Expression of the HMGI(Y) gene products in human neuroblastic tumours correlates with differentiation status.
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HMGI and HMGY are splicing variants of the HMGI(Y) gene and together with HMGI-C, belong to a family of DNA binding proteins involved in maintaining active chromatin conformation and in the regulation of gene transcription. The expression of the HMGI(Y) gene is maximal during embryonic development, declines in adult differentiated tissues and is reactivated in most transformed cells in vitro and in many human cancers in vivo. The HMGI(Y) genomic locus is frequently rearranged in mesenchymal tumours, suggesting a biological role for HMGI(Y) gene products in tumour biology. HMGIs are both target and modulators of retinoic acid activity. In fact, HMGI(Y) gene expression is differentially regulated by retinoic acid in retinoid-sensitive and -resistant neuroblastoma cells, while HMGI-C participates in conferring retinoic acid resistance in some neuroblastoma cells. In this paper we show that HMGI and HMGY isoforms are equally regulated by retinoic acid in neuroblastoma cell lines at both RNA and protein levels. More importantly our immunohistochemical analysis shows that, although HMGI(Y) is expressed in all neuroblastic tumours, consistently higher levels are observed in less differentiated neuroblastomas compared to more differentiated ganglioneuromas, indicating that HMGI(Y) expression should be evaluated as a potential diagnostic and prognostic marker in neuroblastic tumours. (+info)
FRA-1 expression in hyperplastic and neoplastic thyroid diseases.
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fra-1 gene overexpression has been shown to represent a general event in thyroid cell transformation in vitro and in vivo. Moreover, inhibition of FRA-1 protein synthesis by stable transfection with a fra-1 antisense construct significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role of the fra-1 gene product in the process of cellular transformation. In the attempt to define the potential use of FRA-1 protein detection in the diagnosis of thyroid diseases, we analyzed Fra-1 expression by a combination of immunohistochemistry and reverse transcription-PCR (RT-PCR) assay in 174 samples of thyroid nodules (22 nodular hyperplasias, 102 follicular adenomas, 34 papillary carcinomas, 12 follicular carcinomas, and 4 anaplastic carcinomas) representative of the spectrum of thyroid tumor pathology. FRA-1 protein was abundant in all of the carcinoma samples (50/50, 100%), with an intense staining in the nucleus and the cytoplasm. Positive staining was also found in most of the adenomas (90 of 102; 88%), but in this case, the staining was restricted to the nucleus. Similar results were obtained from the analysis of thyroid goiters; however, the number of positive cases is lower than adenomas (8 of 22; 36%); moreover, the staining was not observed in all of the cells. Conversely, no FRA-1 protein was detectable in 12 normal thyroid tissue samples used as controls. RT-PCR analysis confirmed a higher fra-1 expression in papillary and follicular carcinomas compared with goiters and adenomas. fra-1 expression was also analyzed on 10 fine needle aspiration biopsy (FNAB) samples by RT-PCR. fra-1-specific mRNA was detected in seven of the eight FNABs corresponding to thyroid nodules that were eventually diagnosed as adenomas (three of four) and carcinomas (four of four) after surgery. Conversely, no fra-1 gene expression was observed in two FNABs derived from normal thyroid. Further studies are required before suggesting FRA-1 protein detection as a useful tool for the diagnosis of hyperplastic and neoplastic disorders of the thyroid gland. (+info)
PU.1-mediated transcription is enhanced by HMG-I(Y)-dependent structural mechanisms.
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The ets transcription factor PU.1 is an important regulator of the immunoglobulin heavy chain gene intronic enhancer, or mu enhancer. However, PU.1 is only one component of the large multiprotein complex required for B cell-specific enhancer activation. The transcriptional coactivator HMG-I(Y), a protein demonstrated to physically interact with PU.1, increases PU.1 affinity for the mu enhancer muB element, indicating that HMG-I(Y) may play a role in the transcriptionally active mu enhanceosome. Increased PU.1 affinity is not mediated by HMG-I(Y)-induced changes in DNA structure. Investigation of alternative mechanisms to explain the HMG-I(Y)-mediated increase in PU.1/mu enhancer binding demonstrated, by trypsin and chymotrypsin mapping, that interaction between PU.1 and HMG-I(Y) in solution induces a structural change in PU.1. In the presence of HMG-I(Y) and wild-type mu enhancer DNA, PU.1 becomes more chymotrypsin resistant, suggesting an additional change in PU.1 structure upon HMG-I(Y)-induced PU.1/DNA binding. From these results, we suggest that increased DNA affinity under limiting PU.1 concentrations is mediated by an HMG-I(Y)-induced structural change in PU.1. In functional assays, HMG-I(Y) further augments transcriptional synergy between PU.1 and another member of the ets family, Ets-1, indicating that HMG-I(Y) is a functional component of the active enhancer complex. These studies suggest a new mechanism for HMG-I(Y)-mediated coactivation; HMG-I(Y) forms protein-protein interactions with a transcription factor, which alters the three-dimensional structure of the factor, resulting in enhanced DNA binding and transcriptional activation. This mechanism may be important for transcriptional activation under conditions of limiting transcription factor concentration, such as at the low levels of PU.1 expressed in B cells. (+info)
Architectural transcription factor HMGI(Y) promotes tumor progression and mesenchymal transition of human epithelial cells.
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Numerous studies have demonstrated that overexpression or aberrant expression of the HMGI(Y) family of architectural transcription factors is frequently associated with both neoplastic transformation of cells and metastatic tumor progression. Little is known, however, about the molecular roles played by the HMGI(Y) proteins in these events. Here we report that human breast epithelial cells harboring tetracycline-regulated HMGI(Y) transgenes acquire the ability to form both primary and metastatic tumors in nude mice only when the transgenes are actively expressed. Unexpectedly, the HMG-Y, rather than the HMG-I, isoform of these proteins is the most effective elicitor of both neoplastic transformation and metastatic progression in vivo. Furthermore, expression of either antisense or dominant-negative HMGI(Y) constructs inhibits both the rate of proliferation of tumor cells and their ability to grow anchorage independently in soft agar. Array analysis of transcription profiles demonstrates that the HMG-I and HMG-Y isoform proteins each modulate the expression of distinctive constellations of genes known to be involved in signal transduction, cell proliferation, tumor initiation, invasion, migration, induction of angiogenesis, and colonization. Immunohistochemical analyses of tumors formed in nude mice indicate that many have undergone an epithelial-mesenchymal transition in vivo. Together, these findings demonstrate that overexpression of the HMGI(Y) proteins, more specifically, the HMG-Y isoform protein, is causally associated with both neoplastic transformation and metastatic progression and suggest that induction of integrins and their signaling pathways may play significant molecular roles in these biological events. (+info)
A link between apoptosis and degree of phosphorylation of high mobility group A1a protein in leukemic cells.
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Nuclear phosphoprotein HMGA1a, high mobility group A1a, (previously HMGI) has been investigated during apoptosis. A change in the degree of phosphorylation of HMGA1a has been observed during apoptosis induced in four leukemic cell lines (HL60, K562, NB4, and U937) by drugs (etoposide, camptothecin) or herpes simplex virus type-1. Both hyper-phosphorylation and de-phosphorylation of HMGA1a have been ascertained by liquid chromatography-mass spectrometry. Hyper-phosphorylation (at least five phosphate groups/HMGA1a molecule) occurs at the early apoptotic stages and is probably related to HMGA1a displacement from DNA and chromatin release from the nuclear scaffold. De-phosphorylation (one phosphate or no phosphate groups/HMGA1a molecule) accompanies the later formation of highly condensed chromatin in the apoptotic bodies. We report for the first time a direct link between the degree of phosphorylation of HMGA1a protein and apoptosis according to a process that involves the entire amount of HMGA1a present in the cells and, consequently, whole chromatin. At the same time we report that variously phosphorylated forms of HMGA1a protein are also mono-methylated. (+info)
Base-pair substitutions in avian sarcoma virus U5 and U3 long terminal repeat sequences alter the process of DNA integration in vitro.
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We have described a reconstituted avian sarcoma virus (ASV) concerted DNA integration system with specially designed mini-donor DNA containing a supF transcription unit, a supercoiled plasmid acceptor, purified bacterially expressed ASV integrase (IN), and human high-mobility-group protein I(Y). Integration in this system is dependent upon the mini-donor DNA having IN recognition sequences at both ends and upon both ends of the same donor integrating into the acceptor DNA. The integrated DNA product exhibits all of the features associated with integration of viral DNA in vivo (P. Hindmarsh et al., J. Virol., 73:2994-3003, 1999). Individual integrants are isolated from bacteria containing drug-resistant markers with amber mutations. This system was used to evaluate the importance of sequences in the terminal U5 and U3 long terminal repeats at positions 5 and/or 6, adjacent to the conserved CA dinucleotide. Base-pair substitutions introduced at these positions in U5 result in significant reductions in recovered integrants from bacteria, due to increases in one-ended insertion events. Among the recovered integrants from reactions with mutated U5 but not U3 IN recognition sequences were products that contain large deletions in the acceptor DNA. Base-pair substitutions at positions 5 and 6 in U3 mostly reduce the efficiency of integration of the modified donor. Together, these results indicate that sequences directly 5' to the conserved CA dinucleotide are very important for the process of concerted DNA integration. Furthermore, IN interacts with U3 and U5 termini differently, and aberrant end-processing events leading to nonconcerted DNA integration are more common in U5 than in U3. (+info)
Transcriptional regulation of human insulin receptor gene by the high-mobility group protein HMGI(Y).
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We have previously identified two closely related nuclear binding proteins that specifically interact with two unique functional AT-rich sequences of the 5' regulatory region of the human insulin receptor gene. Expression of these nuclear binding proteins increases during myocyte and adipocyte differentiation, and in other tissues appears to correlate with insulin receptor content. We have hypothesized, therefore, that insulin receptor expression in the insulin target tissues is regulated at least in part by these nuclear proteins. Here we show data on purification and biochemical characterization of these DNA binding proteins. Using a conventional chromatographic purification procedure combined with electrophoresis mobility shift assay and immunoblot analyses, a unique approximately 15 kDa protein, either identical to or highly related to the architectural transcription factor HMGI(Y), has now been identified, suggesting an essential role for HMGI(Y) in regulating insulin receptor gene transcription. Direct evidence of HMGI(Y) insulin receptor promoter interactions is provided by functional analysis with the CAT reporter gene and by hormone binding studies in cells expressing HMGI(Y) antisense RNA. In these experiments, antisense HMGI(Y) specifically inhibits insulin receptor promoter function and insulin receptor protein expression, indicating that HMGI(Y) is required for proper transcription of insulin receptor gene. Moreover, our data consistently support the hypothesis that a putative defect in this nuclear binding protein may cause insulin receptor dysfunction with subsequent impairment of insulin signaling and action. (+info)
Maintenance of Epstein-Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1.
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EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP, enables plasmids to persist in dividing human cells as multicopy episomes that attach to chromosomes during mitosis. In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the basic domains of EBNA-1 within amino acids 1-89 and 323-386 as critical for chromosome binding. In contrast, the EBNA-1 C terminus (amino acids 379-641), which includes the nuclear localization signal and DNA-binding domain, does not associate with mitotic chromosomes or retain oriP plasmid DNA in dividing cell nuclei, but does enable the accumulation of replicated oriP-containing plasmid DNA in transient replication assays. The importance of chromosome association in episome maintenance was evaluated by replacing EBNA-1 amino acids 1-378 with cell proteins that have similar chromosome binding characteristics. High-mobility group-I amino acids 1-90 or histone H1-2 could substitute for EBNA-1 amino acids 1-378 in mediating more efficient accumulation of replicated oriP plasmid, association with mitotic chromosomes, nuclear retention, and long-term episome persistence. These data strongly support the hypothesis that mitotic chromosome association is a critical factor for episome maintenance. The replacement of 60% of EBNA-1 with cell protein is a significant step toward eliminating the need for noncellular protein sequences in the maintenance of episomal DNA in human cells. (+info)