RESUMEN
Human centromeres form primarily on α-satellite DNA but sporadically arise de novo at naive ectopic loci, creating neocentromeres. Centromere inheritance is driven primarily by chromatin containing the histone H3 variant CENP-A. Here, we report a chromosome engineering system for neocentromere formation in human cells and characterize the first experimentally induced human neocentromere at a naive locus. The spontaneously formed neocentromere spans a gene-poor 100-kb domain enriched in histone H3 lysine 9 trimethylated (H3K9me3). Long-read sequencing revealed this neocentromere was formed by purely epigenetic means and assembly of a functional kinetochore correlated with CENP-A seeding, eviction of H3K9me3 and local accumulation of mitotic cohesin and RNA polymerase II. At formation, the young neocentromere showed markedly reduced chromosomal passenger complex (CPC) occupancy and poor sister chromatin cohesion. However, long-term tracking revealed increased CPC assembly and low-level transcription providing evidence for centromere maturation over time.
Asunto(s)
Centrómero/metabolismo , Emparejamiento Base/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteína A Centromérica/química , Proteína A Centromérica/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Epigénesis Genética , Genoma Humano , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Metilación , Dominios Proteicos , ARN Polimerasa II/metabolismo , Transcripción Genética , CohesinasRESUMEN
The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.
Asunto(s)
Autoantígenos/genética , Centrómero/fisiología , Proteínas Cromosómicas no Histona/genética , Epigénesis Genética , Histonas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Adenoviridae/genética , Autoantígenos/metabolismo , Centrómero/ultraestructura , Proteína A Centromérica , Proteína B del Centrómero/genética , Proteína B del Centrómero/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/ultraestructura , Células Epiteliales/citología , Células Epiteliales/metabolismo , Vectores Genéticos , Histonas/metabolismo , Humanos , Estructura Terciaria de Proteína , Retina/citología , Retina/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transducción de SeñalRESUMEN
Centromeres are the site of kinetochore formation during mitosis. Centromere protein A (CENP-A), the centromere-specific histone H3 variant, is essential for the epigenetic maintenance of centromere position. Previously we showed that newly synthesized CENP-A is targeted to centromeres exclusively during early G1 phase and is subsequently maintained across mitotic divisions. Using SNAP-based fluorescent pulse labeling, we now demonstrate that cell cycle-restricted chromatin assembly at centromeres is unique to CENP-A nucleosomes and does not involve assembly of other H3 variants. Strikingly, stable retention is restricted to the CENP-A/H4 core of the nucleosome, which we find to outlast general chromatin across several cell divisions. We further show that cell cycle timing of CENP-A assembly is independent of centromeric DNA sequences and instead is mediated by the CENP-A targeting domain. Unexpectedly, this domain also induces stable transmission of centromeric nucleosomes, independent of the CENP-A deposition factor HJURP. This demonstrates that intrinsic properties of the CENP-A protein direct its cell cycle-restricted assembly and induces quantitative mitotic transmission of the CENP-A/H4 nucleosome core, ensuring long-term stability and epigenetic maintenance of centromere position.
Asunto(s)
Autoantígenos/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Fase G1 , Nucleosomas/metabolismo , Autoantígenos/genética , Línea Celular , Centrómero/genética , Centrómero/metabolismo , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Modelos Genéticos , Nucleosomas/genética , Estabilidad Proteica , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The TTAGGG motif is common to two seemingly unrelated dimensions of chromatin function-the vertebrate telomere repeat and the promoter regions of many Schizosaccharomyces pombe genes, including all of those encoding canonical histones. The essential S. pombe protein Teb1 contains two Myb-like DNA binding domains related to those found in telomere proteins and binds the human telomere repeat sequence TTAGGG. Here, we analyse Teb1 binding throughout the genome and the consequences of reduced Teb1 function. Chromatin immunoprecipitation (ChIP)-on-chip analysis reveals robust Teb1 binding at many promoters, notably including all of those controlling canonical histone gene expression. A hypomorphic allele, teb1-1, confers reduced binding and reduced levels of histone transcripts. Prompted by previously suggested connections between histone expression and centromere identity, we examined localization of the centromeric histone H3 variant Cnp1 and found reduced centromeric binding along with reduced centromeric silencing. These data identify Teb1 as a master regulator of histone levels and centromere identity.
Asunto(s)
Centrómero/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Factores de Transcripción/metabolismo , Southern Blotting , Western Blotting , Centrómero/metabolismo , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Cartilla de ADN/genética , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica/genética , Análisis por Micromatrices , Mutagénesis , Telómero/metabolismoRESUMEN
All living organisms require accurate mechanisms to faithfully inherit their genetic material during cell division. The centromere is a unique locus on each chromosome that supports a multiprotein structure called the kinetochore. During mitosis, the kinetochore is responsible for connecting chromosomes to spindle microtubules, allowing faithful segregation of the duplicated genome. In most organisms, centromere position and function is not defined by the local DNA sequence context but rather by an epigenetic chromatin-based mechanism. Centromere protein A (CENP-A) is central to this process, as chromatin assembled from this histone H3 variant is essential for assembly of the centromere complex, as well as for its epigenetic maintenance. As a major determinant of centromere function, CENP-A assembly requires tight control, both in its specificity for the centromere and in timing of assembly. In the last few years, there have been several new insights into the molecular mechanism that allow this process to occur. We will review these here and discuss the general implications of the mechanism of cell cycle coupling of centromere inheritance.
Asunto(s)
Autoantígenos/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Epigénesis Genética , Animales , Secuencia de Bases , Puntos de Control del Ciclo Celular , Proteína A Centromérica , Ensamble y Desensamble de Cromatina , Segregación Cromosómica , Hongos/metabolismo , Histonas/metabolismo , Humanos , Patrón de Herencia , Modelos Genéticos , Mapeo de Interacción de Proteínas , Especificidad por SustratoRESUMEN
Whereas mammalian cells harbor two double strand telomeric repeat binding factors, TRF1 and TRF2, the fission yeast Schizosaccharomyces pombe has been thought to harbor solely the TRF1/TRF2 ortholog Taz1p to perform comparable functions. Here we report the identification of telomeric repeat binding factor 1 (Tbf1), a second TRF1/TRF2 ortholog in S. pombe. Like the Taz1p, the identified Tbf1p shares amino acid sequence similarity, as well as structural and functional characteristics, with the mammalian TRF1 and TRF2 proteins. This family of proteins shares a common architecture with two separate structural domains. An N-terminal domain is necessary and sufficient for the formation of homodimers, and a C-terminal MYB/homeodomain mediates sequence specific recognition of double-stranded telomeric DNA. The identified Tbf1p binds S. pombe telomeric DNA with high sequence specificity in vitro. Targeted deletion of the tbf1 gene reveals that it is essential for survival, and overexpression of the tbf1 gene leads to telomere elongation in vivo, which is dependent upon the MYB domain. These data suggest that fission yeast, like mammals, have two factors that bind double-stranded telomeric DNA and perform distinct roles in telomere length regulation.