RESUMEN
Centromeres are essential for chromosome segregation in most animals and plants yet are among the most rapidly evolving genome elements. The mechanisms underlying this paradoxical phenomenon remain enigmatic. Here, we report that human centromeres innately harbor a striking enrichment of DNA breaks within functionally active centromere regions. Establishing a single-cell imaging strategy that enables comparative assessment of DNA breaks at repetitive regions, we show that centromeric DNA breaks are induced not only during active cellular proliferation but also de novo during quiescence. Markedly, centromere DNA breaks in quiescent cells are resolved enzymatically by the evolutionarily conserved RAD51 recombinase, which in turn safeguards the specification of functional centromeres. This study highlights the innate fragility of centromeres, which may have been co-opted over time to reinforce centromere specification while driving rapid evolution. The findings also provide insights into how fragile centromeres are likely to contribute to human disease.
Asunto(s)
Centrómero , ADN , Animales , Humanos , Centrómero/genética , Centrómero/metabolismo , Proteína A Centromérica , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Recombinación GenéticaRESUMEN
Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. A 16-subunit complex, the constitutive centromere associated network (CCAN), connects between the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, and the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has implications for the mechanism of specific recognition of the CENP-A nucleosome. A model consistent with our structure depicts the CENP-C-bound nucleosome as connected to the CCAN through extended, flexible regions of CENP-C. An alternative model identifies both CENP-C and CENP-N as specificity determinants but requires CENP-N to bind CENP-A in a mode distinct from the classical nucleosome octamer.
Asunto(s)
Cinetocoros , Nucleosomas , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Microscopía por Crioelectrón , Humanos , Cinetocoros/metabolismo , Nucleosomas/genéticaRESUMEN
Centromeres are essential to genome inheritance, serving as the site of kinetochore assembly and coordinating chromosome segregation during cell division. Abnormal centromere function is associated with birth defects, infertility, and cancer. Normally, centromeres are assembled and maintained at the same chromosomal location. However, ectopic centromeres form spontaneously at new genomic locations and contribute to genome instability and developmental defects as well as to acquired and congenital human disease. Studies in model organisms have suggested that certain regions of the genome, including pericentromeres, heterochromatin, and regions of open chromatin or active transcription, support neocentromere activation. However, there is no universal mechanism that explains neocentromere formation. This review focuses on recent technological and intellectual advances in neocentromere research and proposes future areas of study. Understanding neocentromere biology will provide a better perspective on chromosome and genome organization and functional context for information generated from the Human Genome Project, ENCODE, and other large genomics consortia.
Asunto(s)
Centrómero , Cromatina , Centrómero/genética , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Cromatina/genética , Epigénesis Genética , Epigenómica , HumanosRESUMEN
The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3-H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3-H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3-H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.
Asunto(s)
Proteína A Centromérica , Inestabilidad Cromosómica , Histonas , Humanos , Proteína A Centromérica/metabolismo , Proteína A Centromérica/genética , Histonas/metabolismo , Histonas/genética , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Componente 2 del Complejo de Mantenimiento de Minicromosoma/genética , Células HeLa , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Centrómero/metabolismoRESUMEN
Centromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily conserved histone H3 variant, which directs kinetochore assembly and hence centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity-selected solubilized fission yeast CENP-ACnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. Chromatin association of Hap2 is Ies4-dependent. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naïve centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 facilitates transcription from centromere DNA. These analyses suggest that Hap2-Ino80 destabilizes H3 nucleosomes on centromere DNA through transcription-coupled histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.
Asunto(s)
Cromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transcripción Genética/fisiología , Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , ADN de Hongos/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Factores de Transcripción/metabolismoRESUMEN
CENP-A is an essential histone variant that replaces the canonical H3 at the centromeres and marks these regions epigenetically. The CENP-A nucleosome is the specific building block of centromeric chromatin, and it is recognized by CENP-C and CENP-N, two components of the constitutive centromere-associated network (CCAN), the first protein layer of the kinetochore. Recent proposals of the yeast and human (h)CCAN structures position the assembly on exposed DNA, suggesting an elusive spatiotemporal recognition. We summarize the data on the structural organization of the CENP-A nucleosome and the binding of CENP-C and CENP-N. The latter posits an apparent contradiction in engaging the CENP-A nucleosome versus the CCAN. We propose a reconciliatory model for the assembly of CCAN on centromeric chromatin.
Asunto(s)
Cinetocoros , Nucleosomas , Humanos , Proteína A Centromérica , Cromatina , Saccharomyces cerevisiaeRESUMEN
Centromere protein A (CENP-A) nucleosomes containing the centromere-specific histone H3 variant CENP-A represent an epigenetic mark that specifies centromere position. The Mis18 complex is a licensing factor for new CENP-A deposition via the CENP-A chaperone, Holliday junction recognition protein (HJURP), on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP-C-like motif, and our previous study suggested that ggKNL2 directly binds to the CENP-A nucleosome to recruit HJURP/CENP-A to the centromere. However, the molecular basis for CENP-A nucleosome recognition by ggKNL2 has remained unclear. Here, we present the cryo-EM structure of the chicken CENP-A nucleosome in complex with a ggKNL2 fragment containing the CENP-C-like motif. Chicken KNL2 distinguishes between CENP-A and histone H3 in the nucleosome using the CENP-C-like motif and its downstream region. Both the C-terminal tail and the RG-loop of CENP-A are simultaneously recognized as CENP-A characteristics. The CENP-A nucleosome-ggKNL2 interaction is thus essential for KNL2 functions. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 changes its binding partner at the centromere during chicken cell cycle progression.
Asunto(s)
Histonas , Nucleosomas , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Microscopía por Crioelectrón , Histonas/metabolismo , Proteínas de Unión al ADN/química , Animales , PollosRESUMEN
Maintenance of genome stability relies on functional centromeres for correct chromosome segregation and faithful inheritance of the genetic information. The human centromere is the primary constriction within mitotic chromosomes made up of repetitive alpha-satellite DNA hierarchically organized in megabase-long arrays of near-identical higher order repeats (HORs). Centromeres are epigenetically specified by the presence of the centromere-specific histone H3 variant, CENP-A, which enables the assembly of the kinetochore for microtubule attachment. Notably, centromeric DNA is faithfully inherited as intact haplotypes from the parents to the offspring without intervening recombination, yet, outside of meiosis, centromeres are akin to common fragile sites (CFSs), manifesting crossing-overs and ongoing sequence instability. Consequences of DNA changes within the centromere are just starting to emerge, with unclear effects on intra- and inter-generational inheritance driven by centromere's essential role in kinetochore assembly. Here, we review evidence of meiotic selection operating to mitigate centromere drive, as well as recent reports on centromere damage, recombination and repair during the mitotic cell division. We propose an antagonistic pleiotropy interpretation to reconcile centromere DNA instability as both driver of aneuploidy that underlies degenerative diseases, while also potentially necessary for the maintenance of homogenized HORs for centromere function. We attempt to provide a framework for this conceptual leap taking into consideration the structural interface of centromere-kinetochore interaction and present case scenarios for its malfunctioning. Finally, we offer an integrated working model to connect DNA instability, chromatin, and structural changes with functional consequences on chromosome integrity.
Asunto(s)
Centrómero , ADN Satélite , Humanos , ADN Satélite/genética , Centrómero/genética , Cromatina , ADN , Meiosis/genéticaRESUMEN
The kinetochore is an essential structure for chromosome segregation. Although the kinetochore is usually formed on a centromere locus, it can be artificially formed at a non-centromere locus by protein tethering. An artificial kinetochore can be formed by tethering of CENP-C or CENP-I, members of the constitutive centromere-associated network (CCAN). However, how CENP-C or CENP-I recruit the centromere-specific histone CENP-A to form an artificial kinetochore remains unclear. In this study, we analyzed this issue using the tethering assay combined with an auxin-inducible degron (AID)-based knockout method in chicken DT40 cells. We found that tethering of CENP-C or CENP-I induced CENP-A incorporation at the non-centromeric locus in the absence of Knl2 (or MIS18BP1), a component of the Mis18 complex, and that Knl2 tethering recruited CENP-A in the absence of CENP-C. We also showed that CENP-C coimmunoprecipitated with HJURP, independently of Knl2. Considering these results, we propose that CENP-C recruits CENP-A by HJURP binding to form an artificial kinetochore. Our results suggest that CENP-C or CENP-I exert CENP-A recruitment activity, independently of Knl2, for artificial kinetochore formation in chicken DT40 cells. This gives us a new insight into mechanisms for CENP-A incorporation.
Asunto(s)
Proteína A Centromérica , Centrómero , Cinetocoros , Proteína A Centromérica/metabolismo , Segregación Cromosómica , Animales , PollosRESUMEN
Chromosome segregation errors caused by centromere malfunction can lead to chromosome instability and aneuploidy. In Caenorhabditis elegans, the Argonaute protein CSR-1 is essential for proper chromosome segregation, though the specific mechanisms are not fully understood. Here we investigated how CSR-1 regulates centromere and kinetochore function in C. elegans embryos. We found that the depletion of CSR-1 results in defects in mitotic progression and chromosome positioning relative to the spindle pole. CSR-1 knockdown does not affect centromeric histone H3 variant CENP-A/HCP-3 mRNA and protein levels, but increases the localization of HCP-3 and some kinetochore proteins onto the mitotic chromosomes. Such elevation of chromatin HCP-3 localization depends on the CSR-1 RNAi pathway upstream factor EGO-1 and CSR-1's PIWI domain activity. Our results suggest that CSR-1 restricts HCP-3 level at the holocentromeres, prevents erroneous kinetochore assembly, and thereby promotes accurate chromosome segregation. Our work sheds light on CSR-1's role in regulating deposition of HCP-3 on chromatin and centromere function in the embryos.
RESUMEN
The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18ß and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18ß and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18ß, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.
Asunto(s)
Proteína A Centromérica , Centrómero , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/química , Humanos , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Unión Proteica , Ciclo Celular/genética , Modelos Moleculares , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Nucleosomas/metabolismo , Nucleosomas/química , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
The enrichment of histone H3 variant CENP-A is the epigenetic mark of centromere and initiates the assembly of the kinetochore at centromere. The kinetochore is a multi-subunit complex that ensures accurate attachment of microtubule centromere and faithful segregation of sister chromatids during mitosis. As a subunit of kinetochore, CENP-I localization at centromere also depends on CENP-A. However, whether and how CENP-I regulates CENP-A deposition and centromere identity remains unclear. Here, we identified that CENP-I directly interacts with the centromeric DNA and preferentially recognizes AT-rich elements of DNA via a consecutive DNA-binding surface formed by conserved charged residues at the end of N-terminal HEAT repeats. The DNA binding-deficient mutants of CENP-I retained the interaction with CENP-H/K and CENP-M, but significantly diminished the centromeric localization of CENP-I and chromosome alignment in mitosis. Moreover, the DNA binding of CENP-I is required for the centromeric loading of newly synthesized CENP-A. CENP-I stabilizes CENP-A nucleosomes upon binding to nucleosomal DNA instead of histones. These findings unveiled the molecular mechanism of how CENP-I promotes and stabilizes CENP-A deposition and would be insightful for understanding the dynamic interplay of centromere and kinetochore during cell cycle.
Asunto(s)
Centrómero , Proteínas Cromosómicas no Histona , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Centrómero/genética , Centrómero/metabolismo , Histonas/genética , Histonas/metabolismo , Nucleosomas/genética , ADN/genética , Mitosis , Autoantígenos/metabolismoRESUMEN
Centromeres are highly specialised chromosome domains defined by the presence of an epigenetic mark, the specific histone H3 variant called CENP-A (centromere protein A). They constitute the genomic regions on which kinetochores form and when defective cause segregation defects that can lead to aneuploidy and cancer. Here, we discuss how CENP-A is established and maintained to propagate centromere identity while subjected to dynamic chromatin remodelling during essential cellular processes like DNA repair, replication, and transcription. We highlight parallels and identify conserved mechanisms between different model organism with a particular focus on 1) the establishment of CENP-A at centromeres, 2) CENP-A maintenance during transcription and replication, and 3) the mechanisms that help preventing CENP-A localization at non-centromeric sites. We then give examples of how timely loading of new CENP-A to the centromere, maintenance of old CENP-A during S-phase and transcription, and removal of CENP-A at non-centromeric sites are coordinated and controlled by an intricate network of factors whose identity is slowly being unravelled.
Asunto(s)
Cromatina , Histonas , Histonas/genética , Histonas/metabolismo , Proteína A Centromérica/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Centrómero/metabolismo , Proteínas de Ciclo Celular/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismoRESUMEN
Centromeres are chromatin structures specialized in sister chromatid cohesion, kinetochore assembly, and microtubule attachment during chromosome segregation. The regional centromere of vertebrates consists of long regions of highly repetitive sequences occupied by the Histone H3 variant CENP-A, and which are flanked by pericentromeres. The three-dimensional organization of centromeric chromatin is paramount for its functionality and its ability to withstand spindle forces. Alongside CENP-A, key contributors to the folding of this structure include components of the Constitutive Centromere-Associated Network (CCAN), the protein CENP-B, and condensin and cohesin complexes. Despite its importance, the intricate architecture of the regional centromere of vertebrates remains largely unknown. Recent advancements in long-read sequencing, super-resolution and cryo-electron microscopy, and chromosome conformation capture techniques have significantly improved our understanding of this structure at various levels, from the linear arrangement of centromeric sequences and their epigenetic landscape to their higher-order compaction. In this review, we discuss the latest insights on centromere organization and place them in the context of recent findings describing a bipartite higher-order organization of the centromere.
Asunto(s)
Centrómero , Cromatina , Proteínas Cromosómicas no Histona , Vertebrados , Centrómero/metabolismo , Centrómero/ultraestructura , Animales , Cromatina/metabolismo , Cromatina/genética , Cromatina/ultraestructura , Cromatina/química , Humanos , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Vertebrados/genética , Proteína A Centromérica/metabolismo , Proteína A Centromérica/genética , Cohesinas , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Proteína B del Centrómero/metabolismo , Proteína B del Centrómero/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/ultraestructura , Adenosina TrifosfatasasRESUMEN
The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Microscopía por Crioelectrón/métodos , Cinetocoros/metabolismo , Nucleosomas/metabolismo , Animales , Centrómero/ultraestructura , Proteína A Centromérica/ultraestructura , Pollos , Proteínas Cromosómicas no Histona/ultraestructura , Cinetocoros/ultraestructura , Modelos Moleculares , Nucleosomas/ultraestructura , Fosforilación , Conformación ProteicaRESUMEN
Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to non-centromeric regions contributes to CIN in yeasts, flies and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers, the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) in HeLa cells. Among the top five candidates in the screen - the depletion of which showed increased nuclear YFP-CENP-A fluorescence - were the histone chaperones CHAF1B (or p60) and CHAF1A (or p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibited CENP-A mislocalization, CIN phenotypes and increased enrichment of CENP-A in chromatin fractions. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.
Asunto(s)
Proteínas Cromosómicas no Histona , Histonas , Humanos , Histonas/genética , Proteína A Centromérica/genética , Células HeLa , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromatina , Centrómero/metabolismo , Chaperonas Moleculares/metabolismo , Inestabilidad Cromosómica , Autoantígenos/genética , Factor 1 de Ensamblaje de la Cromatina/genéticaRESUMEN
Chromosome segregation during the cell cycle is an evolutionarily conserved, fundamental biological process. Dynamic interaction between spindle microtubules and the kinetochore complex that assembles on centromere DNA is required for faithful chromosome segregation. The first artificial minichromosome was constructed by cloning the centromere DNA of the budding yeast Saccharomyces cerevisiae. Since then, centromeres have been identified in >60 fungal species. The DNA sequence and organization of the sequence elements are highly diverse across these fungal centromeres. In this article, we provide a comprehensive view of the evolution of fungal centromeres. Studies of this process facilitated the identification of factors influencing centromere specification, maintenance, and propagation through many generations. Additionally, we discuss the unique features and plasticity of centromeric chromatin and the involvement of centromeres in karyotype evolution. Finally, we discuss the implications of recurrent loss of RNA interference (RNAi) and/or heterochromatin components on the trajectory of the evolution of fungal centromeres and propose the centromere structure of the last common ancestor of three major fungal phyla-Ascomycota, Basidiomycota, and Mucoromycota.
Asunto(s)
División Celular , Centrómero/genética , Centrómero/metabolismo , Evolución Molecular , Hongos/genética , Segregación Cromosómica , Hongos/clasificación , Heterocromatina/genética , Cariotipo , Cinetocoros/metabolismo , Interferencia de ARNRESUMEN
Chromatin featuring the H3 variant CENP-A at the centromere is critical for its mitotic function and epigenetic maintenance. Assembly of centromeric chromatin is restricted to G1 phase through inhibitory action of Cdk1/2 kinases in other phases of the cell cycle. Here, we identify the two key targets sufficient to maintain cell-cycle control of CENP-A assembly. We uncovered a single phosphorylation site in the licensing factor M18BP1 and a cyclin A binding site in the CENP-A chaperone, HJURP, that mediated specific inhibitory phosphorylation. Simultaneous expression of mutant proteins lacking these residues results in complete uncoupling from the cell cycle. Consequently, CENP-A assembly is fully recapitulated under high Cdk activities, indistinguishable from G1 assembly. We find that Cdk-mediated inhibition is exerted by sequestering active factors away from the centromere. Finally, we show that displacement of M18BP1 from the centromere is critical for the assembly mechanism of CENP-A.
Asunto(s)
Autoantígenos/metabolismo , Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Autoantígenos/genética , Proteína Quinasa CDC2 , Centrómero/genética , Proteína A Centromérica , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Ciclina A/genética , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutación , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , TransfecciónRESUMEN
In mammals, centromere definition involves the histone variant CENP-A (centromere protein A), deposited by its chaperone, HJURP (Holliday junction recognition protein). Alterations in this process impair chromosome segregation and genome stability, which are also compromised by p53 inactivation in cancer. Here we found that CENP-A and HJURP are transcriptionally up-regulated in p53-null human tumors. Using an established mouse embryonic fibroblast (MEF) model combining p53 inactivation with E1A or HRas-V12 oncogene expression, we reproduced a similar up-regulation of HJURP and CENP-A. We delineate functional CDE/CHR motifs within the Hjurp and Cenpa promoters and demonstrate their roles in p53-mediated repression. To assess the importance of HJURP up-regulation in transformed murine and human cells, we used a CRISPR/Cas9 approach. Remarkably, depletion of HJURP leads to distinct outcomes depending on their p53 status. Functional p53 elicits a cell cycle arrest response, whereas, in p53-null transformed cells, the absence of arrest enables the loss of HJURP to induce severe aneuploidy and, ultimately, apoptotic cell death. We thus tested the impact of HJURP depletion in pre-established allograft tumors in mice and revealed a major block of tumor progression in vivo. We discuss a model in which an "epigenetic addiction" to the HJURP chaperone represents an Achilles' heel in p53-deficient transformed cells.
Asunto(s)
Autoantígenos/metabolismo , Transformación Celular Neoplásica/genética , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes p53/genética , Oncogenes/genética , Secuencias de Aminoácidos/genética , Animales , Autoantígenos/genética , Línea Celular , Células Cultivadas , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Proteínas de Unión al ADN/genética , Femenino , Eliminación de Gen , Inestabilidad Genómica/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos AnimalesRESUMEN
Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.