RESUMO
The shelterin protein complex is required for telomere protection in various eukaryotic organisms. In mammals, the shelterin subunit TRF2 is specialized in preventing ATM activation at telomeres and chromosome end fusion in somatic cells. Here, we demonstrate that the zebrafish ortholog of TRF2 (encoded by the terfa gene) is protecting against unwanted ATM activation genome-wide. The terfa-compromised fish develop a prominent and specific embryonic neurodevelopmental failure. The heterozygous fish survive to adulthood but exhibit a premature aging phenotype. The recovery from embryonic neurodevelopmental failure requires both ATM inhibition and transcriptional complementation of neural genes. Furthermore, restoring the expression of TRF2 in glial cells rescues the embryonic neurodevelopment phenotype. These results indicate that the shelterin subunit TRF2 evolved in zebrafish as a general factor of genome maintenance and transcriptional regulation that is required for proper neurodevelopment and normal aging. These findings uncover how TRF2 links development to aging by separate functions in gene expression regulation and genome stability control.
Assuntos
Proteína 2 de Ligação a Repetições Teloméricas , Peixe-Zebra , Envelhecimento/genética , Animais , Mamíferos/genética , Complexo Shelterina , Telômero , Proteína 2 de Ligação a Repetições Teloméricas/genética , Peixe-Zebra/genéticaRESUMO
Zebrafish is broadly used as a model organism in gene loss-of-function studies in vivo, but its employment in vitro is greatly limited by the lack of efficient gene knockdown approaches in zebrafish cell lines such as ZF4. In this article, we attempted to induce silencing of telomere associated genes in ZF4 by applying the frequently-used siRNA transfection technology and a novel moiety-linked morpholino (vivo-MO). By proceeding with integrated optimization of siRNAs transfection and vivo-MOs treatment, we compared five transfection reagents and vivo-MOs simultaneously to evaluate the efficiency of terfa silencing in ZF4. 48 h after siRNAs transfection, Lipofectamine™ 3000 and X-tremeGENE™ HP leaded to knockdown in 35% and 43% of terfa transcription, respectively, while vivo-MO-terfa modulated 58% down-expression of zfTRF2 in contrast to vivo-MO-ctrl 72 h after treatment. Further siRNAs transfection targeting telomere associated genes by X-tremeGENE™ HP showed silencing in 40-68% of these genes without significant cytotoxicity and off-target effect. Our results confirmed the feasibility of gene loss-of-function studies in a zebrafish cell line, offered a systematic optimizing strategy to employ gene silencing experiments, and presented Lipofectamine™ 3000, X-tremeGENE™ HP and vivo-morpholinos as candidate gene silencing approaches for zebrafish in vitro gene loss-of-function studies. Successfully knockdown of shelterin genes further opened a new field for telomeric study in zebrafish.
Assuntos
Técnicas de Silenciamento de Genes/métodos , Telômero/genética , Telômero/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica/efeitos dos fármacos , Proteínas Monoméricas de Ligação ao GTP/genética , Morfolinos/farmacologia , Complexo Shelterina/genética , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Transfecção/métodos , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Two articles in Cell Research focus on the structure-function relationships in the shelterin complex that binds to telomeres and is essential for their stability and functions. These studies concerning both mammalian and Schizosaccharomyces pombe proteins reveal unexpected structural conservation of a motif called TRFH (Telomeric Repeat Factors Homology) domain between several subunits in these complexes, providing a rationale for further dissection of the role of telomeres in chromosome stability, aging and cancer, and encouraging us to revisit the evolution of telomere proteins.