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

Editorial: Hormone Receptors and Breast Cancer

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

A novel HMGA1-CCNE2-YAP axis regulates breast cancer aggressiveness

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

Transforming growth factor-β employs HMGA2 to elicit epithelial–mesenchymal transition

Epithelial–mesenchymal transition (EMT) occurs during embryogenesis, carcinoma invasiveness, and metastasis and can be elicited by transforming growth factor-β (TGF-β) 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-β, 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-β 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

High Mobility Group I Proteins Interfere with the Homeodomains Binding to DNA

Homeodomains (HDs) constitute the DNA binding domain of several transcription factors that control cell differentiation and development in a wide variety of organisms. Most HDs recognize sequences that contain a 5'-TAAT-3' core motif. However, the DNA binding specificity of HD-containing proteins does not solely determine their biological effects, and other molecular mechanisms should be responsible for their ultimate functional activity. Interference by other factors in the HD/DNA interaction could be one of the processes by which HD-containing proteins achieve the functional complexity required for their effects on the expression of target genes. Using gel-retardation assay, we demonstrate that two members of the high mobility group I (HMGI) family of nuclear proteins (HMGI-C and HMGY) can bind to a subset of HD target sequences and inhibit HDs from binding to the same sequences. The inhibition of the HD/DNA interaction occurs while incubating HMGI-C with DNA either before or after the addition of the HD. The reduced half-life of the HD.DNA complex in the presence of HMGI-C, and the shift observed in the CD spectra recorded upon HMGI-C binding to DNA, strongly suggest that structural modifications of the DNA are responsible for the inhibition of the HD.DNA complex formation. Moreover, by co-transfection experiments we provide evidence that this inhibition can occur also in vivo. The data reported here would suggest that HMGI proteins may be potential regulators of the function of HD-containing proteins and that they are able to interfere with the access of the HD to their target genes

Mass spectrometric analysis of the HMGY protein from Lewis lung carcinoma. Identification of phosphorylation sites.

The primary structure of the Lewis lung carcinoma protein HMGY belonging to the nuclear group of proteins HMGI (high mobility group I) was determined using electrospray and fast atom bombardment mass spectrometry. It was demonstrated that the sequence of the tumor protein corresponds to the amino acid sequence derived from the cDNA from cultured cells and that the N-terminal serine residue is N-acetylated. Moreover, the two high performance liquid chromatography-purified forms Y1 and Y2 of the protein HMGY were shown to differ at the level of serine phosphorylation, since they contain three phosphate and two phosphate groups, respectively, in the C-terminal region. No other modification was detected in the remaining part of the molecule

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

The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function

High Mobility Group A (HMGA) is a family of architectural nuclear factors which play an important role in neoplastic transformation. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes including transcription. HMGA localization is exclusively nuclear but, to date, the mechanism of nuclear import for these proteins remains unknown. Here, we report the identification and characterization of a nuclear localization signal (NLS) for HMGA2, a member of the HMGA family. The NLS overlaps with the second of the three AT-hooks, the DNA-binding domains characteristic for this group of proteins. The functionality of this NLS was demonstrated by its ability to target a heterologous β-galactosidase/green fluorescent protein fusion protein to the nucleus. Mutations to alanine of basic residues within the second AT-hook resulted in inhibition of HMGA2 nuclear localization and impairment of its function in activating the cyclin A promoter. In addition, HMGA2 was shown to directly interact with the nuclear import receptor importin-α2 via the second AT-hook. HMGA proteins are overexpressed and rearranged in a variety of tumors; our findings can thus help elucidating their role in neoplastic transformation

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