First body of evidence suggesting a role of a tankyrase-binding motif (TBM) of vinculin (VCL) in epithelial cells

Background: Adherens junctions (AJ) are involved in cancer, infections and neurodegeneration. Still, their composition has not been completely disclosed. Poly(ADP-ribose) polymerases (PARPs) catalyze the synthesis of poly(ADP-ribose) (PAR) as a posttranslational modification. Four PARPs synthesize PAR, namely PARP-1/2 and Tankyrase-1/2 (TNKS). In the epithelial belt, AJ are accompanied by a PAR belt and a subcortical F-actin ring. F-actin depolymerization alters the AJ and PAR belts while PARP inhibitors prevent the assembly of the AJ belt and cortical actin. We wondered which PARP synthesizes the belt and which is the PARylation target protein. Vinculin (VCL) participates in the anchorage of F-actin to the AJ, regulating its functions, and colocalized with the PAR belt. TNKS has been formerly involved in the assembly of epithelial cell junctions. Hypothesis: TNKS poly(ADP-ribosylates) (PARylates) epithelial belt VCL, affecting its functions in AJ, including cell shape maintenance. Materials and Methods: Tankyrase-binding motif (TBM) sequences in hVCL gene were identified and VCL sequences from various vertebrates, Drosophila melanogaster and Caenorhabditis elegans were aligned and compared. Plasma membrane-associated PAR was tested by immunocytofluorescence (ICF) and subcellular fractionation in Vero cells while TNKS role in this structure and cell junction assembly was evaluated using specific inhibitors. The identity of the PARylated proteins was tested by affinity precipitation with PAR-binding reagent followed by western blots. Finally, MCF-7 human breast cancer epithelial cells were subjected to transfection with Tol2-plasmids, carrying a dicistronic expression sequence including Gallus gallus wt VCL (Tol-2-GgVCL), or the same VCL gene with a point mutation in TBM-II (Tol2-GgVCL/*TBM) under the control of a β-actin promoter, plus green fluorescent protein following an internal ribosome entry site (IRES-GFP) to allow the identification of transfected cells without modifying the transfected protein of interest. Results and discussion: In this work, some of the hypothesis predictions have been tested. We have demonstrated that: (1) VCL TBMs were conserved in vertebrate evolution while absent in C. elegans; (2) TNKS inhibitors disrupted the PAR belt synthesis, while PAR and an endogenous TNKS pool were associated to the plasma membrane; (3) a VCL pool was covalently PARylated; (4) transfection of MCF-7 cells leading to overexpression of Gg-VCL/*TBM induced mesenchymal-like cell shape changes. This last point deserves further investigation, bypassing the limits of our transient transfection and overexpression system. In fact, a 5th testable prediction would be that a single point mutation in VCL TBM-II under endogenous expression control would induce an epithelial to mesenchymal transition (EMT). To check this, a CRISPR/Cas9 substitution approach followed by migration, invasion, gene expression and chemo-resistance assays should be performed.Fil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Valsecchi, Wanda Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Fernandez Villamil, Silvia Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Lafon Hughes, Laura I.. Universidad de la Republica. Centro Universitario del Litoral Norte.; Urugua

PARP Inhibitor Olaparib Causes No Potentiation of the Bleomycin Effect in VERO Cells, Even in the Presence of Pooled ATM, DNA-PK, and LigIV Inhibitors

Poly(ADP-ribosyl)polymerase (PARP) synthesizes poly(ADP-ribose) (PAR), which is anchored to proteins. PAR facilitates multiprotein complexes’ assembly. Nuclear PAR affects chromatin’s structure and functions, including transcriptional regulation. In response to stress, particularly genotoxic stress, PARP activation facilitates DNA damage repair. The PARP inhibitor Olaparib (OLA) displays synthetic lethality with mutated homologous recombination proteins (BRCA-1/2), base excision repair proteins (XRCC1, Polβ), and canonical nonhomologous end joining (LigIV). However, the limits of synthetic lethality are not clear. On one hand, it is unknown whether any limiting factor of homologous recombination can be a synthetic PARP lethality partner. On the other hand, some BRCA-mutated patients are not responsive to OLA for still unknown reasons. In an effort to help delineate the boundaries of synthetic lethality, we have induced DNA damage in VERO cells with the radiomimetic chemotherapeutic agent bleomycin (BLEO). A VERO subpopulation was resistant to BLEO, BLEO + OLA, and BLEO + OLA + ATM inhibitor KU55933 + DNA-PK inhibitor KU-0060648 + LigIV inhibitor SCR7 pyrazine. Regarding the mechanism(s) behind the resistance and lack of synthetic lethality, some hypotheses have been discarded and alternative hypotheses are suggested.Fil: Perini, Valentina. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Schacke, Michelle. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Liddle, Pablo. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Keszenman, Deborah J.. Universidad de la Republica. Centro Universitario del Litoral Norte.; UruguayFil: Lafon Hughes, Laura. Instituto de Investigaciones Biológicas "Clemente Estable"; Urugua

Is eastern Mongolia drying? A long-term perspective of a multidecadal trend

Temperatures in semiarid Mongolia have rapidly risen over the past few decades, and increases in drought, urban development, mining, and agriculture have intensified demands on limited water resources. Understanding long-term streamflow variation is critical for Mongolia, particularly if alterations in streamflow are being considered and because of the potential negative impacts of drought on the animal agriculture sector. Here, we present a temporally and spatially improved streamflow reconstruction for the Kherlen River. We have added 11 new records in comparison with two in the original 2001 reconstruction. This new reconstruction extends from 1630 to 2007 and places the most recent droughts in a multicentennial perspective. We find that variations in streamflow have been much greater in the past than in the original study. There was higher variability in the mid to late 1700s, ranging from severe and extended drought conditions from 1723 to 1739 and again in 1768–1778 to two decadal length episodes of very wet conditions in the mid 1700s and late 1700s. Reduced amplitude is seen in the mid-1800s, and several pluvial events are reconstructed for the 1900s. Although recent droughts are severe and disturbing economic and ecological systems in Mongolia and it appears that eastern Mongolia is drying, the drying trend since the late 1900s might in fact be accentuated by a change from a particularly wet era in Mongolia. The recent drought might be a return to more characteristic hydroclimatic conditions of the past four centuries in Mongolia

Novel Cinnamyl Hydroxyamides and 2-Aminoanilides as Histone Deacetylase Inhibitors: Apoptotic Induction and Cytodifferentiation Activity

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Contributions of viral oncogenes of HPV‑18 and hypoxia to oxidative stress and genetic damage in human keratinocytes

Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the evelopment of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression.PEDECIBA. ANII

Genome-Wide Integration on Transcription Factors, Histone Acetylation and Gene Expression Reveals Genes Co-Regulated by Histone Modification Patterns

N-terminal tails of H2A, H2B, H3 and H4 histone families are subjected to posttranslational modifications that take part in transcriptional regulation mechanisms, such as transcription factor binding and gene expression. Regulation mechanisms under control of histone modification are important but remain largely unclear, despite of emerging datasets for comprehensive analysis of histone modification. In this paper, we focus on what we call genetic harmonious units (GHUs), which are co-occurring patterns among transcription factor binding, gene expression and histone modification. We present the first genome-wide approach that captures GHUs by combining ChIP-chip with microarray datasets from Saccharomyces cerevisiae. Our approach employs noise-robust soft clustering to select patterns which share the same preferences in transcription factor-binding, histone modification and gene expression, which are all currently implied to be closely correlated. The detected patterns are a well-studied acetylation of lysine 16 of H4 in glucose depletion as well as co-acetylation of five lysine residues of H3 with H4 Lys12 and H2A Lys7 responsible for ribosome biogenesis. Furthermore, our method further suggested the recognition of acetylated H4 Lys16 being crucial to histone acetyltransferase ESA1, whose essential role is still under controversy, from a microarray dataset on ESA1 and its bypass suppressor mutants. These results demonstrate that our approach allows us to provide clearer principles behind gene regulation mechanisms under histone modifications and detect GHUs further by applying to other microarray and ChIP-chip datasets. The source code of our method, which was implemented in MATLAB (http://www.mathworks.com/), is available from the supporting page for this paper: http://www.bic.kyoto-u.ac.jp/pathway/natsume/hm_detector.htm

Figure S2: Controls proving the poly-ADP-ribose (PAR) identity in mice sciatic nerves

Background Poly-ADP-ribose (PAR) is a polymer synthesized by poly-ADP-ribose polymerases (PARPs) as a postranslational protein modification and catabolized mainly by poly-ADP-ribose glycohydrolase (PARG). In spite of the existence of cytoplasmic PARPs and PARG, research has been focused on nuclear PARPs and PAR, demonstrating roles in the maintenance of chromatin architecture and the participation in DNA damage responses and transcriptional regulation. We have recently detected non-nuclear PAR structurally and functionally associated to the E-cadherin rich zonula adherens and the actin cytoskeleton of VERO epithelial cells. Myelinating Schwann cells (SC) are stabilized by E-cadherin rich autotypic adherens junctions (AJ). We wondered whether PAR would map to these regions. Besides, we have demonstrated an altered microfilament pattern in peripheral nerves of Trembler-J (Tr-J) model of CMT1-E. We hypothesized that cytoplasmic PAR would accompany such modified F-actin pattern. Methods Wild-type (WT) and Tr-J mice sciatic nerves cryosections were subjected to immunohistofluorescence with anti-PAR antibodies (including antibody validation), F-actin detection with a phalloidin probe and DAPI/DNA counterstaining. Confocal image stacks were subjected to a colocalization highlighter and to semi-quantitative image analysis. Results We have shown for the first time the presence of PAR in sciatic nerves. Cytoplasmic PAR colocalized with F-actin at non-compact myelin regions in WT nerves. Moreover, in Tr-J, cytoplasmic PAR was augmented in close correlation with actin. In addition, nuclear PAR was detected in WT SC and was moderately increased in Tr-J SC. Discussion The presence of PAR associated to non-compact myelin regions (which constitute E-cadherin rich autotypic AJ/actin anchorage regions) and the co-alterations experienced by PAR and the actin cytoskeleton in epithelium and nerves, suggest that PAR may be a constitutive component of AJ/actin anchorage regions. Is PAR stabilizing the AJ-actin complexes? This question has strong implications in structural cell biology and cell signaling networks. Moreover, if PAR played a stabilizing role, such stabilization could participate in the physiological control of axonal branching. PARP and PAR alterations exist in several neurodegenerative pathologies including Alzheimer’s, Parkinson’s and Hungtington’s diseases. Conversely, PARP inhibition decreases PAR and promotes neurite outgrowth in cortical neurons in vitro. Coherently, the PARP inhibitor XAV939 improves myelination in vitro, ex vivo and in vivo. Until now such results have been interpreted in terms of nuclear PARP activity. Our results indicate for the first time the presence of PARylation in peripheral nerve fibers, in a healthy environment. Besides, we have evidenced a PARylation increase in Tr-J, suggesting that the involvement of cytoplasmic PARPs and PARylation in normal and neurodegenerative conditions should be re-evaluated