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International audience; Parkin (PK) is an E3-ligase harboring tumor suppressor properties that has been associated to various cancer types including glioblastoma (GBM). However, PK is also a transcription factor (TF), the contribution of which to GBM etiology remains to be established. Methods: The impact of PK on GBM cells proliferation was analyzed by real-time impedance measurement and flow cytometry. Cyclins A and B proteins, promoter activities and mRNA levels were measured by western blot, luciferase assay and quantitative real-time PCR. Protein-protein and protein-promoter interactions were performed by co-immunoprecipitation and by ChIP approaches. The contribution of endogenous PK to tumor progression in vivo was performed by allografts of GL261 GBM cells in wild-type and PK knockout mice. Results: We show that overexpressed and endogenous PK control GBM cells proliferation by modulating the S and G2/M phases of the cell cycle via the trans-repression of cyclin A and cyclin B genes. We establish that cyclin B is regulated by both E3-ligase and TF PK functions while cyclin A is exclusively regulated by PK TF function. PK invalidation leads to enhanced tumor progression in immunocompetent mice suggesting an impact of PK-dependent tumor environment to tumor development. We show that PK is secreted by neuronal cells and recaptured by tumor cells. Recaptured PK lowered cyclins levels and decreased GBM cells proliferation. Further, PK expression is decreased in human GBM biopsies and its expression is inversely correlated to both cyclins A and B expressions. Conclusion: Our work demonstrates that PK tumor suppressor function contributes to the control of tumor by its cellular environment. It also shows a key role of PK TF function in GBM development via the control of cyclins in vitro and in vivo. It suggests that therapeutic strategies aimed at controlling PK shuttling to the nucleus may prove useful to treat GBM.

Cancer heterogeneity is one of the factors that constitute an obstacle towards an efficient targeting of this multifaceted disease. Molecular information can help in classifying cancer subtypes and in providing clinicians with novel targeted therapeutic opportunities. In this regard, classification of breast cancer into intrinsic subtypes based on molecular profiling represents a valuable prototype. The High Mobility Group A (HMGA) chromatin architectural factors (HMGA1a, HMGA1b, and HMGA2) have a relevant and causal role in breast cancer onset and development, by influencing virtually all cancer hallmarks. The regulation of HMGA expression is under the control of major pathways involved in cell proliferation and survival, as well as in other cancer-related processes, thereby suggesting, for the HMGA members, a high degree of homology and overlapping activities. Despite of this evidence, HMGA proteins display also specific functions. In this review, we provide an overview of (i) the pathways involved in HMGA transcriptional and post-transcriptional regulation, (ii) the utilization of HMGA as molecular markers, and (iii) the biological role of HMGA in the context of breast cancer. We focus on the potential significance of HMGA in governing the onset and development of this tumour, as well as on the potential of these factors as novel specific targets for preventing and treating strategies. The emerging picture is a highly interconnected triad of proteins that could mutually influence each other, either in a competitive or cooperative manner, and that, in our opinion, should be considered as a unified and integrated protein system.

Plasticity is an essential condition for cancer cells to invade surrounding tissues. The nucleus is the most rigid cellular organelle and it undergoes substantial deformations to get through environmental constrictions. Nuclear stiffness mostly depends on the nuclear lamina and chromatin, which in turn might be affected by nuclear architectural proteins. Among these is the HMGA1 (High Mobility Group A1) protein, a factor that plays a causal role in neoplastic transformation and that is able to disentangle heterochromatic domains by H1 displacement. Here we made use of atomic force microscopy to analyze the stiffness of breast cancer cellular models in which we modulated HMGA1 expression to investigate its role in regulating nuclear plasticity. Since histone H1 is the main modulator of chromatin structure and HMGA1 is a well-established histone H1 competitor, we correlated HMGA1 expression and cellular stiffness with histone H1 expression level, post-translational modifications, and nuclear distribution. Our results showed that HMGA1 expression level correlates with nuclear stiffness, is associated to histone H1 phosphorylation status, and alters both histone H1 chromatin distribution and expression. These data suggest that HMGA1 might promote chromatin relaxation through a histone H1-mediated mechanism strongly impacting on the invasiveness of cancer cells.

Breast cancer (BC) is the most commonly diagnosed cancer among women worldwide and one of the leading cause of cancer-related deaths. The majority of BCs arise from epithelial cells, either in the ducts or lobules, as the result of genetic and epigenetic alterations, which lead to aberrant growth control and disruption of intracellular signaling. Because of this, BC is considered a heterogeneous disease with multiple sub-types, with cells of distinct origin and function. In summary, these articles contribute to understanding the topic and gathering information about the molecular mechanisms that are involved in BC development and progression. A better knowledge of the mechanisms involved in the pathogenesis of BC could enable us to discovery new biomarkers for patient stratification and identify novel therapeutic targets.

Background Breast cancer is the most common malignancy in women worldwide. Among the breast cancer subtypes, triple-negative breast cancer (TNBC) is the most aggressive and the most difficult to treat. One of the master regulators in TNBC progression is the architectural transcription factor HMGA1. This study aimed to further explore the HMGA1 molecular network to identify molecular mechanisms involved in TNBC progression. Methods RNA from the MDA-MB-231 cell line, silenced for HMGA1 expression, was sequenced and, with a bioinformatic analysis, molecular partners HMGA1 could cooperate with in regulating common downstream gene networks were identified. Among the putative partners, the FOXM1 transcription factor was selected. The relationship occurring between HMGA1 and FOXM1 was explored by qRT-PCR, co-immunoprecipitation and protein stability assays. Subsequently, the transcriptional activity of HMGA1 and FOXM1 was analysed by luciferase assay on the VEGFA promoter. The impact on angiogenesis was assessed in vitro, evaluating the tube formation ability of endothelial cells exposed to the conditioned medium of MDA-MB-231 cells silenced for HMGA1 and FOXM1 and in vivo injecting MDA-MB-231 cells, silenced for the two factors, in zebrafish larvae. Results Here, we discover FOXM1 as a novel molecular partner of HMGA1 in regulating a gene network implicated in several breast cancer hallmarks. HMGA1 forms a complex with FOXM1 and stabilizes it in the nucleus, increasing its transcriptional activity on common target genes, among them, VEGFA, the main inducer of angiogenesis. Furthermore, we demonstrate that HMGA1 and FOXM1 synergistically drive breast cancer cells to promote tumor angiogenesis both in vitro in endothelial cells and in vivo in a zebrafish xenograft model. Moreover, using a dataset of breast cancer patients we show that the co-expression of HMGA1, FOXM1 and VEGFA is a negative prognostic factor of distant metastasis-free survival and relapse-free survival. Conclusions This study reveals FOXM1 as a crucial interactor of HMGA1 and proves that their cooperative action supports breast cancer aggressiveness, by promoting tumor angiogenesis. Therefore, the possibility to target HMGA1/FOXM1 in combination should represent an attractive therapeutic option to counteract breast cancer angiogenesis. Electronic supplementary material The online version of this article (10.1186/s13046-019-1307-8) contains supplementary material, which is available to authorized users.

Chromatin accessibility plays a critical factor in regulating gene expression in cancer cells. Several factors, including the High Mobility Group A (HMGA) family members, are known to participate directly in chromatin relaxation and transcriptional activation. The HMGA1 oncogene encodes an architectural chromatin transcription factor that alters DNA structure and interacts with transcription factors favouring their landing onto transcription regulatory sequences. Here, we provide evidence of an additional mechanism exploited by HMGA1 to modulate transcription. We demonstrate that, in a triple-negative breast cancer cellular model, HMGA1 sustains the action of epigenetic modifiers and in particular it positively influences both histone H3S10 phosphorylation by ribosomal protein S6 kinase alpha-3 (RSK2) and histone H2BK5 acetylation by CREB-binding protein (CBP). HMGA1, RSK2, and CBP control the expression of a set of genes involved in tumor progression and epithelial to mesenchymal transition. These results suggest that HMGA1 has an effect on the epigenetic status of cancer cells and that it could be exploited as a responsiveness predictor for epigenetic therapies in triple-negative breast cancers.

HMGA1 (high mobility group A1) is a nonhistone architectural chromosomal protein that functions mainly as a dynamic regulator of chromatin structure and gene transcription. As such, HMGA1 is involved in a variety of fundamental cellular processes, including gene expression, epigenetic regulation, cell differentiation and proliferation, as well as DNA repair. In the last years, many reports have demonstrated a role of HMGA1 in the transcriptional regulation of several genes implicated in glucose homeostasis. Initially, it was proved that HMGA1 is essential for normal expression of the insulin receptor (INSR), a critical link in insulin action and glucose homeostasis. Later, it was demonstrated that HMGA1 is also a downstream nuclear target of the INSR signaling pathway, representing a novel mediator of insulin action and function at this level. Moreover, other observations have indicated the role of HMGA1 as a positive modulator of the Forkhead box protein O1 (FoxO1), a master regulatory factor for gluconeogenesis and glycogenolysis, as well as a positive regulator of the expression of insulin and of a series of circulating proteins that are involved in glucose counterregulation, such as the insulin growth factor binding protein 1 (IGFBP1), and the retinol binding protein 4 (RBP4). Thus, several lines of evidence underscore the importance of HMGA1 in the regulation of glucose production and disposal. Consistently, lack of HMGA1 causes insulin resistance and diabetes in humans and mice, while variations in the HMGA1 gene are associated with the risk of type 2 diabetes and metabolic syndrome, two highly prevalent diseases that share insulin resistance as a common pathogenetic mechanism. This review intends to give an overview about our current knowledge on the role of HMGA1 in glucose metabolism. Although research in this field is ongoing, many aspects still remain elusive. Future directions to improve our insights into the pathophysiology of glucose homeostasis may include epigenetic studies and the use of “omics” strategies. We believe that a more comprehensive understanding of HMGA1 and its networks may reveal interesting molecular links between glucose metabolism and other biological processes, such as cell proliferation and differentiation.

Diaryldienone derivatives with accessible β-carbons show strong anti-neoplastic properties, related to their ability to make covalent adducts with free thiols by Michael addition, and low toxicity in vivo. Accumulation of poly-ubiquitylated proteins, activation of the unfolded protein response (UPR) and induction of cell death are universal hallmarks of their activities. These compounds have been characterized as inhibitors of isopeptidases, a family of cysteine-proteases, which de-conjugate ubiquitin and ubiquitin-like proteins from their targets. However, it is unclear whether they can also react with additional proteins. In this work, we utilized the biotin-conjugated diaryldienone-derivative named 2c, as a bait to purify novel cellular targets of these small molecules. Proteomic analyses have unveiled that, in addition to isopeptidases, these inhibitors can form stable covalent adducts with different intracellular proteins, thus potentially impacting on multiple functions of the cells, from cytoskeletal organization to metabolism. These widespread activities can explain the ability of diaryldienone derivatives to efficiently trigger different cell death pathways.

The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways. In this work, we provide evidences linking HMGA1 with Non-Homologous End Joining DNA repair. We show that HMGA1 is in complex with and is a substrate for DNA-PK. HMGA1 enhances Ligase IV activity and it counteracts the repressive histone H1 activity towards DNA ends ligation. Moreover, breast cancer cells overexpressing HMGA1 show a faster recovery upon induction of DNA double-strand breaks, which is associated with a higher survival. These data suggest that resistance to DNA-damaging agents in cancer cells could be partially attributed to HMGA1 overexpression thus highlighting the relevance of considering HMGA1 expression levels in the selection of valuable and effective pharmacological regimens.

High Mobility Group A are non-histone nuclear proteins that regulate chromatin plasticity and accessibility, playing an important role both in physiology and pathology. Their activity is controlled by transcriptional, post-transcriptional and post-translational mechanisms. In this study we provide evidence for a novel modulatory mechanism for HMGA functions. We show that HMGAs are complexed in vivo with the histone chaperone nucleophosmin (NPM1), that this interaction requires the histone-binding domain of NPM1 and that NPM1 modulates both DNA-binding affinity and specificity of HMGAs. By focusing on two human genes whose expression is directly regulated by HMGA1, the Insulin receptor (INSR) and the Insulin-like growth factor-binding protein 1 (IGFBP1) genes, we demonstrated that occupancy of their promoters by HMGA1 was NPM1-dependent, reflecting a mechanism in which the activity of these cis-regulatory elements is directly modulated by NPM1 leading to changes in gene expression. HMGAs need short stretches of AT-rich nucleosome-free regions to bind to DNA. Therefore, many putative HMGA binding sites are present within the genome. Our findings indicate that NPM1, by exerting a chaperoning activity towards HMGAs, may act as a master regulator in the control of DNA occupancy by these proteins and hence in HMGA-mediated gene expression.

High Mobility Group A1 (HMGA1) is an architectural chromatin factor that promotes neoplastic transformation and progression. However, the mechanism by which HMGA1 exerts its oncogenic function is not fully understood. Here, we show that cyclin E2 (CCNE2) acts downstream of HMGA1 to regulate the motility and invasiveness of basal-like breast cancer cells by promoting the nuclear localization and activity of YAP, the downstream mediator of the Hippo pathway. Mechanistically, the activity of MST1/2 and LATS1/2, the core kinases of the Hippo pathway, are required for the HMGA1- and CCNE2-mediated regulation of YAP localization. In breast cancer patients, high levels of HMGA1 and CCNE2 expression are associated with the YAP/TAZ signature, supporting this connection. Moreover, we provide evidence that CDK inhibitors induce the translocation of YAP from the nucleus to the cytoplasm, resulting in a decrease in its activity. These findings reveal an association between HMGA1 and the Hippo pathway that is relevant to stem cell biology, tissue homeostasis, and cancer.

Type 2 diabetes mellitus is a widespread disease, affecting millions of people globally. Although genetics and environmental factors seem to have a role, the cause of this metabolic disorder is largely unknown. Here we report a genetic flaw that markedly reduced the intracellular expression of the high mobility group A1 (HMGA1) protein, and adversely affected insulin receptor expression in cells and tissues from four subjects with insulin resistance and type 2 diabetes. Restoration of HMGA1 protein expression in subjects' cells enhanced INSR gene transcription, and restored cell-surface insulin receptor protein expression and insulin-binding capacity. Loss of Hmga1 expression, induced in mice by disrupting the Hmga1 gene, considerably decreased insulin receptor expression in the major targets of insulin action, largely impaired insulin signaling and severely reduced insulin secretion, causing a phenotype characteristic of human type 2 diabetes.

We report for the first time that IFNG gene expression requires high mobility group (HMG)A1, the architectural transcription factor mediating enhanceosome formation. This finding is supported by our direct studies of T cells isolated from the HMGA1-transgenic mice displaying an up-regulation of IFN-gamma production and of HMGA1-deficient mice exhibited a decreased IFN-gamma induction. In parallel transfection studies in EL4 cells, we observed elevated IFNG gene promoter activity in cells stably over-expressing HMGA1 and a reduction of such activity in cells expressing dominant-negative HMGA1. In vitro binding assays further demonstrated a specific interaction of HMGA1 to defined regions of the IFNG gene proximal promoter.

High mobility group A 1 (HMGA1) proteins are chromatinic factors, which are absent or expressed at very low levels in normal adult tissues, while they are over-expressed in several human malignant tumors. In this study, HMGA1 protein expression was investigated by immunohistochemistry in a series of 44 epithelial ovarian specimens, which included four normal ovarian tissues, 29 primary invasive carcinomas, one metastatic ovarian tumor and 10 low malignant potential (LMP) tumors. HMGA1 staining was not detected in normal ovarian surface epithelium, which is the area from which ovarian adenocarcinoma frequently arises. HMGA1 proteins were expressed at low levels in some LMP tumors, whereas they were present in abundance in most of the primary ovarian adenocarcinomas. RT-PCR and western blot analysis correlated with immunohistochemical data. We demonstrated that the suppression of HMGA1 protein synthesis by an adenovirus carrying the HMGA1 gene in antisense orientation (Ad-Yas-GFP) inhibited the growth of two human ovarian carcinoma cell lines (OVCAR-5 and OVCAR-8). These results confirm HMGA1 over-expression as a general feature of human malignant neoplasias, including ovarian cancer and suggest that suppression of HMGA1 protein synthesis by an antisense adenoviral vector may represent a new and promising gene therapy for the treatment of ovarian cancer.

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.

// Silvia Pegoraro 1 , Gloria Ros 1 , Silvano Piazza 2 , Roberta Sommaggio 3 , Yari Ciani 1,2 , Antonio Rosato 3,4 , Riccardo Sgarra 1 , Giannino Del Sal 1,2 , and Guidalberto Manfioletti 1,* 1 Dipartimento di Scienze della Vita, Universita degli Studi di Trieste, Trieste, Italy 2 Laboratorio Nazionale CIB, (LNCIB), Area Science Park, Trieste, Italy 3 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy 4 Istituto Oncologico Veneto IRCCS, Padova, Italy Correspondence: Guidalberto Manfioletti, email: // Keywords : Epithelial to mesenchymal transition, stemness, HMGA1, breast cancer, invasion, metastasis, gene-signature Received : June 28, 2013 Accepted : July 7, 2013 Published : July 9, 2013 Abstract Breast cancer is a heterogeneous disease that progresses to the critical hallmark of metastasis. In the present study, we show that the High Mobility Group A1 (HMGA1) protein plays a fundamental role in this process in basal-like breast cancer subtype. HMGA1 knockdown induces the mesenchymal to epithelial transition and dramatically decreases stemness and self-renewal. Notably, HMGA1 depletion in basal-like breast cancer cell lines reduced migration and invasion in vitro and the formation of metastases in vivo . Mechanistically, HMGA1 activated stemness and key migration-associated genes which were linked to the Wnt/beta-catenin, Notch and Pin1/mutant p53 signalling pathways. Moreover, we identified a specific HMGA1 gene expression signature that was activated in a large subset of human primary breast tumours and was associated with poor prognosis. Taken together, these data provide new insights into the role of HMGA1 in the acquisition of aggressive features in breast cancer.

High-mobility group AT-hook 1 (HMGA1) protein is an important nuclear factor that activates gene transcription by binding to AT-rich sequences in the promoter region of DNA. We previously demonstrated that HMGA1 is a key regulator of the insulin receptor (INSR) gene and individuals with defects in HMGA1 have decreased INSR expression and increased susceptibility to type 2 diabetes mellitus. In addition, there is evidence that intracellular regulatory molecules that are employed by the INSR signaling system are involved in post-translational modifications of HMGA1, including protein phosphorylation. It is known that phosphorylation of HMGA1 reduces DNA-binding affinity and transcriptional activation. In the present study, we investigated whether activation of the INSR by insulin affected HMGA1 protein phosphorylation and its regulation of gene transcription. Collectively, our findings indicate that HMGA1 is a novel downstream target of the INSR signaling pathway, thus representing a new critical nuclear mediator of insulin action and function.

The IGF-I receptor (IGF-IR) is often overexpressed in cancer and is believed to play a crucial role in cancer progression. High Mobility Group A1 (HMGA1) is a non-histone chromatin protein that has the ability to regulate gene expression through DNA binding and involvement in enhanceosome complexes. HMGA1 is expressed at low level in adult differentiated cells, whereas it is expressed at high level in embryonic and malignant cells. We evaluated whether the HMGA1 aberrant expression has a role in IGF-IR overexpression in cancer. We found that HMGA1 silencing induces a marked decrease in IGF-IR expression in various human cancer cell lines. Conversely, forced HMGA1 overexpression in cells with low endogenous HMGA1 levels was associated with IGF-IR upregulation. HMGA1 silencing reduced igf-ir promoter activity whereas forced HMGA1 expression increased it. Using the chromatin immunoprecipitation assay, HMGA1 protein was found to bind to the igf-ir promoter. Moreover, HMGA1 was found to associate with both p53 and Sp1, two major regulators of igf-ir gene transcription and to antagonise the p53 inhibitory activity while enhancing the Sp1 stimulatory activity. Our data indicate, therefore, that HMGA1 protein is a positive regulator of IGF-IR expression and that HMGA1 overexpression may contribute to IGF-IR dysregulation in cancer cells.

Summary: Retinoblastoma (RB) is the commonest primaryintraocular tumor in children. Overexpression of the high mobilitygroup (HMG) A2 protein has been observed in a variety ofmalignant tumors and often correlates with poor prognosis. Westudied the expression of HMGA2 in primary tumor samples andcorrelated with clinicopathologic features such as invasion,differentiation, and laterality of the tumors. Among 64 tumors,there were 29 tumors with invasion of the optic nerve, choroid,and/or orbit and 35 tumors without invasion. HMGA2 immuno-reactivity was evaluated on archival paraffin sections and theresults confirmed by Western blotting on 12 fresh tumor samples.Among 29 tumors with invasion, HMGA2 was strongly positive(++) in 10 tumors, moderately positive (+) in 11 tumors. Among35 tumors without invasion, HMGA2 was strongly positive (++)in 6 tumors, moderately positive (+) in 6 tumors. Tumorswith invasion showed significantly higher expression of HMGA2compared with tumors without invasion (P

Epithelial-mesenchymal transition (EMT) occurs during embryogenesis, carcinoma invasiveness, and metastasis and can be elicited by transforming growth factor-beta (TGF-beta) signaling via intracellular Smad transducers. The molecular mechanisms that control the onset of EMT remain largely unexplored. Transcriptomic analysis revealed that the high mobility group A2 (HMGA2) gene is induced by the Smad pathway during EMT. Endogenous HMGA2 mediates EMT by TGF-beta, whereas ectopic HMGA2 causes irreversible EMT characterized by severe E-cadherin suppression. HMGA2 provides transcriptional input for the expression control of four known regulators of EMT, the zinc-finger proteins Snail and Slug, the basic helix-loop-helix protein Twist, and inhibitor of differentiation 2. We delineate a pathway that links TGF-beta signaling to the control of epithelial differentiation via HMGA2 and a cohort of major regulators of tumor invasiveness and metastasis. This network of signaling/transcription factors that work sequentially to establish EMT suggests that combinatorial detection of these proteins could serve as a new tool for EMT analysis in cancer patients.Epithelial-mesenchymal transition (EMT) occurs during embryogenesis, carcinoma invasiveness, and metastasis and can be elicited by transforming growth factor-beta (TGF-beta) signaling via intracellular Smad transducers. The molecular mechanisms that control the onset of EMT remain largely unexplored. Transcriptomic analysis revealed that the high mobility group A2 (HMGA2) gene is induced by the Smad pathway during EMT. Endogenous HMGA2 mediates EMT by TGF-beta, whereas ectopic HMGA2 causes irreversible EMT characterized by severe E-cadherin suppression. HMGA2 provides transcriptional input for the expression control of four known regulators of EMT, the zinc-finger proteins Snail and Slug, the basic helix-loop-helix protein Twist, and inhibitor of differentiation 2. We delineate a pathway that links TGF-beta signaling to the control of epithelial differentiation via HMGA2 and a cohort of major regulators of tumor invasiveness and metastasis. This network of signaling/transcription factors that work sequentially to establish EMT suggests that combinatorial detection of these proteins could serve as a new tool for EMT analysis in cancer patients.

HMGA2 belongs, together with HMGA1a and HMGA1b, to the HMGA family of nuclear proteins, whose expression has functional implications in the pathogenesis of several human tumors (2, 20, 47, 50). A strong association between the expression of these proteins and the transformed phenotype has been observed since they were first described, in transformed rat thyroid cells (24, 25), and was later found in several human neoplasias (e.g., colon, prostate, cervical, and thyroid carcinomas) (4, 10, 17, 51). Their expression is very low, if not absent, in adult tissues and is restricted to embryogenesis (11, 28, 32, 55). The first evidence of a direct role played by these factors in tumorigenesis came from transfection of an antisense construct for HMGA2 in normal rat thyroid cells that prevented the neoplastic transformation induced by myeloproliferative sarcoma virus and kirsten murine sarcoma virus (7). More recently, the increased expression of all three HMGA family members was shown to lead to transformation with anchorage-independent cell growth (53, 54), and the overexpression of HMGA1 was also shown to promote tumor progression in human breast epithelial cells (41). Of additional interest, rearrangements of the HMGA2 gene, resulting in the loss of the acidic C-terminal tail, have been frequently detected in benign human tumors of mesenchimal origin (50). Indeed, transgenic mice expressing the truncated HMGA2 protein develop tumors such as lipomas and natural killer lymphomas (1, 3, 6, 18), while mice expressing wild-type HMGA2 develop pituitary adenomas (19). Despite this evidence, the molecular events and the precise role played by HMGA2 in cell proliferation and tumorigenesis still need to be defined. HMGA proteins contain about 100 amino acid residues and have three DNA-binding domains which have been named AT hooks because of their ability to interact with the narrow minor groove of AT-rich DNA sequences (42). These nuclear proteins, by binding to DNA and/or to transcription factors, can organize the assembly of nucleoprotein-DNA transcriptional complexes (called enhanceosomes) at the level of enhancers or promoters, resulting in enhancement or repression of transcription. For this reason, they are referred to as architectural transcription factors (42). Since a critical step in the assembly of enhanceosomes is the ability of HMGA to establish protein-protein interactions with other nuclear factors, we used different approaches to search for new HMGA2-interacting proteins and identified the transcription factor p120E4F. p120E4F is a ubiquitously expressed GLI-Kruppel-related mammalian transcription factor which was first identified as a cellular factor binding to regulatory regions of the adenovirus E4 promoter and which is responsible for E4 regulation of expression in the course of adenoviral infection (21). p120E4F is likely to play a key role in mammalian cell cycle control. In fact, ectopic expression of p120E4F leads to growth suppression, an effect which is mediated by the interaction with the tumor suppressors pRB (15), p14ARF (43), and p53 (46). Moreover, overexpression of p120E4F in NIH 3T3 cells inhibits progression from G1 to S phase by a mechanism that involves the repression of cyclin A (16). This effect is mediated by p120E4F binding to a cyclic AMP-responsive element (CRE) which is required for full transcriptional activation of cyclin A gene (5, 13). The CRE site is also recognized by CREB and ATF family members, raising the question of how the access of these factors to DNA is modulated. Here we report that HMGA2 associates in vitro and in vivo with p120E4F. Ectopic expression of HMGA2 activates the cyclin A promoter, counteracting the repressing activity of p120E4F exerted through the CRE element. Moreover, we show that HMGA2 is able to induce the expression of the endogenous cyclin A gene. Finally, chromatin immunoprecipitation experiments indicate that association of HMGA2 with the promoter is regulated during the cell cycle, positively correlating with transcriptional activation. The identification of cyclin A, a key factor in cell cycle control, as a functional cellular target for HMGA2 provides for the first time a mechanism that might reveal the oncogenic functions of this factor.

HMGA2 is an architectural nuclear factor which plays an important role in development and tumorigenesis, but mechanisms regulating its expression are largely unknown. The proximal promoters of the mouse and human genes coding for HMGA2 contain a conserved polypyrimidine/polypurine (ppyr/ppur) element which constitutes a multiple binding site for Sp1 and Sp3 transcription factors. In the present study we report that this region can adopt a single-stranded DNA conformation, as demonstrated in vitro by S1 nuclease sensitivity on supercoiled plasmids, indicative of an intramolecular triple-helical H-DNA structure. Moreover, we find that PTB (polypyrimidine tract binding protein), a member of the hnRNP family, binds the pyrimidine strand of Hmga2 as well as similar ppyr/ppur elements of the c-Ki-ras (R.Y) and c-myc P1 promoters. Transfection experiments indicate that non-B-DNA conformers of the ppyr/ppur tract of the Hmga2 promoter contribute to positive transcriptional activity. We propose a transcriptional mechanism, acting on the Hmga2 non-B-DNA structure and functioning through interconversion between double-stranded and single-stranded DNA, that seems to be adopted by an increasing number of genes, mainly growth-related.

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.

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.

SUMMARY 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 II � . 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. (J Histochem Cytochem 46: 863–864, 1998)

The effect of triplex-forming oligonucleotides (TFO) on the transcription activity of T7 RNA polymerase has been investigated by an in vitro assay. The TFOs, either containing only phosphate (PO2) or phosphate and phosphorothioate (POS) internucleotide linkages, were targeted to a 30-bp homopurine. homopyrimidine (R.Y) site cloned in plasmid Bluescript KS+ about four helical turns downstream from the T7 RNA promoter. Band-shift and ultraviolet absorption melting experiments showed that the designed pyrimidine PO2 and POS TFOs form stable triple-helical complexes with the R.Y target duplex (the delta GTFO values of triplex formation vary from -42 to -63 kJ/mol). The triple-helical complexes resulting from POS oligonucleotides were less stable (by 4-12 kJ/mol) than those obtained with PO2 analogues, the magnitude of destabilization being dependent on the number of POS groups present in the third strand. The designed TFOs were shown to efficiently repress bacteriophage T7 RNA polymerase transcription under different experimental conditions. The repression depended on pH, TFO concentration and temperature. When the TFO/template ratio was fixed to 100, a strong repressive effect was observed with normal and phosphorothioate pyrimidine TFOs, also under physiological conditions. In contrast, a purine-rich oligonucleotide containing 44% of guanine residues promoted only a weak transcription inhibition, even at a TFO/template ratio as high as 750. Both PO2- and POS-containing pyrimidine TFOs produced their strong repressive effect on T7 RNA polymerase transcription even when they were added to the reaction mixture simultaneously with the polymerase. A mechanism of transcription repression is discussed. The data reported in this paper are useful for designing oligonucleotides acting as artificial repressors in the antigene strategy and indicate that the R.Y target need not to be precisely confined to the promoter.

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.