Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary.

BACKGROUND: Ovarian retinoid homeostasis plays an important role in the physiological function of the ovary. Retinol-binding protein 4 (RBP4) acts as the mediator for the systemic and intercellular transport of retinol and is heavily involved in cellular retinol influx, efflux, and exchange. However, the expression patterns and regulatory mechanisms of Rbp4 in the ovary remain unclear. METHODS: The expression pattern of ovarian Rbp4 was examined in immature mice during different developmental stages and in adult mice during different stages of the estrous cycle. The potential regulation and mechanisms of ovarian Rbp4 expression by estrogen and related gonadotropins in mouse ovaries were also investigated. RESULTS: The present study demonstrated that the ovarian expression of Rbp4 remained constant before puberty and increased significantly in the peripubertal period. In adult female mice, the expression of Rbp4 increased at proestrus and peaked at estrus at both the mRNA and protein levels. The protein distribution of RBP4 was mainly localized in the granulosa cell and theca cell layer in follicles. In addition, the expression of Rbp4 was significantly induced by follicle-stimulating hormone (FSH) or FSH + luteinizing hormone (LH) in combination in immature mouse (3 weeks old) ovaries in vivo and in granulosa cells cultured in vitro, both at the mRNA and protein levels. In contrast, treatment with LH or 17ß-estradiol did not exhibit any observable effects on ovarian Rbp4 expression. Transcription factors high-mobility group AT-hook 1 (HMGA1), steroidogenic factor 1 (SF-1), and liver receptor homolog 1 (LRH-1) (which have been previously shown to be involved in activation of Rbp4 transcription), also responded to FSH stimulation. In addition, H-89, an inhibitor of protein kinase A (PKA), and the depletion of HMGA1, SF-1, and LRH-1 by small interfering RNAs (siRNAs), resulted in a dramatic loss of the induction of Rbp4 expression by FSH at both the mRNA and protein levels. CONCLUSIONS: These data indicate that the dynamic expression of Rbp4 is mainly regulated by FSH through the cAMP-PKA pathway, involving transcriptional factors HMGA1, SF-1, and LRH-1, in the mouse ovary during different stages of development and the estrous cycle.

Potential Role for a ß-Catenin Coactivator (High-Mobility Group AT-Hook 1 Protein) during the Latency-Reactivation Cycle of Bovine Herpesvirus 1.

The latency-related (LR) RNA encoded by bovine herpesvirus 1 (BoHV-1) is abundantly expressed in latently infected sensory neurons. Although the LR gene encodes several products, ORF2 appears to mediate important steps during the latency-reactivation cycle because a mutant virus containing stop codons at the amino terminus of ORF2 does not reactivate from latency in calves. We recently found that the Wnt/ß-catenin signaling pathway is regulated during the BoHV-1 latency-reactivation cycle (Y. Liu, M. Hancock, A. Workman, A. Doster, and C. Jones, J Virol 90:3148-3159, 2016). In the present study, a ß-catenin coactivator, high-mobility group AT-hook 1 protein (HMGA1), was detected in significantly more neurons in the trigeminal ganglia of latently infected calves than in those of uninfected calves. Consequently, we hypothesized that HMGA1 cooperates with ORF2 and ß-catenin to maintain latency. In support of this hypothesis, coimmunoprecipitation studies demonstrated that ORF2 stably interacts with a complex containing ß-catenin and/or HMGA1 in transfected mouse neuroblastoma (Neuro-2A) cells. Confocal microscopy provided evidence that ORF2 was relocalized by HMGA1 and ß-catenin in Neuro-2A cells. ORF2 consistently enhanced the ability of HMGA1 to stimulate ß-catenin-dependent transcription, suggesting that interactions between ORF2 and a complex containing ß-catenin and HMGA1 have functional significance. An ORF2 stop codon mutant, an ORF2 nuclear localization mutant, or a mutant lacking the 5 protein kinase A or C phosphorylation sites interfered with its ability to stimulate ß-catenin-dependent transcription. Since the canonical Wnt/ß-catenin signaling pathway promotes neurogenesis (synapse formation and remodeling) and inhibits neurodegeneration, interactions between ORF2, HMGA1, and ß-catenin may be important for certain aspects of the latency-reactivation cycle.IMPORTANCE The lifelong latency of bovine herpesvirus 1 (BoHV-1) requires that significant numbers of infected sensory neurons survive infection and maintain normal functions. Consequently, we hypothesize that viral products expressed during latency cooperate with neuronal factors to maintain latency. Our studies revealed that a ß-catenin coactivator, high-mobility group AT-hook 1 protein (HMGA1), was readily detected in a subset of trigeminal ganglion neurons in latently infected calves but not in uninfected calves. A viral protein (ORF2) expressed in latently infected neurons interacted with ß-catenin and HMGA1 in transfected cells, which resulted in the nuclear localization of ß-catenin. This interaction correlated with the ability of ORF2 to stimulate the coactivator functions of HMGA1. These findings are significant because the canonical Wnt/ß-catenin signaling pathway promotes neurogenesis and inhibits neurodegeneration.

The Epstein-Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins.

Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate-dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation.

The impairment of the High Mobility Group A (HMGA) protein function contributes to the anticancer activity of trabectedin.

Trabectedin (Ecteinascidin-743 or ET-743) is a novel antitumour agent of marine origin with potent antitumour activity both in vitro and in vivo. It interacts with the minor groove of DNA, interfering with transcriptional activity and DNA repair pathways. Here, we report a novel mechanism by which trabectedin exerts its cytotoxic effects on carcinoma cells. It is based on its ability to impair the function of the High-Mobility Group A (HMGA) proteins. These proteins have a key role in cell transformation, and their overexpression is a common feature of human malignant neoplasias, representing a poor prognostic index often correlated to anti-cancer drug resistance. They bind the minor groove of DNA, alter chromatin structure and, thus, regulate the transcription of several genes by enhancing or suppressing the activity of transcription factors. We first report that trabectedin has a higher cytotoxic effect on thyroid and colon carcinoma cells expressing abundant levels of HMGAs in comparison with cells not expressing them. Then, we have shown that trabectedin treatment displaces HMGA proteins from the HMGA-responsive promoters, including ATM promoter, impairing their transcriptional activity. Finally, we report a synergism between Ionising Radiations and trabectedin treatment restricted to the HMGA-overexpressing cancer cells. This result might have important clinical implications since it would suggest the use of trabectedin for the treatment of neoplasias expressing abundant HMGA levels that are frequently associated to chemoresistance and poor prognosis.

HMGA regulates the global chromatin state and neurogenic potential in neocortical precursor cells.

Neural precursor cells (NPCs) in the mouse neocortex generate various neuronal and glial cell types in a developmental stage­dependent manner. Most NPCs lose their neurogenic potential during development, although the underlying mechanisms of this process are not fully understood. We found that the chromatin of mouse NPCs gradually becomes more condensed and less dynamic on a global scale during neocortical development. Furthermore, we found high mobility group A (HMGA) proteins to be essential for the open chromatin state of NPCs at early developmental stages. Knockdown of HMGA proteins in early-stage NPCs reduced their neurogenic potential. Conversely, overexpression of HMGA proteins conferred neurogenic potential on late-stage NPCs, an effect that was antagonized by coexpression of a histone H1 mutant that inhibits chromatin opening. Thus, HMGA proteins contribute to the neurogenic potential of NPCs in the early stages of neocortical development, possibly through induction of an open chromatin state.

Core promoter-selective function of HMGA1 and Mediator in Initiator-dependent transcription.

The factors and mechanisms underlying the differential activity and regulation of eukaryotic RNA polymerase II on different types of core promoters have remained elusive. Here we show that the architectural factor HMGA1 and the Mediator coregulator complex cooperate to enhance basal transcription from core promoters containing both a TATA box and an Initiator (INR) element but not from "TATA-only" core promoters. INR-dependent activation by HMGA1 and Mediator requires the TATA-binding protein (TBP)-associated factors (TAFs) within the TFIID complex and counteracts negative regulators of TBP/TATA-dependent transcription such as NC2 and Topoisomerase I. HMGA1 interacts with TFIID and Mediator and is required for the synergy of TATA and INR elements in mammalian cells. Accordingly, natural HMGA1-activated genes in embryonic stem cells tend to have both TATA and INR elements in a synergistic configuration. Our results suggest a core promoter-specific regulation of Mediator and the basal transcription machinery by HMGA1.

HMGA proteins promote ATM expression and enhance cancer cell resistance to genotoxic agents.

DNA-damaging therapies represent a keystone in cancer treatment. Unfortunately, many tumors often relapse because of a group of cancer cells, which are resistant to conventional therapies. High-mobility group A (HMGA) proteins has a key role in cell transformation, and their overexpression is a common feature of human malignant neoplasias, representing a poor prognostic index often correlated to anti-cancer drug resistance. Our previous results demonstrated that HMGA1 is a substrate of ataxia-telangiectasia mutated (ATM), the main cellular sensor of genotoxic stress. Here we also report thatHMGA2, the other member of the HMGA family, is a novel substrate of ATM. Interestingly, we found that HMGA proteins positively regulate ATM gene expression. Moreover, induction of ATM kinase activity by DNA-damaging agents enhances HMGA-dependent transcriptional activation of ATM promoter, suggesting that ATM expression is modulated by a DNA-damage- and HMGA-dependent positive feedback loop. Finally, inhibition of HMGA expression in mouse embryonic fibroblasts and in cancer cells strongly reduces ATM protein levels, impairing the cellular DNA-damage response and enhancing the sensitivity to DNA-damaging agents. These findings indicate this novel HMGA-ATM pathway as a new potential target to improve the effectiveness of conventional anti-neoplastic treatments on the genotoxic-drug resistant cancer cells.

The HMGA proteins: a myriad of functions (Review).

The 'high mobility group' HMGA protein family consists of four members: HMGA1a, HMGA1b and HMGA1c, which result from translation of alternative spliced forms of one gene and HMGA2, which is encoded for by another gene. HMGA proteins are characterized by three DNA-binding domains, called AT-hooks, and an acidic carboxy-terminal tail. HMGA proteins are architectural transcription factors that both positively and negatively regulate the transcription of a variety of genes. They do not display direct transcriptional activation capacity, but regulate gene expression by changing the DNA conformation by binding to AT-rich regions in the DNA and/or direct interaction with several transcription factors. In this way, they influence a diverse array of normal biological processes including cell growth, proliferation, differentiation and death. Both HMGA1 and HMGA2 are hardly detectable in normal adult tissue but are abundantly and ubiquitously expressed during embryonic development. In malignant epithelial tumors as well as in leukemia, however, expression of HMGA1 is again strongly elevated to embryonic levels thus leading to ectopic expression of (fetal) target genes. HMGA2 overexpression also has a causal role in inducing neoplasia. Besides overexpression of full length HMGA proteins in different tumors, the HMGA genes are often involved in chromosomal rearrangements. Such translocations are mostly detected in benign tumors of mesenchymal origin and are believed to be one of the most common chromosomal rearrangements in human neoplasia. To provide clarity in the abundance of articles on this topic, this review gives a general overview of the nuclear functions and regulation of the HMGA genes and corresponding proteins.

The Mia/Cd-rap gene expression is downregulated by the high-mobility group A proteins in mouse pituitary adenomas.

The high-mobility group A (HMGA) family of proteins orchestrates the assembly of nucleoprotein structures playing important roles in gene transcription, recombination, and chromatin structure through a complex network of protein-DNA and protein-protein interactions. Recently, we have generated transgenic mice carrying wild type or truncated HMGA2 genes under the transcriptional control of the cytomegalovirus promoter. These mice developed pituitary adenomas secreting prolactin and GH mainly due to an increased E2F1 activity, directly consequent to the HMGA2 overexpression. To identify other genes involved in the process of pituitary tumorigenesis induced by the HMGA2 gene, in this study we have analyzed the gene expression profile of three HMGA2-pituitary adenomas in comparison with a pool of ten normal pituitary glands from control mice, using the Affymetrix MG MU11K oligonucleotide array representing approximately 13,000 unique genes. We have identified 82 transcripts that increased and 72 transcripts that decreased at least four-fold in all the mice pituitary adenomas analyzed compared with normal pituitary glands. Among these genes, we focused our attention on the Mia/Cd-rap gene, whose expression was essentially suppressed in all of the pituitary adenomas tested by the microarray. We demonstrated that the HMGA proteins directly bind to the promoter of the Mia/Cd-rap gene and are able to downregulate its expression. In order to understand a possible role of Mia/Cd-rap in pituitary cell growth, we performed a colony assay in GH3 and GH4 cells. Interestingly, Mia/Cd-rap expression inhibits their proliferation, suggesting a potential tumor suppressor role of Mia/Cd-rap in pituitary cells.

HMG chromosomal proteins in development and disease.

The high mobility group (HMG) proteins are a superfamily of abundant and ubiquitous nuclear proteins that bind to DNA and nucleosomes and induce structural changes in the chromatin fiber. They are important in chromatin dynamics and influence DNA processing in the context of chromatin. Results emerging from studies of human disease, genetically modified mice and cells with altered HMG expression indicate that the expression of the HMG proteins is developmentally regulated and that changes in HMG protein levels alter the cellular phenotype and can lead to developmental abnormalities and disease. Here, we focus on the biological function of HMG proteins and highlight their possible roles in cellular differentiation and in the etiology of various diseases.

Transactivation functions of the tumor-specific HMGA2/LPP fusion protein are augmented by wild-type HMGA2.

The gene encoding the architectural transcription factor HMGA2 is frequently rearranged in several benign tumors of mesenchymal origin. The lipoma preferred partner (LPP) gene is the most frequent translocation partner of HMGA2 in a subgroup of lipomas, which are benign tumors of adipose tissue. In these lipomas, HMGA2/LPP fusion transcripts are expressed, which encode for the three AT-hooks of HMGA2 followed by the two most carboxyl-terminal LIM domains (protein-protein interaction domains) of LPP. Identical fusion transcripts are also expressed in other benign mesenchymal tumors. Previous studies revealed that the LIM domains of LPP have transcriptional activation capacity in GAL4-based luciferase reporter assays. Here, we show that the HMGA2/LPP fusion protein retains the transactivation functions of the LPP LIM domains and thus functions as transcription factor. The HMGA2/LPP fusion protein activates transcription from the well-characterized PRDII element, which is a part of the IFN-beta enhancer and which is known to bind to HMGA2. We also show that HMGA2/LPP activates transcription from the BAT-1 element of the rhodopsin promoter, a HMGA1-binding element. HMGA1 is a closely related family member of HMGA2. Finally, in a number of lipomas, HMGA2/LPP and HMGA2 are coexpressed, and HMGA2 augments the transactivation functions of HMGA2/LPP. These results support the concept that the transactivation functions of the novel HMGA2/LPP transcription factor contribute to lipomagenesis.

IFN-gamma gene expression is controlled by the architectural transcription factor HMGA1.

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.

Loss of Hmga1 gene function affects embryonic stem cell lympho-hematopoietic differentiation.

By interacting with transcription machinery, high-mobility group A 1 (HMGA1) proteins alter the chromatin structure and thereby regulate the transcriptional activity of several genes. To assess their role in development, we studied the in vitro differentiation of embryonic stem (ES) cells that bear one or both disrupted Hmga1 alleles. Here, we report that Hmga1 null ES cells generate fewer T-cell precursors than do wild-type ES cells. Indeed, they preferentially differentiate to B cells, probably consequent to decreased interleukin 2 expression and increased interleukin 6 expression. Moreover, a lack of HMGA1 expression induces changes in hemopoietic differentiation, i.e., a reduced monocyte/macrophage population and an increase in megakaryocyte precursor numbers, erythropoiesis, and globin gene expression. Re-expression of the Hmga1 gene in Hmga1 null ES cells restores the wild-type phenotype. The effect on megakaryocyte/erythrocyte lineages seems, at least in part, mediated by the GATA-1 transcription factor, a key regulator of red blood cell differentiation. In fact, we found that Hmga1-/- ES cells overexpress GATA-1 and that HMGA1 proteins directly control GATA-1 transcription. Taken together, these data indicate that HMGA1 proteins play a prime role in lymphohematopoietic differentiation.