Increase of HMGA1a protein methylation is a distinctive characteristic of leukaemic cells induced to undergo apoptosis.
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Apoptosis↗
Biomedical subjects
G Manfioletti
Publications and source records attributed to G Manfioletti.
High mobility group HMGI(Y) protein expression in human colorectal hyperplastic and neoplastic diseases.
HMGI(Y) proteins are overexpressed in experimental and human malignancies, including colon, prostate and thyroid carcinomas. To determine at which step of the carcinogenic process HMGI(Y) induction occurs, we analysed the expression of the HMGI(Y) proteins in hyperplastic, preneoplastic and neoplastic tissues of colorectal origin by immunohistochemistry. All the colorectal carcinomas were HMGI(Y)-positive, whereas no expression was detected in normal colon mucosa tissue. HMGI(Y) expression in adenomas was closely correlated with the degree of cellular atypia. Only 2 of the 18 non-neoplastic polyps tested were HMGI(Y)-positive. These data indicate that HMGI(Y) protein induction is associated with the early stages of neoplastic transformation of colon cells and only rarely with colon cell hyperproliferation.
Adenoma↗
A link between apoptosis and degree of phosphorylation of high mobility group A1a protein in leukemic cells.
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.
Amino Acid Sequence↗
Role of the high mobility group A proteins in human lipomas.
The HMGA family is comprised of four proteins: HMGA1a, HMGA1b, HMGA1c and HMGA2. The first three proteins are products of the same gene, HMGA1, generated through an alternative splicing mechanism. The HMGA proteins are involved in the regulation of chromatin structure and HMGA DNA-binding sites have been identified in functional regions of many gene promoters. Rearrangements of the HMGA2 gene have been frequently detected in human benign tumors of mesenchymal origin including lipomas. 12q13-15 chromosomal translocations involving the HMGA2 gene locus, account for these rearrangements. The HMGA proteins have three AT-hook domains and an acidic C-terminal tail. The HMGA2 modifications consist in the loss of the C-terminal tail and fusion with ectopic sequences. A pivotal role of the HMGA2 rearrangements in the process of lipomagenesis is suggested by experiments showing that transgenic mice carrying a truncated HMGA2 gene showed a giant phenotype together with abdominal/pelvic lipomatosis. As HMGA2 null mice showed a great reduction in fat tissue, a positive role of the HMGA2 gene in adipocytic cell proliferation is proposed. More recently, similar alterations of the HMGA1 gene have been described. As the block of the HMGA1 protein synthesis induces an increase in growth rate of the pre-adipocytic cell line 3T3-L1, we suggest a negative role of the HMGA1 proteins in adipocytic cell growth and, therefore, we propose that adipocytic cell growth derives from the balance of the HMGA1 and HMGA2 protein functions.
Adipocytes↗
Transcriptional regulation of human insulin receptor gene by the high-mobility group protein HMGI(Y).
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.
5' Untranslated Regions↗
Genomic organization and chromosome mapping of the human homeobox gene HHEX.
In the present study, we report the genomic reconstruction of the human homeobox-containing gene HHEX by the use of the data available in public databases. This analysis allowed characterization of the gene organization showing that it is very similar to the mouse gene. Moreover the gene was mapped using FISH to 10q24.
Animals↗
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.
Animals↗
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.
Amino Acid Sequence↗
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.
Adipocytes↗
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.
Amino Acid Sequence↗
HMGI-C and HMGI(Y) immunoreactivity correlates with cytogenetic abnormalities in lipomas, pulmonary chondroid hamartomas, endometrial polyps, and uterine leiomyomas and is compatible with rearrangement of the HMGI-C and HMGI(Y) genes.
High-mobility group (HMG) proteins are nonhistone nuclear proteins that play an important role in the regulation of chromatin structure and function. HMGI-C and HMGI(Y) are members of the HMGI family of HMG proteins, and their expression in adult tissues generally correlates with malignant tumor phenotypes. However, HMGI-C and HMGI(Y) dysregulation as a result of specific rearrangements involving 12q15 and 6p21, the respective chromosomal sites in which the HMGI-C and HMGI(Y) genes are located, is also identified in a variety of common benign mesenchymal tumors, such as lipomas and uterine leiomyomata. The general prevalence of HMGI-C and HMGI(Y) protein expression and its correlation with chromosomal alterations in these benign tumors are unknown. We analyzed 95 human tumors (20 lipomas, 21 pulmonary chondroid hamartomas, 26 uterine leiomyomata, and 28 endometrial polyps) representing a selection of the benign lesions in which karyotypic alterations involving the chromosomal regions 12q15 and 6p21 are frequently detected. All cases were successfully karyotyped and some of them analyzed by fluorescent in situ hybridization with probes spanning the HMGI-C and HMGI(Y) genes. The results of this study demonstrate that expression of HMGI-C or HMGI(Y) is a common occurrence in lipomas, pulmonary chondroid hamartomas, leiomyomata, and endometrial polyps; that it correlates with 12q15 and 6p21 chromosomal alterations (p < 0.001); and that it is compatible with rearrangement of the HMGI-C and HMGI(Y) genes. The expression pattern and cellular localization of the immunoreactivity support the view that in biphasic lesions composed of a mixture of both stromal and epithelial cells, such as pulmonary chondroid hamartoma and endometrial polyps, the mesenchymal component is the site of the HMGI genetic alterations.
Chromosome Aberrations↗
Expression of the HMGI(Y) gene products in human neuroblastic tumours correlates with differentiation status.
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.
Antineoplastic Agents↗
Sp1 and CTF/NF-1 transcription factors are involved in the basal expression of the Hmgi-c proximal promoter.
HMGI-C is a nuclear architectural factor which is expressed during embryogenesis but not in adult tissues while it becomes re-expressed following neoplastic transformation. In this paper we identify the promoter region of the mouse Hmgi-c gene and by stepwise deletion of the 5' sequences we map the promoter activity of the most abundant transcript to a very short fragment containing a long polypyrimidine/polypurine (ppyr/ppur) tract. We demonstrate that this tract is a multiple binding site for the transcription factors Sp1 and Sp3 and that in Drosophila SL2 cells, Sp1 activates the Hmgi-c promoter. In addition, another transcription factor, CTF/NF-1, binds the proximal promoter immediately downstream of this region and its mutation decreases transcription in NIH-3T3 cells. This study identifies factors responsible for the basal activity of Hmgi-c gene and provides a foundation for further analysis of the mechanism of its regulation.
Animals↗
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.
Animals↗
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.
Adenocarcinoma, Follicular↗
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.
Animals↗
NF-kappaB mediated transcriptional activation is enhanced by the architectural factor HMGI-C.
High mobility group I proteins (HMGI, HMGY and HMGI-C) are a family of low molecular mass non-histone nuclear proteins which constitute an important component of the active chromatin structure. Two members of this family, HMGI and HMGY, have been demonstrated to contribute to the transcriptional regulation of several promoters by interacting with the DNA and with different transcription factors. On the contrary, very little is known about the third member, HMGI-C, which plays an important role during embryonic growth and in the process of cell transformation, its gene being rearranged in a large number of mesenchimal tumors. In this paper we show for the first time that HMGI-C is also able to function as architectural factor, enhancing the activity of a transcription factor, NF-kappaB, through the PRDII element of the beta-interferon enhancer. Moreover we show that this enhancement is absolutely dependent on the binding of HMGI-C to its target sequence. The demonstration that HMGI-C is able to modulate transcription is thus an important initial step in the identification of genes regulated by this factor.
3T3 Cells↗
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.
Antibodies, Monoclonal↗