Massimo P. Crippa

Incorporation of chromosomal proteins HMG-14/HMG-17 into nascent nucleosomes induces an extended chromatin conformation and enhances the utilization of active transcription complexes.

The role of chromosomal proteins HMG‐14 and HMG‐17 in the generation of transcriptionally active chromatin was studied in a Xenopus laevis egg extract which supports complementary DNA strand synthesis and chromatin assembly. Chromosomal proteins HMG‐14/HMG‐17 enhanced transcription from a chromatin template carrying a 5S rRNA gene, but not from a DNA template. The transcriptional potential of chromatin was enhanced only when these proteins were incorporated into the template during, but not after, chromatin assembly. HMG‐14 and HMG‐17 stimulate transcription by increasing the activity, and not the number, of transcribed templates. They unfold the chromatin template without affecting the nucleosomal repeat or decreasing the content of histone B4. We suggest that HMG‐14/HMG‐17 enhance transcription by inducing an extended conformation in the chromatin fiber, perhaps due to interactions with histone tails in nucleosomes. By disrupting the higher order chromatin structure HMG‐14/HMG‐17 increase the accessibility of target sequences to components of the transcriptional apparatus.

Poised Transcription Factories Prime Silent uPA Gene Prior to Activation

The association of poised genes with transcription factories may contribute to rapid transcriptional activation in response to stimuli and to silencing when genes are located at the interior of their chromosome territories.

Deposition of chromosomal protein HMG-17 during replication affects the nucleosomal ladder and transcriptional potential of nascent chromatin

A cell‐free system from Xenopus eggs was used to study the role of chromosomal protein HMG‐17 in the generation of the chromatin structure of transcriptionally active genes. Addition of HMG‐17 protein to the extracts, which do not contain structural homologs of the HMG‐14/−17 protein family, indicates the protein is incorporated into the nascent template during replication, prior to completion of chromatin assembly. The protein binds to and stabilizes the structure of the nucleosomal core thereby improving the apparent periodicity of the nucleosomal spacing of nascent chromatin. Assembly of HMG‐17 into the nascent chromatin structure significantly increased the transcription potential of the 5S RNA gene and satellite I chromatin. Kinetic studies indicate that the increase in transcriptional potential is observed only when HMG‐17 is incorporated into nucleosomes during chromatin assembly.

Pertussis Toxin B-Oligomer Suppresses IL-6 Induced HIV-1 and Chemokine Expression in Chronically Infected U1 Cells via Inhibition of Activator Protein 1

Pertussis toxin B-oligomer (PTX-B) inhibits HIV replication in T lymphocytes and monocyte-derived macrophages by interfering with multiple steps of the HIV life cycle. PTX-B prevents CCR5-dependent (R5) virus entry in a noncompetitive manner, and it also exerts suppressive effects on both R5- and CXCR4-dependent HIV expression at a less-characterized postentry level. We demonstrate in this study that PTX-B profoundly inhibits HIV expression in chronically infected promonocytic U1 cells stimulated with several cytokines and, particularly, the IL-6-mediated effect, a cytokine that triggers viral production in these cells independently of NF-κB activation. From U1 cells we have subcloned a cell line, named U1-CR1, with increased responsiveness to IL-6. In these cells, PTX-B neither down-regulated the IL-6R nor prevented IL-6 induced signaling in terms of STAT3 phosphorylation and DNA binding. In contrast, PTX-B inhibited AP-1 binding to target DNA and modified its composition with a proportional increases in FosB, Fra2, and ATF2. PTX-B inhibited IL-6-induced HIV-1 long-terminal repeat-driven transcription from A, C, E, and F viral subtypes, which contain functional AP-1 binding sites, but failed to inhibit transcription from subtypes B and D LTR devoid of these sites. In addition, PTX-B inhibited the secretion of IL-6-induced, AP-1-dependent genes, including urokinase-type plasminogen activator, CXCL8/IL-8, and CCL2/monocyte chemotactic protein-1. Thus, PTX-B suppression of IL-6 induced expression of HIV and cellular genes in chronically infected promonocytic cells is strongly correlated to inhibition of AP-1.

Prep1 Directly Regulates the Intrinsic Apoptotic Pathway by Controlling Bcl-XL Levels

ABSTRACT The Prep1 homeodomain transcription factor is essential in embryonic development. Prep1 hypomorphic mutant mouse (Prep1i/i) embryos (embryonic day 9.5) display an increased terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction compared to wild-type (WT) littermates. Prep1i/i mouse embryo fibroblasts (MEFs) show an increased basal level of annexin V binding activity, reduction of the mitochondrial-membrane potential, and increased caspase 9 and 3 activation, indicating increased apoptosis. Prep1i/i MEFs also respond faster than WT MEFs to genotoxic stress, indicating increased activation of the intrinsic apoptotic pathways. We did not observe an increase in p53 or an abnormal p53 response to apoptotic stimuli. However, hypomorphic MEFs have decreased endogenous levels of antiapoptotic Bcl-XL mRNA and protein, and Bcl-x overexpression rescues the defect of Prep1i/i MEFs. Using transient transfections and chromatin immunoprecipitation, we identified the Bcl-x promoter as a novel target of Prep1. Thus, Prep1 directly controls mitochondrial homeostasis (and the apoptotic potential) by modulating Bcl-x gene expression.

Down syndrome fibroblasts and mouse Prep1-overexpressing cells display increased sensitivity to genotoxic stress

PREP1 (PKNOX1) maps in the Down syndrome (DS) critical region of chromosome 21, is overexpressed in some DS tissues and might be involved in the DS phenotype. By using fibroblasts from DS patients and by overexpressing Prep1 in F9 teratocarcinoma and Prep1i/i MEF to single out the role of the protein, we report that excess Prep1 increases the sensitivity of cells to genotoxic stress and the extent of the apoptosis directly correlates with the level of Prep1. The apoptotic response of Prep1-overexpressing cells is mediated by the pro-apoptotic p53 protein that we show is a direct target of Prep1, as its depletion reverts the apoptotic phenotype. The induction of p53 overcomes the anti-apoptotic role of Bcl-XL, previously shown to be also a Prep1 target, the levels of which are increased in Prep1-overexpressing cells as well. Our results provide a rationale for the involvement of PREP1 in the apoptotic phenotype of DS tissues and indicate that differences in Prep1 level can have drastic effects.

A Transcription-dependent Micrococcal Nuclease-resistant Fragment of the Urokinase-type Plasminogen Activator Promoter Interacts with the Enhancer

We show the interaction between the enhancer and the minimal promoter of urokinase-type plasminogen activator gene during active transcription by coupling micrococcal nuclease digestion of cross-linked, sonicated chromatin, and chromatin immunoprecipitation. This approach allowed the precise identification of the interacting genomic fragments, one of which is resistant to micrococcal nuclease cleavage. The interacting fragments form a single transcriptional control unit, as indicated by their common protein content. Furthermore, we show that the enhancer-MP interaction persists during the early stages of transcription and is lost upon α-amanitin treatment, indicating the requirement for active transcription. Our results support a looping model of interaction between the enhancer and the MP of the urokinase-type plasminogen activator gene.

High-mobility group box 1 protein orchestrates responses to tissue damage via inflammation, innate and adaptive immunity, and tissue repair

A single protein, HMGB1, directs the triggering of inflammation, innate and adaptive immune responses, and tissue healing after damage. HMGB1 is the best characterized damage‐associated molecular pattern (DAMP), proteins that are normally inside the cell but are released after cell death, and allow the immune system to distinguish between antigens that are dangerous or not. Notably, cells undergoing severe stress actively secrete HMGB1 via a dedicated secretion pathway: HMGB1 is relocated from the nucleus to the cytoplasm and then to secretory lysosomes or directly to the extracellular space. Extracellular HMGB1 (either released or secreted) triggers inflammation and adaptive immunological responses by switching among multiple oxidation states, which direct the mutually exclusive choices of different binding partners and receptors. Immune cells are first recruited to the damaged tissue and then activated; thereafter, HMGB1 supports tissue repair and healing, by coordinating the switch of macrophages to a tissue‐healing phenotype, activation and proliferation of stem cells, and neoangiogenesis. Inevitably, HMGB1 also orchestrates the support of stressed but illegitimate tissues: tumors. Concomitantly, HMGB1 enhances the immunogenicity of mutated proteins in the tumor (neoantigens), promoting anti‐tumor responses and immunological memory. Tweaking the activities of HMGB1 in inflammation, immune responses and tissue repair could bring large rewards in the therapy of multiple medical conditions, including cancer.

Human malignant mesothelioma is recapitulated in immunocompetent BALB/c mice injected with murine AB cells

Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment.

High resolution in vitro bioluminescence imaging using a multimodal optical system

Bioluminescence in vitro studies are usually performed with dedicated microscopes. In this work, we developed a novel image recovery algorithm and a multimodal system prototype to perform bioluminescence microscopy. We performed a feasibility study using GEANT4 Monte Carlo (MC) simulation of bioluminescent cells acquired at low SNR frames and processed using a Super Resolution Regularization Algorithm (SRRA). The method was also tested using in vitro cell acquisition. The results obtained with MC simulations showed an improvement in the spatial resolution from 90 μ m to 10 μ m and from 110 μ m to 13 μ m for in vitro imaging of mesothelioma cells.