The D4Z4 Macrosatellite Repeat Acts as a CTCF and A-Type Lamins-Dependent Insulator in Facio-Scapulo-Humeral Dystrophy

Both genetic and epigenetic alterations contribute to Facio-Scapulo-Humeral Dystrophy (FSHD), which is linked to the shortening of the array of D4Z4 repeats at the 4q35 locus. The consequence of this rearrangement remains enigmatic, but deletion of this 3.3-kb macrosatellite element might affect the expression of the FSHD-associated gene(s) through position effect mechanisms. We investigated this hypothesis by creating a large collection of constructs carrying 1 to >11 D4Z4 repeats integrated into the human genome, either at random sites or proximal to a telomere, mimicking thereby the organization of the 4q35 locus. We show that D4Z4 acts as an insulator that interferes with enhancer–promoter communication and protects transgenes from position effect. This last property depends on both CTCF and A-type Lamins. We further demonstrate that both anti-silencing activity of D4Z4 and CTCF binding are lost upon multimerization of the repeat in cells from FSHD patients compared to control myoblasts from healthy individuals, suggesting that FSHD corresponds to a gain-of-function of CTCF at the residual D4Z4 repeats. We propose that contraction of the D4Z4 array contributes to FSHD physio-pathology by acting as a CTCF-dependent insulator in patients

Effet de position télomérique et criblage moléculaire (vers de nouvelles cibles télomériques)

Notre laboratoire s intéresse depuis de nombreuses années à l implication des télomères en pathologie humaine. Les télomères sont des structures nucléoprotéiques présentes aux extrémités des chromosomes. Ils jouent un rôle important dans le bon fonctionnement et la durée de vie des cellules, et ils nécessitent, de ce fait, une régulation et une protection stricte. De plus, dans différents organismes, les télomères sont associés à la répression des gènes proximaux. Ce phénomène épigénétique appelé effet de position télomérique (TPE) implique la propagation d une structure chromatinienne répressive. Très étudié chez S. cerevisiae, cet effet a aussi été démontré chez l homme et dépend de protéines télomériques et de remodelages chromatiniens. Ainsi, le traitement des cellules humaines par des agents modifiant l architecture chromatinienne tels que l inhibiteur des histones déacetylases, la Trichostatine A, permet de lever le TPE et d induire l expression d un gène en position subtélomérique. En revanche, la 5-aza-2 -deoxycytidine, un inhibiteur de la méthylation de l ADN, n abroge pas ce type de silencing suggérant l implication de modifications spécifiques de ces régions dont l organisation nucléosomale est particulière. Afin d identifier de nouveaux composant chimiques capables d inhiber les effets de position télomériques dans les cellules humaines, nous avons effectué le criblage d une banque de molécules bioactives Prestwick Chemical Library sur la plateforme du Centre pour les Molécules Bioactives (CMBA) au CEA de Grenoble. Les criblages primaires et secondaires de cette chimiothèque ont permis à nos collaborateurs de nous livrer une liste de 15 touches confirmées, inhibitrices de la répression transcriptionnelle dépendante des télomères et observée expérimentalement grâce au gène rapporteur de la luciférase. Après avoir testé l ensemble de ces molécules dans notre laboratoire, deux polyphénols (Acacetine et Chrysine) étaient capables de lever spécifiquement le TPE sans modifier l expression du gène rapporteur placé hors contrôle télomérique. Nous nous sommes dès lors demandé à quel niveau ces deux molécules agissent sur le TPE (raccourcissement ou prolongement des tailles des télomères, perte ou gain de protéines associées aux télomères et/ou création ou pas d un dysfonctionnement télomérique). L ensemble de ces analyses a montré que le traitement des cellules humaines avec ces deux molécules est accompagné par un raccourcissement de la taille des télomères, un dysfonctionnement télomérique et la création de fusion entre chromatides sœurs. La découverte de ces molécules ouvre de nouvelles perspectives sur le traitement des maladies associées à des mécanismes télomériques, le cancer mais aussi des maladies rares comme la dystrophie facio-scapulo-Humérale, dont nous avons parallèlement étudié les mécanismes moléculaires.Telomeres are protective structures present at the ends of linear chromosomes and consist of simple repeated DNA sequences and specialized proteins. They are essential for the stable maintenance of eukaryotic chromosomes and they can regulate the lifespan of cells. In addition to their essential functions, telomeres are, in diverse organisms, specialized sites with regard to gene expression. Indeed, the transcription of genes located next to telomeres is repressed: this phenomenon was termed telomere position effect (TPE). As observed in the yeast, S. cerevisiae -where TPE is best characterized- TPE in human cells depends of both telomere length and architecture. The treatment of cells with Trichostatin A, an inhibitor of class I and II histone deacetylases antagonizes TPE while treating the cells with 5-aza-2 -deoxycytidin, a demethylating agent, has no apparent effect on telomeric repression. Overall, position effects at human chromosome ends are dependent on a specific higher-order organization of the telomeric chromatin. In order to identify new molecules able to modulate TPE in human cells, we carried out the screening of the Prestwick Chemical Library , a bank of bioactive molecules, on the CMBA-CEA platform in Grenoble. A total of 15 molecules were identified as anti-TPE factors after primary and secondary screening based on the increase in fluorescence measurement. After testing all of these molecules in our laboratory, two polyphenols, Acacetin and Chrysin, were retained as potent inhibitors of TPE and further characterized as drugs targeting telomeres specifically. We then asked whether TPE reversal was dependent on telomere length. The incubation in the presence of Acacetin and Chrysin affects telomere length suggesting that increased expression of the subtelomeric reporters is associated with a shortening of telomeres. To check whether these molecules revert telomeric silencing, by affecting telomere integrity, we analyzed chromosome stability in cells treated with Acacetin or Chrysin. The two molecules exhibit a rapid increase in the frequency of telomere induced DNA damage foci, suggesting that these drugs trigger telomeric dysfunction. Drug treatment also significantly increases the frequency of telomeric abnormalities observed in metaphase with a significant increase of end-to-end fusions between sisters chromatid. The discovery of these new compounds targeting specifically chromosome ends opens new avenue for the targeted treatment of diseases linked to telomeric pathways such as cancer but also rare diseases such as Facio-Scapulo-Humeral dystrophy for which we investigated the molecular mechanisms.LYON-ENS Sciences (693872304) / SudocSudocFranceF

The Proto-oncogene c-Kit Inhibits Tumor Growth by Behaving as a Dependence Receptor

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FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC

RNA polymerase V (Pol V) long noncoding RNAs (lncRNAs) have been proposed to guide ARGONAUTE4 (AGO4) to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we provide evidence, based on laser UV-assisted zero-length cross-linking, for functionally relevant AGO4–DNA interaction at RdDM targets. We further demonstrate that Pol V lncRNAs or the act of their transcription are required to lock Pol V holoenzyme into a stable DNA-bound state that allows AGO4 recruitment via redundant glycine–tryptophan/tryptophan–glycine AGO hook motifs present on both Pol V and its associated factor, SPT5L. We propose a model in which AGO4–DNA interaction could be responsible for the unique specificities of RdDM

Acacetin and Chrysin, Two Polyphenolic Compounds, Alleviate Telomeric Position Effect in Human Cells

We took advantage of the ability of human telomeres to silence neighboring genes (telomere position effect or TPE) to design a high-throughput screening assay for drugs altering telomeres. We identified, for the first time, that two dietary flavones, acacetin and chrysin, are able to specifically alleviate TPE in human cells. We further investigated their influence on telomere integrity and showed that both drugs drastically deprotect telomeres against DNA damage response. However, telomere deprotection triggered by shelterin dysfunction does not affect TPE, indicating that acacetin and chrysin target several functions of telomeres. These results show that TPE-based screening assays represent valuable methods to discover new compounds targeting telomeres

Netrin-1 and Its Receptor DCC Are Causally Implicated in Melanoma Progression

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Replication Timing of Human Telomeres Is Chromosome Arm-Specific, Influenced by Subtelomeric Structures and Connected to Nuclear Localization

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of β-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4-associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm-specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing

Identification of a perinuclear positioning element in human subtelomeres that requires A-type lamins and CTCF

The localization of genes within the nuclear space is of paramount importance for proper genome functions. However, very little is known on the cis-acting elements determining subnuclear positioning of chromosome segments. We show here that the D4Z4 human subtelomeric repeat localizes a telomere at the nuclear periphery. This perinuclear activity lies within an 80 bp sequence included within a region known to interact with CTCF and A-type Lamins. We further show that a reduced level of either CTCF or A-type Lamins suppresses the perinuclear activities of D4Z4 and that an array of multimerized D4Z4 sequence, which has lost its ability to bind CTCF and A-type Lamins, is not localized at the periphery. Overall, these findings reveal the existence of an 80 bp D4Z4 sequence that is sufficient to position an adjacent telomere to the nuclear periphery in a CTCF and A-type lamins-dependent manner. Strikingly, this sequence includes a 30 bp GA-rich motif, which binds CTCF and is present at several locations in the human genome