary, and skin carcinomas. Northern (RNA) blot and run-on analyses demonstrated that the induction of HMGI genes in transformed thyroid cells occurs at the transcriptional level. An antisense methodology to block HMGI-C protein synthesis was then used to analyze the role of this protein in the process of thyroid cell transformation. Transfection of an antisense construct for the HMGI-C cDNA into normal thyroid cells, followed by infection with transforming myeloproliferative sarcoma virus or Kirsten murine sarcoma virus, generated cell lines that expressed significant levels of the retroviral transforming oncogenes v-mos or v-ras-Ki and removed the dependency on thyroid-stimulating hormones. However, in contrast with untransfected cells or cells transfected with the sense construct, those containing the antisense construct did not demonstrate the appearance of any malignant phenotypic markers (growth in soft agar and tumorigenicity in athymic mice). A great reduction of the HMGI-C protein levels and the absence of the HMGI(Y) proteins was observed in the HMGI-C antisense-transfected, virally infected cells. Therefore, the HMGI-C protein seems to play a key role in the transformation of these thyroid cells.

Expression and function of the homeodomain-containing protein Hex in thyroid cells.

The homeodomain-containing protein Hex (also named Prh) is expressed in primitive endoderm (during the early phases of development), in some endoderm-derived tissues and in endothelial and hematopoietic precursors. Hex expression is exting-uished during terminal differentiation of endothelial and hematopoietic cells as well as in adult lung. Previous investigations have demonstrated that Hex is expressed during early thyroid gland development. No information has been reported on Hex expression in adult thyroid gland or on the function of this protein in follicular thyroid cells. These issues represent the focus of the present study. We demonstrate that Hex mRNA is present in rat and human adult thyroid gland as well as in differentiated follicular thyroid cell lines. In FRTL-5 cells TSH reduces Hex expression. In thyroid cell lines transformed by several oncogenes Hex expression is completely abolished. By using co-transfection assays we demonstrate that Hex is a repressor of the thyroglobulin promoter and that it is able to abolish the activating effects of both TTF-1 and Pax8. These data would suggest that Hex may play an important role in thyroid cell differentiation. Protein-DNA interaction experiments indicate that Hex is able to bind sites of the thyroglobulin promoter containing either the core sequence 5'-TAAT-3' or 5'-CAAG-3'. The DNA binding specificity of the Hex homeodomain, therefore, is more 'relaxed' than that observed in the majority of other homeo-domains.

Human colorectal carcinomas express high levels of high mobility group HMGI(Y) proteins.

A correlation has previously been demonstrated between the presence of the three HMGI proteins (HMGI, HMGY, and HMGI-C) and the expression of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells; this being subsequently extended to experimental thyroid, lung, prostate, mammary, and skin carcinomas. Recently, we have demonstrated that expression of HMGI and HMGY proteins, coded for by the HMGI(Y) gene, is associated with the malignant phenotype of human thyroid neoplasias. Here, we show that HMGI(Y) gene expression is present both at the RNA and protein level in human colorectal carcinoma cell lines and tissues examined in this study. Conversely, no HMGI(Y) proteins were detected in normal intestinal mucosa. Therefore, these results suggest an involvement of HMGI and HMGY proteins overexpression in colorectal tumorigenesis.

Expression of HMGI(Y) proteins in squamous intraepithelial and invasive lesions of the uterine cervix.

The expression of nuclear proteins high mobility group (HMG) I and HMGY was investigated in intraepithelial and invasive lesions of the uterine cervix. Human carcinoma cell lines C-41, ME-180, and CaSki were used for testing protein expression in neoplastic cells from the cervix. Morphological grading of the dysplasias (CIN 1, CIN 2, and CIN 3) and invasive carcinomas from formalin-fixed paraffin-embedded samples parallels the degree of nuclear immunostaining obtained using a polyclonal antibody raised against the amino-terminal region of HMGI(Y) proteins. The immunostaining obtained with HMGI(Y) antibody was compared with that observed using the antibody Ki-67, and the results were similar. We suggest the use of HMGI(Y) antibody in clinical oncology as a useful marker of intraepithelial lesions and invasive carcinomas.

Detection of high mobility group I HMGI(Y) protein in the diagnosis of thyroid tumors: HMGI(Y) expression represents a potential diagnostic indicator of carcinoma.

Hyperplastic or neoplastic proliferative lesions of thyroid follicular epithelium consist of a spectrum, ranging from nodular hyperplasia to undifferentiated (anaplastic) carcinoma, and usually present as palpable thyroid nodules. Thyroid nodules are a common occurrence in the general population, but only a small proportion of them are eventually diagnosed as carcinoma. The difficulty in objectively identifying those thyroid nodules that are malignant to avoid unnecessary surgery, combined with the range and effectiveness of the available therapeutic options in those patients who do, indeed, have thyroid carcinoma, has prompted the search for tumor markers and prognostic indicators. The high mobility group I (HMGI) proteins represent a class of nuclear proteins involved in the regulation of chromatin structure and function. HMGI(Y), one of the members of this class, is expressed at high levels during embryogenesis and in malignant tumors but at generally low levels in normal adult human tissues. Previous work on a limited number of thyroid samples suggested that the detection of the HMGI(Y) proteins may provide a clinically useful diagnostic tool. To verify this assumption, we analyzed HMGI(Y) expression by a combination of immunohistochemistry and reverse transcription-PCR in 358 thyroid tissue samples that were representative of the spectrum of thyroid tumor pathology. HMGI(Y) was detectable in 18 of 19 follicular carcinomas, 92 of 96 papillary carcinomas, and 11 of 11 undifferentiated (anaplastic) carcinomas but in only 1 of 20 hyperplastic nodules, 44 of 200 follicular adenomas, and 0 of 12 normal tissue samples. The correlation between HMGI(Y) expression and a diagnosis of carcinoma was highly significant (P < 0.0001). We also prospectively collected and analyzed for HMGI(Y) expression by immunohistochemistry and reverse transcription-PCR in 12 fine needle aspiration biopsies from 10 patients who subsequently underwent surgical removal of a solitary thyroid nodule. HMGI(Y) was detectable only in the four fine needle aspiration biopsies, corresponding to the thyroid nodules that were definitively diagnosed as carcinomas after surgery (two follicular carcinomas and two papillary carcinomas). The remaining eight samples (six follicular adenomas and two samples consisting of normal follicular cells) were negative. The findings of this study confirm the differential expression of HMGI(Y) in thyroid neoplasia and indicate the HMGI(Y) protein as a potential marker for thyroid carcinoma.

The expression of the high mobility group HMGI (Y) proteins correlates with the malignant phenotype of human thyroid neoplasias.

High Mobility Group I (HMGI) proteins are nuclear proteins involved in the regulation of chromatin structure and function. Elevated expression of the HMGI proteins (HMGI, HMGY and HMGI-C) has been correlated with the presence of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells, and in several experimental carcinomas. Here, we demonstrate that HMGI and HMGY proteins are expressed in human thyroid carcinomas and thyroid carcinoma cell lines, but not in adenomas, goiters, normal thyroid tissues and cells. These results indicate a correlation between HMGI and HMGY expression and the malignant phenotype of thyroid neoplasias, suggesting that these proteins may be used as markers in thyroid cancer.

The architectural transcription factor high mobility group I(Y) participates in photoreceptor-specific gene expression.

The nonhistone chromosomal proteins high mobility group I(Y) [HMG I(Y)] have been shown to function as architectural transcription factors facilitating enhanceosome formation on a variety of mammalian promoters. Specifically, they have been shown to act as a "molecular glue" mediating protein-protein and protein-DNA contacts within the enhanceosome complex. HMG I(Y) proteins are expressed at high levels in embryonic and transformed cells and have been implicated in transcriptional regulation in these cells. Terminally differentiated cells, however, have been reported to express only minimal, if any, HMG I(Y). In contrast to these observations, we show here that adult mouse retinal photoreceptors, which are terminally differentiated cells, express high levels of these proteins. Using retinoblastoma cells as an approximate model, we further demonstrate in transiently transfected cells that inhibition of HMG I(Y) expression and mutation of HMG I(Y) binding sites significantly reduce rhodopsin promoter activity. DNase I footprint analysis indicates that HMG I protein interacts with a discrete site within the rhodopsin proximal promoter. This site overlaps with the binding site for Crx, a paired-like homeodomain transcription factor that is essential for photoreceptor functioning and that when mutated causes several forms of human photoreceptor degeneration. Both biochemical and functional experiments demonstrate that HMG I(Y) physically associate with Crx and that their interaction with DNA is required for high-level transcription of the rhodopsin gene. These data provide the first demonstration that HMG I(Y) can be important for gene activation in terminally differentiated cells.

Isolation and characterization of the gene coding for murine high-mobility-group protein HMGI-C.

The HMGI-C protein is a nuclear factor expressed in human and rodent neoplastic cells which has been shown to be involved in the process of cell transformation. We have previously isolated the cDNA encoding murine HMGI-C and now we report the cloning and analysis of the mouse Hmgi-c gene. The gene is at least 50 kb long, contains five exons, and each of the three DNA-binding domains is encoded by a different exon. The location of exon-intron junctions was determined and shown to follow the GT-AG rule. The sequence revealed that the overall organization is similar to the gene encoding human HMGI(Y), the other member of the HMGI family, suggesting that HMGI genes probably evolved through gene duplication and exon shuffling events from an ancestral gene. A highly homologous pseudogene is also present in the mouse genome. Our results on Hmgi-c structure provide basic information to carry out further studies on the regulation of its expression.

DNA binding of NF-Y: the effect of HMGI proteins depends upon the CCAAT box.

NF-Y is a conserved sequence-specific transcription factor binding to CCAAT boxes. The chromatin-associated HMGI proteins influence promoter activities through positive and negative effects on binding of transcription factors. It was previously shown that HMGI(Y) synergizes the binding of NF-Y to the alpha2-collagen CCAAT box [Currie, R.A. (1997) J. Biol Chem. 272, 30880-30888]. Using recombinant proteins, we confirm that at low concentrations of NF-Y, HMGI(Y) acts synergistically on the alpha2-collagen CCAAT and we extend this observation to HMGI and HMGI-C. However, enhancement of DNA binding to gamma-globin, alpha-globin and MHC class II Ea CCAAT boxes was not observed. At high concentrations, HMGI proteins inhibit binding to alpha2-collagen and to gamma-globin, but not to high affinity Ea or a-globin CCAAT. In none of our experiments did we see a ternary complex between NF-Y, HMGI(Y) and DNA. In protein competition experiments, NF-Y affinity was at least two orders of magnitude higher, even in the context of the suboptimal gamma-globin CCAAT. Our data prove that HMGI proteins have complex positive and negative effects on NF binding to some, but not to all CCAAT boxes, suggesting that this phenomenon is dictated by the sequences flanking the pentanucleotide rather than direct protein-protein interactions.

A novel downstream positive regulatory element mediating transcription of the human high mobility group (HMG) I-C gene.

The high mobility group (HMG) I proteins are small, non-histone chromosomal proteins that promote gene activation during development and within rapidly dividing cells. They do so by facilitating enhanceosome formation on inducible genes, via both protein/DNA and protein/protein interactions. The HMG I-C gene is tightly regulated, normally being expressed exclusively during embryonic development. However, HMG I-C expression is also observed frequently in a number of tumor types, and this expression has been shown to contribute to the malignant transformation process. With the aim of dissecting pathways that lead to aberrant expression of HMG I-C in tumor cells, we have analyzed HMG I-C gene regulation in the human hepatoma cell line PLC/PRF/5. One of the two HMG I-C transcripts detected in this cell line originates from a novel downstream initiation site at nucleotide -161 relative to the first methionine. Transcription from the downstream initiation site is mediated by a PRE located between nt -222 and -217. We show here that the Sp1 and Sp3 transcription factors interact with the PRE and transactivate the HMG I-C promoter in a cooperative fashion. This study provides the first characterization of this downstream HMG I-C promoter.

The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing.

This report describes a common method of obtaining template DNA from phagemids, phages and plasmids. The strategy is based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) for DNA precipitation. By avoiding phase separation, many manipulation steps are reduced. A time-saving modification to perform double-stranded DNA sequencing directly after alkaline-denaturation is also introduced. The protocols described here allow the researcher to obtain template DNA from a variety of initial sources, thus giving reproducible sequencing results when using T7 DNA polymerase.

A fast method for high-quality genomic DNA extraction from whole human blood.

A simple and fast protocol is described for the purification of genomic DNA from 0.3 ml of whole human blood. The recovery of DNA is quantitative and reproducible; the quality is such that it can be used for all relevant molecular biology techniques.

New lambda and plasmid vectors for expression cloning in mammalian cells.

This report describes the construction of a new family of lambda phage and plasmid cloning vectors. lambda GDST3/T7 allows cDNA insertion up to 14 kb; it is derived from lambda NM1151 by the insertion of a multiple cloning site containing eight unique restriction sites. The two asymmetrical SfiI sites are flanked by the T3 and T7 promoters for direct sequencing and in vitro transcription/translation. The same multiple cloning site is also present in both orientations in the eukaryotic expression plasmids, pGDSV3 and pGDSV7. By exploiting the superior discrimination of the signal-to-noise ratio of the lambda vectors for primary screening (by either nucleic acids or antibody probes), relevant cDNAs can thus be efficiently transferred through SfiI sites into the plasmids pGDSV3/7 for functional secondary screening by expression in mammalian cells.

Intranuclear distribution of HMGI/Y proteins. An immunocytochemical study.

The intranuclear distribution of HMGI/Y proteins was analyzed by immunofluorescent staining in several cell lines using a polyclonal antibody that stained a fibrogranular network. In actively growing 3T3 fibroblasts, HMGI/Y proteins were mainly localized to heterochromatin masses, whereas in quiescent cells they were more diffusely distributed. Double labeling experiments showed a co-localization of HMGI/Y with DNA topoisomerase IIalpha. These results are in agreement with previously published biochemical data and indicate a possible involvement of HMGI/Y proteins in several nuclear functions, including chromatin organization and gene expression.

A new and fast method for preparing high quality lambda DNA suitable for sequencing.

A method is described for the rapid purification of high quality lambda DNA. The method can be used from either liquid or plate lysates and on a small scale or a large scale. It relies on the preadsobtion of all polyanions present in the lysate to an "insoluble" anion-exchange matrix (DEAE or TEAE). Phage particles are then disrupted by combined treatment with EDTA/proteinase K and the resulting DNA is precipitated by the addition of the cationic detergent cetyl (or hexadecyl)-trimethyl ammonium bromide-CTAB ("soluble" anion-exchange matrix). The precipitated CTAB-DNA complex is then exchanged to Na-DNA and ethanol precipitated. The resultant purified DNA is suitable for enzymatic reactions and provides a high quality template for dideoxy-sequence analysis.

Architecture of high mobility group protein I-C.DNA complex and its perturbation upon phosphorylation by Cdc2 kinase.

The high mobility group I-C (HMGI-C) protein is an abundant component of rapidly proliferating undifferentiated cells. High level expression of this protein is characteristic for early embryonic tissue and diverse tumors. HMGI-C can function as an architectural factor enhancing the activity of transcription factor NF-kappaB on the beta-interferon promoter. The protein has three minor groove DNA-binding domains (AT-hooks). Here, we describe the complex of HMGI-C with a fragment of the beta-interferon promoter. We show that the protein binds to NRDI and PRDII elements of the promoter with its first and second AT-hook, respectively. Phosphorylation by Cdc2 kinase leads to a partial derailing of the AT-hooks from the minor groove, affecting mainly the second binding domain. In contrast, binding to long AT stretches of DNA involves contacts with all three AT-hooks and is marginally sensitive to phosphorylation. Our data stress the importance of conformation of the DNA binding site and protein phosphorylation for its function.

Transgenic mice expressing a truncated form of the high mobility group I-C protein develop adiposity and an abnormally high prevalence of lipomas.

Chromosomal translocations in human lipomas frequently create fusion transcripts encoding high mobility group (HMG) I-C DNA-binding domains and C-terminal sequences from different presumed transcription factors, suggesting a potential role for HMG I-C in the development of lipomas. To evaluate the role of the HMG I-C component, the three DNA-binding domains of HMG I-C have now been expressed in transgenic mice. Despite the ubiquitous expression of the truncated HMG I-C protein, the transgenic mice develop a selective abundance of fat tissue early in life, show marked adipose tissue inflammation, and have an abnormally high incidence of lipomas. These findings demonstrate that the DNA-binding domains of HMG I-C, in the absence of a C-terminal fusion partner, are sufficient to perturb adipogenesis and predispose to lipomas. We provide data supporting the central utility of this animal model as a tool to understand the molecular mechanisms underlying the development of one of the most common kind of human benign tumors.