RESUMEN
Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric alpha-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.
Asunto(s)
Autoantígenos , Centrómero/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN Satélite , Proteínas de Unión al ADN , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Bases , División Celular , Línea Celular Transformada , Células Cultivadas , Centrómero/química , Proteína B del Centrómero , Proteínas Cromosómicas no Histona/genética , Cromosomas Artificiales de los Mamíferos , Cromosomas Humanos Par 21/química , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 21/metabolismo , ADN Satélite/síntesis química , ADN Satélite/genética , ADN Satélite/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Hibridación Fluorescente in Situ , Mitosis , Mutación Puntual , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing alpha-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input alpha-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.
Asunto(s)
Anafase/genética , Cromosomas Artificiales Humanos/genética , Metafase/genética , Sitios de Unión , Línea Celular Tumoral , Centrómero/genética , Humanos , Lactosa/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de TiempoAsunto(s)
Ensamble y Desensamble de Cromatina/genética , Cromosomas Artificiales Humanos , Cromosomas Humanos/genética , Cromosomas Humanos/metabolismo , Animales , Autoantígenos/fisiología , División Celular/genética , Centrómero/química , Centrómero/genética , Centrómero/fisiología , Proteína A Centromérica , Proteína B del Centrómero/fisiología , Proteínas Cromosómicas no Histona/fisiología , Cromosomas Artificiales Humanos/genética , Cromosomas Artificiales Humanos/metabolismo , ADN , Epigénesis Genética , Heterocromatina , Humanos , RatonesRESUMEN
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.
Asunto(s)
Proteína B del Centrómero/metabolismo , Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Cromosomas Artificiales Humanos/metabolismo , Cromosomas Artificiales de los Mamíferos/metabolismo , ADN Satélite/metabolismo , Fibroblastos/metabolismo , Animales , Autoantígenos/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Proteína A Centromérica , Proteína B del Centrómero/deficiencia , Proteína B del Centrómero/genética , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Islas de CpG , Metilación de ADN , Embrión de Mamíferos , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Lisina/metabolismo , Metilación , Ratones , Ratones Noqueados , Conformación de Ácido Nucleico , Unión Proteica , Factores de Tiempo , TransfecciónRESUMEN
Potential problems of conventional transgenes include insertional disruption of the host genome and unpredictable, irreproducible expression of the transgene by random integration. Alternatively, human artificial chromosomes (HACs) can circumvent some of the problems. Although several HACs were generated and their mitotic stability was assessed, a practical way for introducing exogenous genes by the HACs has yet to be explored. In this study, we developed a novel HAC from sequence-ready human chromosome 21 by telomere-directed chromosome truncation and added a loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. This 21HAC vector, delivered to a human cell line HT1080 by microcell fusion, bound centromere proteins A, B, and C and was mitotically stable during long-term culture without selection. The EGFP gene inserted in the HAC vector expressed persistently. These results suggest that the HAC vector provides useful system for functional studies of genes in isogenic cell lines.