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The single molecule of DNA that constitutes a eukaryotic chromosome begins and ends with a stretch of repetitive DNA known as a telomere. These sequences appear to be necessary to preserve the integrity of the genetic material through the cell cycle. Telomeric DNA is organized into regions of non-nucleosomal chromatin called the telosome, which can interact with other telosomes and with the nuclear envelope. This review focuses on cytological evidence for these interactions and on recent insights into the molecular organization of the telomeric complex.

Repressor Activator Protein 1 (RAP1) is an essential nuclear protein of the yeast Saccharomyces cerevisiae that recognizes a 13 base-pair (bp) consensus sequence found in numerous upstream activating sequences, at the silencers of transcriptionally repressed mating-type genes, and in telomeric tracts, called (C1-3 A) repeats. RAP1 has been shown to influence transcriptional activation, transcriptional repression, telomere length, circular plasmid segregation and meiotic recombination in vivo. We have studied the structure of the protein-DNA complex reconstituted in vitro with highly purified RAP1, by using DNase I and chemical footprinting. Both full-length RAP1 and its minimal DNA-binding domain of roughly 30 kDa, induce a distortion within the 13 bp recognition site, as demonstrated by reactivity to KMnO4 primarily at nucleotides 8 and 10 in the binding consensus Rc/AAYCCRYNCAYY. Dimethylsulphate reactivity shows that RAP1 binding does not create unpaired regions at its binding site, although the DNA may be locally underwound or aberrantly base-paired at the permanganate reactive nucleotides. In addition to the permanganate-sensitive distortion, the full-length RAP1, but not its DNA-binding domain, induces a bend in DNA 5' of the recognition sequence, altering the electrophoretic mobility of the protein-DNA complex. The KMnO4-reactivity has allowed a precise mapping of RAP1 molecules on telomeric DNA, revealing RAP1 sites as frequently as one per 18 bp of telomeric DNA, or potentially 20 RAP1 molecules bound per average telomeric tract of 370 bp. This suggests that RAP1 plays a major role in organizing yeast telomeres, and is consistent with recently published immunofluorescence studies showing a major fraction of RAP1 at the ends of meiotic chromosomes.

Repressor Activator Protein 1 (RAP1) of Saccharomyces cerevisiae is an abundant nuclear protein implicated in telomere length maintenance, transactivation, and in the establishment of silent chromatin domains. The RAP1 binding site 5' of the yeast HIS4 gene is also a region of hyperrecombination in meiosis. We report here that as RAP1 binds its recognition consensus, it appears to untwist double-stranded DNA, which we detect as the introduction of a negative supercoil in circularization assays. Coincident with the RAP1-dependent untwisting, we observe stimulation of the association of a single-stranded yeast telomeric sequence with its homologous double-stranded sequence in a supercoiled plasmid. This unusual distortion of the DNA double helix by RAP1 may contribute to the RAP1-dependent enhancement of recombination rates and promote non-duplex strand interactions at telomeres.

Cancer stem cells (CSCs) have been identified in several solid malignancies and are now emerging as a plausible target for drug discovery. Beside the questionable existence of CSCs specific markers, the expression of CD133 was reported to be responsible for conferring CSC aggressiveness. Here, we identified two G-rich sequences localized within the introns 3 and 7 of the CD133 gene able to form G-quadruplex (G4) structures, bound and stabilized by small molecules. We further showed that treatment of patient-derived colon CSCs with G4-interacting agents triggers alternative splicing that dramatically impairs the expression of CD133. Interestingly, this is strongly associated with a loss of CSC properties, including self-renewing, motility, tumor initiation and metastases dissemination. Notably, the effects of G4 stabilization on some of these CSC properties are uncoupled from DNA damage response and are fully recapitulated by the selective interference of the CD133 expression. In conclusion, we provided the first proof of the existence of G4 structures within the CD133 gene that can be pharmacologically targeted to impair CSC aggressiveness. This discloses a class of potential antitumoral agents capable of targeting the CSC subpopulation within the tumoral bulk.

Polynucléotides possédant des propriétés insulatrices permettant de protéger en cis l'expression d'un transgène des éléments adjacents dans les cellules d'eucaryotes supérieurs. Ces polynucléotides peuvent notamment être utilisés pour la transgénèse, la thérapie génique et la production de protéines recombinantes.

In an article published in this issue of Developmental Cell , Maria Blasco's group shows that the telomere end-binding protein TPP1 is involved in both end protection and telomerase regulation in vivo. Importantly, they highlight the relevance of telomerase activity in highly proliferative tissues and in reprogramming of cells to induced pluripotency (iPS).

SUMMARYSenescent cells play important physiological- and pathophysiological roles in tumor suppression, tissue regeneration, and aging. While select genetic and epigenetic elements crucial for senescence induction were identified, the dynamics, underlying epigenetic mechanisms, and regulatory networks defining senescence competence, induction and maintenance remain poorly understood, precluding a deliberate therapeutic manipulation of these dynamic processes. Here, we show, using dynamic analyses of transcriptome and epigenome profiles, that the epigenetic state of enhancers predetermines their sequential activation during senescence. We demonstrate that activator protein 1 (AP-1) ‘imprints’ the senescence enhancer landscape effectively regulating transcriptional activities pertinent to the timely execution of the senescence program. We define and validate a hierarchical transcription factor (TF) network model and demonstrate its effectiveness for the design of senescence reprogramming experiments. Together, our findings define the dynamic nature and organizational principles of gene-regulatory elements driving the senescence program and reveal promising inroads for therapeutic manipulation of senescent cells.