TGS1 impacts snRNA 3'-end processing, ameliorates survival motor neuron-dependent neurological phenotypes in vivo and prevents neurodegeneration.

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5'-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3' tails that are often uridylated. snRNAs with defective 3' terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

The S-adenosylmethionine analog sinefungin inhibits the trimethylguanosine synthase TGS1 to promote telomerase activity and telomere lengthening.

Mutations in many genes that control the expression, the function, or the stability of telomerase cause telomere biology disorders (TBDs), such as dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia. Mutations in a subset of the genes associated with TBDs cause reductions of the telomerase RNA moiety hTR, thus limiting telomerase activity. We have recently found that loss of the trimethylguanosine synthase TGS1 increases both hTR abundance and telomerase activity and leads to telomere elongation. Here, we show that treatment with the S-adenosylmethionine analog sinefungin inhibits TGS1 activity, increases the hTR levels, and promotes telomere lengthening in different cell types. Our results hold promise for restoring telomere length in stem and progenitor cells from TBD patients with reduced hTR levels.

Loss of Human TGS1 Hypermethylase Promotes Increased Telomerase RNA and Telomere Elongation.

Biogenesis of the human telomerase RNA (hTR) involves a complex series of posttranscriptional modifications, including hypermethylation of the 5' mono-methylguanosine cap to a tri-methylguanosine cap (TMG). How the TMG cap affects hTR maturation is unknown. Here, we show that depletion of trimethylguanosine synthase 1 (TGS1), the enzyme responsible for cap hypermethylation, increases levels of hTR and telomerase. Diminished trimethylation increases hTR association with the cap-binding complex (CBC) and with Sm chaperone proteins. Loss of TGS1 causes an increase in accumulation of mature hTR in both the nucleus and the cytoplasm compared with controls. In TGS1 mutant cells, increased hTR assembles with telomerase reverse transcriptase (TERT) protein to yield elevated active telomerase complexes and increased telomerase activity, resulting in telomere elongation in cultured human cells. Our results show that TGS1-mediated hypermethylation of the hTR cap inhibits hTR accumulation, restrains levels of assembled telomerase, and limits telomere elongation.

A role for Separase in telomere protection.

Drosophila telomeres are elongated by transposition of specialized retroelements rather than telomerase activity and are assembled independently of the sequence. Fly telomeres are protected by the terminin complex that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. We show that mutations in the Drosophila Separase encoding gene Sse lead not only to endoreduplication but also telomeric fusions (TFs), suggesting a role for Sse in telomere capping. We demonstrate that Separase binds terminin proteins and HP1, and that it is enriched at telomeres. Furthermore, we show that loss of Sse strongly reduces HP1 levels, and that HP1 overexpression in Sse mutants suppresses TFs, suggesting that TFs are caused by a HP1 diminution. Finally, we find that siRNA-induced depletion of ESPL1, the Sse human orthologue, causes telomere dysfunction and HP1 level reduction in primary fibroblasts, highlighting a conserved role of Separase in telomere protection.

AKTIP/Ft1, a New Shelterin-Interacting Factor Required for Telomere Maintenance.

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (human AKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factor identified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere dysfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication.

TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization.

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection.

Chromatin structure in telomere dynamics.

The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a DNA damage response that leads to replicative senescence. In normal human somatic cells, characterized by telomere shortening with each cell division, telomere uncapping is a regulated process associated with cell turnover. Nevertheless, telomere dysfunction has also been associated with genomic instability, cell transformation, and cancer. Despite the essential role telomeres play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure involved in telomere maintenance is still limited. Here we review the recent findings on chromatin modifications associated with the dynamic changes of telomeres from protected to deprotected state and their role in telomere functions.

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.

The human telomeric protein hTRF1 induces telomere-specific nucleosome mobility.

Human telomeres consist of thousands of base pairs of double-stranded TTAGGG repeats, organized by histone proteins into tightly spaced nucleosomes. The double-stranded telomeric repeats are also specifically bound by the telomeric proteins hTRF1 and hTRF2, which are essential for telomere length maintenance and for chromosome protection. An unresolved question is what role nucleosomes play in telomere structure and dynamics and how they interact and/or compete with hTRF proteins. Here we show that hTRF1 specifically induces mobility of telomeric nucleosomes. Moreover, Atomic Force Microscopy (AFM) imaging shows that hTRF1 induces compaction of telomeric DNA only in the presence of a nucleosome, suggesting that this compaction occurs through hTRF1-nucleosome interactions. Our findings reveal an unknown property of hTRF1 that has implications for understanding telomere structure and dynamics.

Telomeric nucleosomes are intrinsically mobile.

Nucleosomes are no longer considered only static basic units that package eukaryotic DNA but they emerge as dynamic players in all chromosomal processes. Regulatory proteins can gain access to recognition sequences hidden by the histone octamer through the action of ATP-dependent chromatin remodeling complexes that cause nucleosome sliding. In addition, it is known that nucleosomes are able to spontaneously reposition along the DNA due to intrinsic dynamic properties, but it is not clear yet to what extent sequence-dependent dynamic properties contribute to nucleosome repositioning. Here, we study mobility of nucleosomes formed on telomeric sequences as a function of temperature and ionic strength. We find that telomeric nucleosomes are highly intrinsically mobile under physiological conditions, whereas nucleosomes formed on an average DNA sequence mostly remain in the initial position. This indicates that DNA sequence affects not only the thermodynamic stability and the positioning of nucleosomes but also their dynamic properties. Moreover, our findings suggest that the high mobility of telomeric nucleosomes may be relevant to the dynamics of telomeric chromatin.

The human telomeric protein TRF1 specifically recognizes nucleosomal binding sites and alters nucleosome structure.

Telomeres are dynamic nucleoprotein structures that cap the ends of eukaryotic chromosomes. In humans, the long (TTAGGG)(n) double-stranded telomeric DNA repeats are bound specifically by the two related proteins TRF1 and TRF2, and are organized in nucleosomes. Whereas the role of TRF1 and TRF2 in telomeric function has been studied extensively, little is known about the involvement of telomeric nucleosomes in telomere structures or how chromatin formation may affect binding of the TRFs. Here, we address the question of whether TRF1 is able to bind to telomeric binding sites in a nucleosomal context. We show that TRF1 is able to specifically recognize telomeric binding sites located within nucleosomes, forming a ternary complex. The formation of this complex is strongly dependent on the orientation of binding sites on the nucleosome surface, rather than on the location of the binding sites with respect to the nucleosome dyad. Strikingly, TRF1 binding causes alterations in nucleosome structure without dissociation of histone subunits. These results indicate that nucleosomes contribute to the establishment of a telomeric capping complex, whose structure and dynamics can be modulated by the binding of telomeric factors.