TRF2 controls telomeric nucleosome organization in a cell cycle phase-dependent manner

Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization

Identification de la séquence de fixation à l'ADN et de recherche de gènes cibles du produit du gène suppresseur de tumeurs HIC1

LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

The Human Candidate Tumor Suppressor Gene HIC1 Recruits CtBP through a Degenerate GLDLSKK Motif

HIC1 (hypermethylated in cancer) and its close relative HRG22 (HIC1-related gene on chromosome 22) encode transcriptional repressors with five C(2)H(2) zinc fingers and an N-terminal BTB/POZ autonomous transcriptional repression domain that is unable to recruit histone deacetylases (HDACs). Alignment of the HIC1 and HRG22 proteins from various species highlighted a perfectly conserved GLDLSKK/R motif highly related to the consensus CtBP interaction motif (PXDLSXK/R), except for the replacement of the virtually invariant proline by a glycine. HIC1 strongly interacts with mCtBP1 both in vivo and in vitro through this conserved GLDLSKK motif, thus extending the CtBP consensus binding site. The BTB/POZ domain does not interact with mCtBP1, but the dimerization of HIC1 through this domain is required for the interaction with mCtBP1. When tethered to DNA by fusion with the Gal4 DNA-binding domain, the HIC1 central region represses transcription through interactions with CtBP in a trichostatin A-sensitive manner. In conclusion, our results demonstrate that HIC1 mediates transcriptional repression by both HDAC-independent and HDAC-dependent mechanisms and show that CtBP is a HIC1 corepressor that is recruited via a variant binding site

Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation

International audienceActivation of the blood vessel endothelium is a critical step during inflammation. Endothelial cells stimulated by pro-inflammatory cytokines play an essential part in the adhesion and extravasation of circulating leukocytes into inflamed tissues. The endothelial egfl7 gene (VE-statin) represses endothelial cell activation in tumors, and prior observations suggested that it could also participate in the regulation of endothelial cell activation during inflammation. We show here that Egfl7 expression is strongly repressed in mouse lung endothelial cells during LPS- and TNFα-induced inflammation in vivo LPS have a limited effect on Egfl7 expression by endothelial cells in vitro, whereas the pro-inflammatory cytokine TNFα strongly represses Egfl7 expression in endothelial cells. TNFα regulates the egfl7 gene promoter through regions located between -7585 and -5550 bp ahead of the main transcription start site and via an NF-κB-dependent mechanism. Conversely, Egfl7 regulates the response of endothelial cells to TNFα by restraining the induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, resulting in a decreased adhesion of leukocytes onto endothelial cells stimulated by TNFα. Egfl7 regulates the expression of these adhesion molecules through the NF-κB and MEK/Erk pathways, in particular by preventing the proteasome-mediated degradation of IkBα both in non-activated endothelial cells and during activation. Egfl7 is thus an endogenous and constitutive repressor of blood vessel endothelial cell activation in normal and inflammatory conditions and participates in a loop of regulation of activation of these cells by pro-inflammatory cytokines

An Acetylation/Deacetylation-SUMOylation Switch through a Phylogenetically Conserved ψKXEP Motif in the Tumor Suppressor HIC1 Regulates Transcriptional Repression Activity▿

Tumor suppressor HIC1 (hypermethylated in cancer 1) is a gene that is essential for mammalian development, epigenetically silenced in many human tumors, and involved in a complex pathway regulating P53 tumor suppression activity. HIC1 encodes a sequence-specific transcriptional repressor containing five Krüppel-like C2H2 zinc fingers and an N-terminal BTB/POZ repression domain. Here, we show that endogenous HIC1 is SUMOylated in vivo on a phylogenetically conserved lysine, K314, located in the central region which is a second repression domain. K314R mutation does not influence HIC1 subnuclear localization but significantly reduces its transcriptional repression potential, as does the mutation of the other conserved residue in the ψKXE consensus, E316A, or the overexpression of the deSUMOylase SSP3/SENP2. Furthermore, HIC1 is acetylated in vitro by P300/CBP. Strikingly, the K314R mutant is less acetylated than wild-type HIC1, suggesting that this lysine is a target for both SUMOylation and acetylation. We further show that HIC1 transcriptional repression activity is positively controlled by two types of deacetylases, SIRT1 and HDAC4, which increase the deacetylation and SUMOylation, respectively, of K314. Knockdown of endogenous SIRT1 by the transfection of short interfering RNA causes a significant loss of HIC1 SUMOylation. Thus, this dual-deacetylase complex induces either a phosphorylation-dependent acetylation-SUMOylation switch through a ψKXEXXSP motif, as previously shown for MEF2, or a phosphorylation-independent switch through a ψKXEP motif, as shown here for HIC1, since P317A mutation severely impairs HIC1 acetylation. Finally, our results demonstrate that HIC1 is a target of the class III deacetylase SIRT1 and identify a new posttranslational modification step in the P53-HIC1-SIRT1 regulatory loop

Anatomy of a Flaring Proto-Planetary Disk Around a Young Intermediate-Mass Star

International audienceAlthough planets are being discovered around stars more massive than the Sun, information about the proto-planetary disks where such planets have built up is sparse. We have imaged mid-infrared emission from polycyclic aromatic hydrocarbons at the surface of the disk surrounding the young intermediate-mass star HD 97048 and characterized the disk. The disk is in an early stage of evolution, as indicated by its large content of dust and its hydrostatic flared geometry, indicative of the presence of a large amount of gas that is well mixed with dust and gravitationally stable. The disk is a precursor of debris disks found around more-evolved A stars such as β-Pictoris and provides the rare opportunity to witness the conditions prevailing before (or during) planet formation

Réalisation des ouvrages d'étanchéité en sol compacté

-Le présent fascicule propose d'harmoniser les pratiques sous forme de recommandations pour la réalisation d'ouvrages d'étanchéité en sol compacté. Le but est de faire connaître ces pratiques et de promouvoir leur utilisation dans toutes les activités de ce secteur. Ce fascicule d'adresse à un large public pour lequel des connaissances de base en matière de géotechnique et de terrassements sont indispensables : personnel des entreprises de travaux à divers niveaux, autorités et organismes de contrôle, bureaux d'études et de conception, exploitants d'ouvrages. Il comprend les informations pour évaluer l'applicabilité des techniques en général ou dans des cas particuliers. Il comporte quatre chapitres techniques, de l'étude à la réalisation des ouvrages, permettant de mener un projet à son terme. Il doit être considéré comme un document de référence à consulter avec d'autres publications de l'AFNOR et du BNTRA et avec le guide technique de réalisation des remblais et des couches de forme (GTR, aux éditions SETRA-LCPC). Ce fascicule définit les conditions de réutilisation de matériaux naturels ou traités ayant vocation à constituer une étanchéité. Ces conditions s'appliquent à la réalisation d'ouvrages ayant une fonction de protection de l'environnement ou une fonction hydraulique tels que barrières étanches, noyaux d'ouvrage hydraulique ou masques d'étanchéité. La méthodologie décrite dans ce fascicule allie les performances de faible perméabilité et les conditions de mise en oeuvre. Elle ne traite pas de la recherche d'autres performances comme la résistance mécanique (stabilité, résistance à l'érosion, ...) ou la résistance chimique des matériaux propres à la conception des ouvrages. Moyennant la recherche de ces autres performances, ces recommandations s'appliquent à la réalisation des ouvrages ou des parties d'ouvrage liées à : - Ouvrages fonction protection de l'environnement - La gestion des déchets dangereux et non dangereux (casiers de stockage, bassin de lixiviats, ...) - La gestion des résidus miniers susceptibles d'avoir un impact sur l'environnement - Des opérations de confinements dans le domaine des sites et sols pollués - Bassins de rétention, de traitement des eaux pour éviter la pollution des nappes - Ouvrages fonction hydraulique - Des ouvrages d'étanchéité en remblai (barrages, digues, bassins, etc.) - Des masques d'étanchéité en argile (digues, bassins et fossés, etc.

Nucleosome density at human telomeres depends on TRF2 expression.

<p>(<b>A</b>) ChIP of C33A cells overexpressing TRF2^FL or TRF2^ΔBΔM and control C33A cells using the indicated antibodies. Slot-blots were hybridized with a labelled Telo repeat probe and an Alu probe. (<b>B</b>) Quantification of the data in (<b>A</b>) expressed as probe/input hybridization signals. Error bars are s.d. of three independent experiments. Asterisks, p<0.05 based on unpaired Student's t-test.</p