Faculty Bibliography

Attachment of telomeres to the nuclear envelope (NE) and their clustering in a chromosomal bouquet during meiotic prophase I is an evolutionary conserved event that promotes chromosome pairing and recombination. In fission yeast, bouquet formation fails when the telomeric protein Rap1 is absent or when the telomeric protein Taz1 fails to recruit Rap1 to telomeres. The mammalian Rap1 orthologue is a component of the shelterin complex and localises to telomeres through an interaction with a Taz1-like telomeric DNA binding factor, TRF2. Here, we investigated the role of mammalian Rap1 in meiotic telomere attachment and clustering by analysing spermatogenesis in Rap1-deficient mice. The results establish that the meiotic three-dimensional nuclear architecture and recombination are not affected by the absence of Rap1. Furthermore, Rap1-deficient meiotic telomeres assemble the SUN1 nuclear membrane protein, attach to the NE, and undergo bouquet formation indistinguishable from the wild-type setting. Thus, the role of Rap1 in meiosis is not conserved between fission yeast and mammals, suggesting that mammals have alternative modes for connecting telomeres to SUN proteins on the meiotic nuclear envelope

Mammalian Rap1, a TRF2-interacting protein in the telomeric shelterin complex, was recently shown to repress homology-directed repair at chromosome ends. In addition, Rap1 plays a role in transcriptional regulation and NFkappaB signaling. Rap1 is unique among the components of shelterin in that it is conserved in budding yeast and has non-telomeric functions. Comparison of mammalian Rap1 to the Rap1 proteins of several budding yeasts and fission yeast reveal both striking similarities and notable differences. The protean nature of Rap1 is best understood by viewing it as an adaptor that can mediate a variety of protein-protein and protein-DNA interactions depending on the organism and the complex in which it is functioning

Shelterin is an essential telomeric protein complex that prevents DNA damage signaling and DNA repair at mammalian chromosome ends. Here we report on the role of the TRF2-interacting factor Rap1, a conserved shelterin subunit of unknown function. We removed Rap1 from mouse telomeres either through gene deletion or by replacing TRF2 with a mutant that does not bind Rap1. Rap1 was dispensable for the essential functions of TRF2--repression of ATM kinase signaling and nonhomologous end joining (NHEJ)--and mice lacking telomeric Rap1 were viable and fertile. However, Rap1 was critical for the repression of homology-directed repair (HDR), which can alter telomere length. The data reveal that HDR at telomeres can take place in the absence of DNA damage foci and underscore the functional compartmentalization within shelterin

Telomeres are thought to be maintained by the preferential recruitment of telomerase to the shortest telomeres. The extension of the G-rich telomeric strand by telomerase is also believed to be coordinated with the complementary synthesis of the C strand by the conventional replication machinery. However, we show that under telomere length-maintenance conditions in cancer cells, human telomerase extends most chromosome ends during each S phase and is not preferentially recruited to the shortest telomeres. Telomerase rapidly extends the G-rich strand following telomere replication but fill-in of the C strand is delayed into late S phase. This late C-strand fill-in is not executed by conventional Okazaki fragment synthesis but by a mechanism using a series of small incremental steps. These findings highlight differences between telomerase actions during steady state versus nonequilibrium conditions and reveal steps in the human telomere maintenance pathway that may provide additional targets for the development of anti-telomerase therapeutics

Telomeres protect chromosome ends through the interaction of telomeric repeats with shelterin, a protein complex that represses DNA damage signaling and DNA repair reactions. The telomeric repeats are maintained by telomerase, which solves the end replication problem. We report that the TTAGGG repeat arrays of mammalian telomeres pose a challenge to the DNA replication machinery, giving rise to replication-dependent defects that resemble those of aphidicolin-induced common fragile sites. Gene deletion experiments showed that efficient duplication of telomeres requires the shelterin component TRF1. Without TRF1, telomeres activate the ATR kinase in S phase and show a fragile-site phenotype in metaphase. Single-molecule analysis of replicating telomeres showed that TRF1 promotes efficient replication of TTAGGG repeats and prevents fork stalling. Two helicases implicated in the removal of G4 DNA structures, BLM and RTEL1, were required to repress the fragile-telomere phenotype. These results identify a second telomere replication problem that is solved by the shelterin component TRF1

A central function of telomeres is to prevent chromosome ends from being recognized as DNA double-strand breaks (DSBs). Several proteins involved in processing DSBs associate with telomeres, but the roles of these factors at telomeres are largely unknown. To investigate whether the Mre11/Rad50/Nbs1 (MRN) complex is involved in the generation of proper 3' G-overhangs at human telomere ends, we used RNA interference to decrease expression of MRN and analysed their effects. Reduction of MRN resulted in a transient shortening of G-overhang length in telomerase-positive cells. The terminal nucleotides of both C- and G-rich strands remain unaltered in Mre11-diminished cells, indicating that MRN is not responsible for specifying the final end-processing event. The reduction in overhang length was not seen in telomerase-negative cells, but was observed after the expression of exogenous telomerase, which suggested that the MRN complex might be involved in the recruitment or action of telomerase

Telomeres protect chromosomes from degradation and loss of vital sequence, block end-end fusion, and allow the cell to distinguish between broken ends and chromosome ends. Mammalian telomeres end in single-stranded (TTAGGG)-rich 3'-overhangs that are tucked back into the preceding double stranded region to form a T-loop. The end structure of mammalian telomeres has just started to be elucidated and through this extra views we highlight one aspect of that structure. We have recently identified the terminal nucleotides of both the C-rich and G-rich telomere strands in human cells and showed that approximately 80% of the C-rich strands terminate precisely in ATC-5', while the last base of the G-strand is less precise. This finding has important implications for the processing events that act on the telomere ends post-replication. While the mechanism behind this phenotype is yet to be unraveled, we discuss potential models that could explain the last base specificity

The hallmarks of telomere dysfunction in mammals are reduced telomeric 3' overhangs, telomere fusions, and cell cycle arrest due to a DNA damage response. Here, we report on the phenotypes of RNAi-mediated inhibition of POT1, the single-stranded telomeric DNA-binding protein. A 10-fold reduction in POT1 protein in tumor cells induced neither telomere fusions nor cell cycle arrest. However, the 3' overhang DNA was reduced and all telomeres elicited a transient DNA damage response in G1, indicating that extensive telomere damage can occur without cell cycle arrest or telomere fusions. RNAi to POT1 also revealed its role in generating the correct sequence at chromosome ends. The recessed 5' end of the telomere, which normally ends on the sequence ATC-5', was changed to a random position within the AATCCC repeat. Thus, POT1 determines the structure of the 3' and 5' ends of human chromosomes, and its inhibition generates a novel combination of telomere dysfunction phenotypes in which chromosome ends behave transiently as sites of DNA damage, yet remain protected from nonhomologous end-joining

Mammalian telomeres end in single-stranded, G-rich 3' overhangs resulting from both the 'end-replication problem' (the inability of DNA polymerase to replicate the very end of the telomeres) and postreplication processing. Telomeric G-rich overhangs are precisely defined in ciliates; the length and the terminal nucleotides are fixed. Human telomeres have very long overhangs that are heterogeneous in size (35-600 nt), indicating that their processing must differ in some respects from model organisms. We developed telomere-end ligation protocols that allowed us to identify the terminal nucleotides of both the C-rich and the G-rich telomere strands. Up to approximately 80% of the C-rich strands terminate in CCAATC-5', suggesting that after replication a nuclease with high specificity or constrained action acts on the C strand. In contrast, the G-terminal nucleotide was less precise than Tetrahymena and Euplotes but still had a bias that changed as a function of telomerase expression

The mammary gland reaches a fully differentiated phenotype at lactation, a stage characterized by the abundant expression of beta-casein. We have investigated the expression and regulation of gap junction proteins (connexins, Cx) during the various developmental stages of mouse mammary gland. Immunohistochemical analysis, with specific antibodies, reveals that Cx26 and Cx32 are expressed and confined to the cell borders of luminal epithelial cells in all developmental stages of the gland. Cx26 and Cx32 expression, at the mRNA and protein levels, increases in pregnancy and peaks in lactation. Whereas Cx43 mRNA decreases in pregnancy and lactation, the functional activity of Cx43 protein, which has been localized to myoepithelial cells, is regulated (through phosphorylation) during pregnancy and peaks during lactation. Cx30 mRNA and proteins have, for the first time, been detected in mammary gland epithelia. Using reverse transcription/polymerase chain reaction and sequencing techniques, we show that Cx30 is abundant in pregnant and lactating mammary gland. Cx30 protein levels have not been detected in the mammary gland prior to day 15 of pregnancy, whereas maximum expression occurs at the onset of lactation. In mouse mammary cells in culture, Cx30 is epithelial-cell-specific and is induced by lactogenic hormones. These data identify a novel player in mammary differentiation and suggest a potential role for Cx30 in the fully differentiated gland