RESUMEN
Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Sitios de Unión/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/clasificación , Proteínas de Caenorhabditis elegans/genética , ADN/genética , Proteínas de Unión al ADN/genética , Células Germinativas/metabolismo , Microscopía Fluorescente/métodos , Complejos Multiproteicos/genética , Mutación , Filogenia , Unión Proteica , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Telómero/genética , Proteínas de Unión a Telómeros/clasificación , Proteínas de Unión a Telómeros/genéticaRESUMEN
Telomeres protect the ends of eukaryotic chromosomes and are essential for cell viability. In mammals, telomere dynamics vary with life history traits (e.g. body mass and longevity), suggesting differential selection depending on physiological characteristics. Telomeres, in analogy to centromeric regions, also represent candidate meiotic drivers and subtelomeric DNA evolves rapidly. We analyzed the evolutionary history of mammalian genes implicated in telomere homeostasis (TEL genes). We detected widespread positive selection and we tested two alternative hypotheses: (i) fast evolution is driven by changes in life history traits; (ii) a conflict with selfish DNA elements at the female meiosis represents the underlying selective pressure. By accounting for the phylogenetic relationships among mammalian species, we show that life history traits do not contribute to shape diversity of TEL genes. Conversely, the evolutionary rate of TEL genes correlates with expression levels during meiosis and episodes of positive selection across mammalian species are associated with karyotype features (number of chromosome arms). We thus propose a telomere drive hypothesis, whereby (sub)telomeres and telomere-binding proteins are engaged in an intra-genomic conflict similar to the one described for centromeres.
Asunto(s)
Evolución Molecular , Expresión Génica , Células Germinativas/metabolismo , Homeostasis del Telómero/genética , Animales , Femenino , Humanos , Cariotipo , Masculino , Mamíferos , Meiosis/genética , Ratones , Filogenia , Proteínas de Unión a Telómeros/clasificación , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.
Asunto(s)
Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas de Unión a Telómeros/genética , Telómero/genética , Animales , Secuencia de Bases , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Ensayo de Cambio de Movilidad Electroforética , Evolución Molecular , Polarización de Fluorescencia , Variación Genética , Humanos , Microscopía Electrónica , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Filogenia , Unión Proteica , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/ultraestructura , Telómero/metabolismo , Telómero/ultraestructura , Proteínas de Unión a Telómeros/clasificación , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión a Telómeros/ultraestructura , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/ultraestructuraRESUMEN
BACKGROUND: Telomeres are nucleoprotein complexes at the end of linear eukaryotic chromosomes which maintain the genome integrity by regulating telomere length, preventing recombination and end to end fusion events. Multiple proteins associate with telomeres and function in concert to carry out these functions. Rap1 interacting factor 1 (Rif1), was identified as a protein involved in telomere length regulation in yeast. Rif1 is conserved upto mammals but its function has diversified from telomere length regulation to maintenance of genome integrity. RESULTS: We have carried out detailed bioinformatic analyses and identified Rif1 homologues in 92 organisms from yeast to human. We identified Rif1 homologues in Drosophila melanogaster, even though fly telomeres are maintained by a telomerase independent pathway. Our analysis shows that Drosophila Rif1 (dRif1) sequence is phylogenetically closer to the one of vertebrates than yeast and has identified a few Rif1 specific motifs conserved through evolution. This includes a Rif1 family specific conserved region within the HEAT repeat domain and a motif involved in protein phosphatase1 docking. We show that dRif1 is nuclear localized with a prominent heterochromatin association and unlike human Rif1, it does not respond to DNA damage by localizing to damaged sites. To test the evolutionary conservation of dRif1 function, we expressed the dRif1 protein in yeast and HeLa cells. In yeast, dRif1 did not perturb yeast Rif1 (yRif1) functions; and in HeLa cells it did not colocalize with DNA damage foci. CONCLUSIONS: Telomeres are maintained by retrotransposons in all Drosophila species and consequently, telomerase and many of the telomere associated protein homologues are absent, including Rap1, which is the binding partner of Rif1. We found that a homologue of yRif1 protein is present in fly and dRif1 has evolutionarily conserved motifs. Functional studies show that dRif1 responds differently to DNA damage, implying that dRif1 may have a different function and this may be conserved in other organisms as well.
Asunto(s)
Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/genética , Telómero/metabolismo , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Biología Computacional , Daño del ADN , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Evolución Molecular , Células HeLa , Humanos , Filogenia , Unión Proteica , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/clasificación , Proteínas Represoras/metabolismo , Retroelementos , Proteínas de Saccharomyces cerevisiae/clasificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/clasificación , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis.