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1.
Trends Genet ; 40(9): 739-746, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38910033

RESUMEN

The emergence of aerobic respiration created unprecedented bioenergetic advantages, while imposing the need to protect critical genetic information from reactive byproducts of oxidative metabolism (i.e., reactive oxygen species, ROS). The evolution of histone proteins fulfilled the need to shield DNA from these potentially damaging toxins, while providing the means to compact and structure massive eukaryotic genomes. To date, several metabolism-linked histone post-translational modifications (PTMs) have been shown to regulate chromatin structure and gene expression. However, whether and how PTMs enacted by metabolically produced ROS regulate adaptive chromatin remodeling remain relatively unexplored. Here, we review novel mechanistic insights into the interactions of ROS with histones and their consequences for the control of gene expression regulation, cellular plasticity, and behavior.


Asunto(s)
Regulación de la Expresión Génica , Histonas , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , Especies Reactivas de Oxígeno , Histonas/metabolismo , Histonas/genética , Procesamiento Proteico-Postraduccional/genética , Regulación de la Expresión Génica/genética , Humanos , Especies Reactivas de Oxígeno/metabolismo , Animales , Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Cromatina/metabolismo
2.
Cell ; 148(3): 394-6, 2012 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-22304909

RESUMEN

The centromere is a classic system to study epigenetic specification, and most research has focused on a specialized histone variant, CENP-A, that is required for kinetochore assembly. Now Nishino et al. reveal a new level of complexity for centromeric chromatin, by showing that the kinetochore complex CENP-T-W-S-X shares structural and functional properties with canonical histones.

3.
EMBO J ; 40(24): e108307, 2021 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-34786730

RESUMEN

Histone chaperones modulate the stability of histones beginning from histone synthesis, through incorporation into DNA, and during recycling during transcription and replication. Following histone removal from DNA, chaperones regulate histone storage and degradation. Here, we demonstrate that UBR7 is a histone H3.1 chaperone that modulates the supply of pre-existing post-nucleosomal histone complexes. We demonstrate that UBR7 binds to post-nucleosomal H3K4me3 and H3K9me3 histones via its UBR box and PHD. UBR7 binds to the non-nucleosomal histone chaperone NASP. In the absence of UBR7, the pool of NASP-bound post-nucleosomal histones accumulate and chromatin is depleted of H3K4me3-modified histones. We propose that the interaction of UBR7 with NASP and histones opposes the histone storage functions of NASP and that UBR7 promotes reincorporation of post-nucleosomal H3 complexes.


Asunto(s)
Autoantígenos/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Células HEK293 , Células HeLa , Código de Histonas , Histonas/química , Humanos , Nucleosomas/metabolismo , Dominios Proteicos
4.
J Cell Sci ; 136(10)2023 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-37129573

RESUMEN

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/genética
5.
Proc Natl Acad Sci U S A ; 119(27): e2111262119, 2022 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-35776542

RESUMEN

All cells contain specialized signaling pathways that enable adaptation to specific molecular stressors. Yet, whether these pathways are centrally regulated in complex physiological stress states remains unclear. Using genome-scale fitness screening data, we quantified the stress phenotype of 739 cancer cell lines, each representing a unique combination of intrinsic tumor stresses. Integrating dependency and stress perturbation transcriptomic data, we illuminated a network of genes with vital functions spanning diverse stress contexts. Analyses for central regulators of this network nominated C16orf72/HAPSTR1, an evolutionarily ancient gene critical for the fitness of cells reliant on multiple stress response pathways. We found that HAPSTR1 plays a pleiotropic role in cellular stress signaling, functioning to titrate various specialized cell-autonomous and paracrine stress response programs. This function, while dispensable to unstressed cells and nematodes, is essential for resilience in the presence of stressors ranging from DNA damage to starvation and proteotoxicity. Mechanistically, diverse stresses induce HAPSTR1, which encodes a protein expressed as two equally abundant isoforms. Perfectly conserved residues in a domain shared between HAPSTR1 isoforms mediate oligomerization and binding to the ubiquitin ligase HUWE1. We show that HUWE1 is a required cofactor for HAPSTR1 to control stress signaling and that, in turn, HUWE1 feeds back to ubiquitinate and destabilize HAPSTR1. Altogether, we propose that HAPSTR1 is a central rheostat in a network of pathways responsible for cellular adaptability, the modulation of which may have broad utility in human disease.


Asunto(s)
Daño del ADN , Aptitud Genética , Proteínas Nucleares , Estrés Fisiológico , Secuencias de Aminoácidos , Animales , Línea Celular Tumoral , Secuencia Conservada , Daño del ADN/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dominios Proteicos , Transducción de Señal/genética , Estrés Fisiológico/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo
6.
Cell ; 137(3): 472-84, 2009 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-19410544

RESUMEN

The centromere is responsible for accurate chromosome segregation. Mammalian centromeres are specified epigenetically, with all active centromeres containing centromere-specific chromatin in which CENP-A replaces histone H3 within the nucleosome. The proteins responsible for assembly of human CENP-A into centromeric nucleosomes during the G1 phase of the cell cycle are shown here to be distinct from the chromatin assembly factors previously shown to load other histone H3 variants. Here we demonstrate that prenucleosomal CENP-A is complexed with histone H4, nucleophosmin 1, and HJURP. Recruitment of new CENP-A into nucleosomes at replicated centromeres is dependent on HJURP. Recognition by HJURP is mediated through the centromere targeting domain (CATD) of CENP-A, a region that we demonstrated previously to induce a unique conformational rigidity to both the subnucleosomal CENP-A heterotetramer and the corresponding assembled nucleosome. We propose HJURP to be a cell-cycle-regulated CENP-A-specific histone chaperone required for centromeric chromatin assembly.


Asunto(s)
Autoantígenos/metabolismo , Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Línea Celular , Centrómero/ultraestructura , Proteína A Centromérica , Fase G1 , Histonas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Nucleofosmina , Nucleosomas/ultraestructura
7.
Mol Cell ; 61(5): 774-787, 2016 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-26942680

RESUMEN

Centromeres are specialized chromatin domains specified by the centromere-specific CENP-A nucleosome. The stable inheritance of vertebrate centromeres is an epigenetic process requiring deposition of new CENP-A nucleosomes by HJURP. We show HJURP is recruited to centromeres through a direct interaction between the HJURP centromere targeting domain and the Mis18α-ß C-terminal coiled-coil domains. We demonstrate Mis18α and Mis18ß form a heterotetramer through their C-terminal coiled-coil domains. Mis18α-ß heterotetramer formation is required for Mis18BP1 binding and centromere recognition. S. pombe contains a single Mis18 isoform that forms a homotetramer, showing tetrameric Mis18 is conserved from fission yeast to humans. HJURP binding disrupts the Mis18α-ß heterotetramer and removes Mis18α from centromeres. We propose stable binding of Mis18 to centromeres in telophase licenses them for CENP-A deposition. Binding of HJURP deposits CENP-A at centromeres and facilitates the removal of Mis18, restricting CENP-A deposition to a single event per cell cycle.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Telofase , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transducción de Señal , Transfección
8.
Chromosoma ; 127(3): 279-290, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29569072

RESUMEN

Centromeres are specialized chromosome domain that serve as the site for kinetochore assembly and microtubule attachment during cell division, to ensure proper segregation of chromosomes. In higher eukaryotes, the identity of active centromeres is marked by the presence of CENP-A (centromeric protein-A), a histone H3 variant. CENP-A forms a centromere-specific nucleosome that acts as a foundation for centromere assembly and function. The posttranslational modification (PTM) of histone proteins is a major mechanism regulating the function of chromatin. While a few CENP-A site-specific modifications are shared with histone H3, the majority are specific to CENP-A-containing nucleosomes, indicating that modification of these residues contribute to centromere-specific function. CENP-A undergoes posttranslational modifications including phosphorylation, acetylation, methylation, and ubiquitylation. Work from many laboratories have uncovered the importance of these CENP-A modifications in its deposition at centromeres, protein stability, and recruitment of the CCAN (constitutive centromere-associated network). Here, we discuss the PTMs of CENP-A and their biological relevance.


Asunto(s)
Proteína A Centromérica/metabolismo , Procesamiento Proteico-Postraduccional , Ciclo Celular , Centrómero/genética , Centrómero/metabolismo , Proteína A Centromérica/química , Histonas/metabolismo , Humanos , Metilación , Mitosis/genética , Unión Proteica , Especificidad de la Especie , Relación Estructura-Actividad
9.
Mol Cell Proteomics ; 15(3): 918-31, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26685127

RESUMEN

The centromere is the locus on the chromosome that acts as the essential connection point between the chromosome and the mitotic spindle. A histone H3 variant, CENP-A, defines the location of the centromere, but centromeric chromatin consists of a mixture of both CENP-A-containing and H3-containing nucleosomes. We report a surprisingly uniform pattern of primarily monomethylation on lysine 20 of histone H4 present in short polynucleosomes mixtures of CENP-A and H3 nucleosomes isolated from functional centromeres. Canonical H3 is not a component of CENP-A-containing nucleosomes at centromeres, so the H3 we copurify from these preparations comes exclusively from adjacent nucleosomes. We find that CENP-A-proximal H3 nucleosomes are not uniformly modified but contain a complex set of PTMs. Dually modified K9me2-K27me2 H3 nucleosomes are observed at the centromere. Side-chain acetylation of both histone H3 and histone H4 is low at the centromere. Prior to assembly at centromeres, newly expressed CENP-A is sequestered for a large portion of the cell cycle (late S-phase, G2, and most of mitosis) in a complex that contains its partner, H4, and its chaperone, HJURP. In contrast to chromatin associated centromeric histone H4, we show that prenucleosomal CENP-A-associated histone H4 lacks K20 methylation and contains side-chain and α-amino acetylation. We show HJURP displays a complex set of serine phosphorylation that may potentially regulate the deposition of CENP-A. Taken together, our findings provide key information regarding some of the key components of functional centromeric chromatin.


Asunto(s)
Autoantígenos/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Ciclo Celular , Proteína A Centromérica , Células HeLa , Humanos , Lisina/metabolismo , Metilación , Serina/metabolismo , Espectrometría de Masa por Ionización de Electrospray
10.
Prog Mol Subcell Biol ; 56: 165-192, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28840237

RESUMEN

Centromeres are chromosomal loci that are defined epigenetically in most eukaryotes by incorporation of a centromere-specific nucleosome in which the canonical histone H3 variant is replaced by Centromere Protein A (CENP-A). Therefore, the assembly and propagation of centromeric nucleosomes are critical for maintaining centromere identify and ensuring genomic stability. Centromeres direct chromosome segregation (during mitosis and meiosis) by recruiting the constitutive centromere-associated network of proteins throughout the cell cycle that in turn recruits the kinetochore during mitosis. Assembly of centromere-specific nucleosomes in humans requires the dedicated CENP-A chaperone HJURP, and the Mis18 complex to couple the deposition of new CENP-A to the site of the pre-existing centromere, which is essential for maintaining centromere identity. Human CENP-A deposition occurs specifically in early G1, into pre-existing chromatin, and several additional chromatin-associated complexes regulate CENP-A nucleosome deposition and stability. Here we review the current knowledge on how new CENP-A nucleosomes are assembled selectively at the existing centromere in different species and how this process is controlled to ensure stable epigenetic inheritance of the centromere.


Asunto(s)
Centrómero/metabolismo , Nucleosomas/metabolismo , Centrómero/química , Proteína A Centromérica/metabolismo , Humanos , Nucleosomas/química
11.
EMBO J ; 32(15): 2113-24, 2013 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-23771058

RESUMEN

The epigenetic mark of the centromere is thought to be a unique centromeric nucleosome that contains the histone H3 variant, centromere protein-A (CENP-A). The deposition of new centromeric nucleosomes requires the CENP-A-specific chromatin assembly factor HJURP (Holliday junction recognition protein). Crystallographic and biochemical data demonstrate that the Scm3-like domain of HJURP binds a single CENP-A-histone H4 heterodimer. However, several lines of evidence suggest that HJURP forms an octameric CENP-A nucleosome. How an octameric CENP-A nucleosome forms from individual CENP-A/histone H4 heterodimers is unknown. Here, we show that HJURP forms a homodimer through its C-terminal domain that includes the second HJURP_C domain. HJURP exists as a dimer in the soluble preassembly complex and at chromatin when new CENP-A is deposited. Dimerization of HJURP is essential for the deposition of new CENP-A nucleosomes. The recruitment of HJURP to centromeres occurs independent of dimerization and CENP-A binding. These data provide a mechanism whereby the CENP-A pre-nucleosomal complex achieves assembly of the octameric CENP-A nucleosome through the dimerization of the CENP-A chaperone HJURP.


Asunto(s)
Autoantígenos/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Multimerización de Proteína/fisiología , Autoantígenos/genética , Centrómero/genética , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Células HEK293 , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Nucleosomas/genética , Estructura Terciaria de Proteína
12.
Proc Natl Acad Sci U S A ; 110(29): 11827-32, 2013 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-23818633

RESUMEN

Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyperoligomerized state exhibited by unmodified CENP-A nucleosome arrays. Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere.


Asunto(s)
Autoantígenos/genética , Autoantígenos/metabolismo , Centrómero/química , Cromatina/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Epigénesis Genética/genética , Conformación Molecular , Procesamiento Proteico-Postraduccional/genética , Autoantígenos/aislamiento & purificación , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteína A Centromérica , Cromatografía Líquida de Alta Presión , Proteínas Cromosómicas no Histona/aislamiento & purificación , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Espectrometría de Masas , Metilación , Proteínas Nucleares/metabolismo , Fosforilación , Ultracentrifugación
13.
Cell Mol Life Sci ; 70(3): 387-406, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22729156

RESUMEN

The centromere is the chromosomal region that directs kinetochore assembly during mitosis in order to facilitate the faithful segregation of sister chromatids. The location of the human centromere is epigenetically specified. The presence of nucleosomes that contain the histone H3 variant, CENP-A, are thought to be the epigenetic mark that indicates active centromeres. Maintenance of centromeric identity requires the deposition of new CENP-A nucleosomes with each cell cycle. During S-phase, existing CENP-A nucleosomes are divided among the daughter chromosomes, while new CENP-A nucleosomes are deposited during early G1. The specific assembly of CENP-A nucleosomes at centromeres requires the Mis18 complex, which recruits the CENP-A assembly factor, HJURP. We will review the unique features of centromeric chromatin as well as the mechanism of CENP-A nucleosome deposition. We will also highlight a few recent discoveries that begin to elucidate the factors that temporally and spatially control CENP-A deposition.


Asunto(s)
Autoantígenos/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Autoantígenos/química , Centrómero/metabolismo , Proteína A Centromérica , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Epigenómica , Histonas/genética , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Nucleosomas/metabolismo , Estructura Terciaria de Proteína , ARN no Traducido/metabolismo , Fase S
14.
PLoS Genet ; 7(9): e1002303, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21980305

RESUMEN

The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability.


Asunto(s)
Inestabilidad Cromosómica/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Centrómero/genética , Centrómero/metabolismo , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Mol Cell Biol ; 44(6): 209-225, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38779933

RESUMEN

Proper chromosome segregation is required to ensure chromosomal stability. The centromere (CEN) is a unique chromatin domain defined by CENP-A and is responsible for recruiting the kinetochore (KT) during mitosis, ultimately regulating microtubule spindle attachment and mitotic checkpoint function. Upregulation of many CEN/KT genes is commonly observed in cancer. Here, we show that although FOXM1 occupies promoters of many CEN/KT genes with MYBL2, FOXM1 overexpression alone is insufficient to drive the FOXM1-correlated transcriptional program. CENP-F is canonically an outer kinetochore component; however, it functions with FOXM1 to coregulate G2/M transcription and proper chromosome segregation. Loss of CENP-F results in altered chromatin accessibility at G2/M genes and reduced FOXM1-MBB complex formation. We show that coordinated CENP-FFOXM1 transcriptional regulation is a cancer-specific function. We observe a small subset of CEN/KT genes including CENP-C, that are not regulated by FOXM1. Upregulation of CENP-C in the context of CENP-A overexpression leads to increased chromosome missegregation and cell death suggesting that escape of CENP-C from FOXM1 regulation is a cancer survival mechanism. Together, we show that FOXM1 and CENP-F coordinately regulate G2/M genes, and this coordination is specific to a subset of genes to allow for maintenance of chromosome instability levels and subsequent cell survival.


Asunto(s)
Centrómero , Proteínas Cromosómicas no Histona , Segregación Cromosómica , Proteína Forkhead Box M1 , Cinetocoros , Proteína Forkhead Box M1/metabolismo , Proteína Forkhead Box M1/genética , Humanos , Cinetocoros/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Centrómero/metabolismo , Segregación Cromosómica/genética , Línea Celular Tumoral , Mitosis/genética , Proteína A Centromérica/metabolismo , Proteína A Centromérica/genética , Transcripción Genética , Regulación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Cromatina/metabolismo , Cromatina/genética , Regiones Promotoras Genéticas/genética , Proteínas de Microfilamentos
16.
Nat Cell Biol ; 8(5): 458-69, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16622419

RESUMEN

The basic element for chromosome inheritance, the centromere, is epigenetically determined in mammals. The prime candidate for specifying centromere identity is the array of nucleosomes assembled with CENP-A, the centromere-specific histone H3 variant. Here, we show that CENP-A nucleosomes directly recruit a proximal CENP-A nucleosome associated complex (NAC) comprised of three new human centromere proteins (CENP-M, CENP-N and CENP-T), along with CENP-U(50), CENP-C and CENP-H. Assembly of the CENP-A NAC at centromeres is dependent on CENP-M, CENP-N and CENP-T. Facilitates chromatin transcription (FACT) and nucleophosmin-1 (previously implicated in transcriptional chromatin remodelling and as a multifunctional nuclear chaperone, respectively) are absent from histone H3-containing nucleosomes, but are stably recruited to CENP-A nucleosomes independent of CENP-A NAC. Seven new CENP-A-nucleosome distal (CAD) centromere components (CENP-K, CENP-L, CENP-O, CENP-P, CENP-Q, CENP-R and CENP-S) are identified as assembling on the CENP-A NAC. The CENP-A NAC is essential, as disruption of the complex causes errors of chromosome alignment and segregation that preclude cell survival despite continued centromere-derived mitotic checkpoint signalling.


Asunto(s)
Autoantígenos/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Nucleosomas/metabolismo , Secuencia de Aminoácidos , Autoantígenos/química , Autoantígenos/aislamiento & purificación , Proteína A Centromérica , Factor 1 de Ensamblaje de la Cromatina , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/aislamiento & purificación , Cromosomas Humanos/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Histonas/química , Humanos , Mitosis/genética , Datos de Secuencia Molecular , Unión Proteica , Transducción de Señal
17.
bioRxiv ; 2023 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-38234763

RESUMEN

Proper chromosome segregation is required to ensure genomic and chromosomal stability. The centromere is a unique chromatin domain present throughout the cell cycle on each chromosome defined by the CENP-A nucleosome. Centromeres (CEN) are responsible for recruiting the kinetochore (KT) during mitosis, ultimately regulating spindle attachment and mitotic checkpoint function. Upregulation of many genes that encode the CEN/KT proteins is commonly observed in cancer. Here, we show although that FOXM1 occupies the promoters of many CEN/KT genes with MYBL2, occupancy is insufficient alone to drive the FOXM1 correlated transcriptional program. We show that CENP-F, a component of the outer kinetochore, functions with FOXM1 to coregulate G2/M transcription and proper chromosome segregation. Loss of CENP-F results in alteration of chromatin accessibility at G2/M genes, including CENP-A, and leads to reduced FOXM1-MBB complex formation. The FOXM1-CENP-F transcriptional coordination is a cancer-specific function. We observed that a few CEN/KT genes escape FOXM1 regulation such as CENP-C which when upregulated with CENP-A, leads to increased chromosome misegregation and cell death. Together, we show that the FOXM1 and CENP-F coordinately regulate G2/M gene expression, and this coordination is specific to a subset of genes to allow for proliferation and maintenance of chromosome stability for cancer cell survival.

18.
bioRxiv ; 2023 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-37808683

RESUMEN

Many Lamin A-associated proteins (LAAP's) that are key constituents of the nuclear envelope (NE), assemble at the "core" domains of chromosomes during NE reformation and mitotic exit. However, the identity and function of the chromosomal core domains remain ill-defined. Here, we show that a distinct section of the core domain overlaps with the centromeres/kinetochores of chromosomes during mitotic telophase. The core domain can thus be demarcated into a kinetochore proximal core (KPC) on one side of the segregated chromosomes and the kinetochore distal core (KDC) on the opposite side, close to the central spindle. We next tested if centromere assembly is connected to NE re-formation. We find that centromere assembly is markedly perturbed after inhibiting the function of LMNA and the core-localized LAAPs, BANF1 and Emerin. We also find that the LAAPs exhibit multiple biochemical interactions with the centromere and inner kinetochore proteins. Consistent with this, normal mitotic progression and chromosome segregation was severely impeded after inhibiting LAAP function. Intriguingly, the inhibition of centromere function also interferes with the assembly of LAAP components at the core domain, suggesting a mutual dependence of LAAP and centromeres for their assembly at the core domains. Finally, we find that the localization of key proteins involved in the centromeric loading of CENP-A, including the Mis18 complex and HJURP were markedly affected in LAAP-inhibited cells. Our evidence points to a model where LAAP assembly at the core domain serves a key function in loading new copies of centromeric proteins during or immediately after mitotic exit.

19.
Mol Biol Cell ; 34(4): br5, 2023 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-36753381

RESUMEN

Centromeres are known to cluster around nucleoli in Drosophila and mammalian cells, but the significance of the nucleoli-centromere interaction remains underexplored. To determine whether the interaction is dynamic under different physiological and pathological conditions, we examined nucleolar structure and centromeres at various differentiation stages using cell culture models and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells. Embryonic stem cells usually have a single large nucleolus, which is clustered with a high percentage of centromeres. As cells differentiate into intermediate states, the nucleolar number increases and the centromere association decreases. In terminally differentiated cells, including myotubes, neurons, and keratinocytes, the number of nucleoli and their association with centromeres are at the lowest. Cancer cells demonstrate the pattern of nucleoli number and nucleoli-centromere association that is akin to proliferative cell types, suggesting that nucleolar reorganization and changes in nucleoli-centromere interactions may play a role in facilitating malignant transformation. This idea is supported in a case of pediatric rhabdomyosarcoma, in which induced differentiation reduces the nucleolar number and centromere association. These findings suggest active roles of nucleolar structure in centromere function and genome organization critical for cellular function in both normal development and cancer.


Asunto(s)
Nucléolo Celular , Neoplasias , Animales , Nucléolo Celular/metabolismo , Centrómero , Núcleo Celular/metabolismo , Mamíferos , Neoplasias/metabolismo
20.
J Cell Biol ; 176(6): 795-805, 2007 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-17339380

RESUMEN

Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A-containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.


Asunto(s)
Autoantígenos/metabolismo , Centrómero/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Mitosis/fisiología , Autoantígenos/análisis , Autoantígenos/fisiología , Centrómero/ultraestructura , Proteína A Centromérica , Cromatina/ultraestructura , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/fisiología , Replicación del ADN , Epigénesis Genética , Fase G1/fisiología , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Genéticos , Proteínas Recombinantes de Fusión/análisis
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