RESUMEN
Recent breakthroughs with synthetic budding yeast chromosomes expedite the creation of synthetic mammalian chromosomes and genomes. Mammals, unlike budding yeast, depend on the histone H3 variant, CENP-A, to epigenetically specify the location of the centromere-the locus essential for chromosome segregation. Prior human artificial chromosomes (HACs) required large arrays of centromeric α-satellite repeats harboring binding sites for the DNA sequence-specific binding protein, CENP-B. We report the development of a type of HAC that functions independently of these constraints. Formed by an initial CENP-A nucleosome seeding strategy, a construct lacking repetitive centromeric DNA formed several self-sufficient HACs that showed no uptake of genomic DNA. In contrast to traditional α-satellite HAC formation, the non-repetitive construct can form functional HACs without CENP-B or initial CENP-A nucleosome seeding, revealing distinct paths to centromere formation for different DNA sequence types. Our developments streamline the construction and characterization of HACs to facilitate mammalian synthetic genome efforts.
Asunto(s)
Centrómero/metabolismo , Cromosomas Artificiales Humanos/metabolismo , ADN Satélite/metabolismo , Sitios de Unión , Línea Celular Tumoral , Centrómero/genética , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Proteína B del Centrómero/deficiencia , Proteína B del Centrómero/genética , Proteína B del Centrómero/metabolismo , Epigénesis Genética , Humanos , Nucleosomas/química , Nucleosomas/metabolismo , Plásmidos/genética , Plásmidos/metabolismoRESUMEN
Centromere-binding protein B (CENP-B) is a widely conserved DNA binding factor associated with heterochromatin and centromeric satellite repeats. In fission yeast, CENP-B homologues have been shown to silence long terminal repeat (LTR) retrotransposons by recruiting histone deacetylases. However, CENP-B factors also have unexplained roles in DNA replication. Here we show that a molecular function of CENP-B is to promote replication-fork progression through the LTR. Mutants have increased genomic instability caused by replication-fork blockage that depends on the DNA binding factor switch-activating protein 1 (Sap1), which is directly recruited by the LTR. The loss of Sap1-dependent barrier activity allows the unhindered progression of the replication fork, but results in rearrangements deleterious to the retrotransposon. We conclude that retrotransposons influence replication polarity through recruitment of Sap1 and transposition near replication-fork blocks, whereas CENP-B counteracts this activity and promotes fork stability. Our results may account for the role of LTR in fragile sites, and for the association of CENP-B with pericentromeric heterochromatin and tandem satellite repeats.
Asunto(s)
Proteína B del Centrómero/metabolismo , Replicación del ADN/genética , Genoma Fúngico/genética , Inestabilidad Genómica/genética , Retroelementos/genética , Schizosaccharomyces/genética , Secuencias Repetidas Terminales/genética , Proteína B del Centrómero/deficiencia , Proteína B del Centrómero/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Secuencia Conservada/genética , Daño del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Recombinación Genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Aneuploidy has long been recognized as one of the hallmarks of cancer. It nonetheless remains uncertain whether aneuploidy occurring early in the development of a cancer is a primary cause of oncogenic transformation, or whether it is an epiphenomenon that arises from a general breakdown in cell cycle control late in tumorigenesis. The accuracy of chromosome segregation is ensured both by the intrinsic mechanics of mitosis and by an error-checking spindle assembly checkpoint. Many cancers show altered expression of proteins involved in the spindle checkpoint or in proteins implicated in other mitotic processes. To understand the role of aneuploidy in the initiation and progression of cancer, a number of spindle checkpoint genes have been disrupted in mice, most through conventional gene targeting (to create germ-line knockouts). We describe the consequence of these mutations with respect to embryonic development, tumor progression and an unexpected link to premature aging; readers are referred elsewhere [1] for a discussion of other cell cycle regulators.
Asunto(s)
Aneuploidia , Inestabilidad Cromosómica/fisiología , Animales , Autoantígenos , Proteínas de Ciclo Celular/fisiología , Proteína A Centromérica , Proteína B del Centrómero/deficiencia , Centrosoma/fisiología , Proteínas Cromosómicas no Histona/deficiencia , Segregación Cromosómica/fisiología , Eliminación de Gen , Genes Supresores de Tumor/fisiología , Humanos , Cinetocoros/metabolismo , Proteínas Mad2 , Ratones , Microtúbulos/metabolismo , Modelos Animales , Neoplasias/etiología , Neoplasias/genética , Proteínas Nucleares/fisiología , Huso AcromáticoRESUMEN
The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.