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1.
J Cell Sci ; 137(3)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38319136

RESUMO

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.


Assuntos
Proteína Centromérica A , Centrômero , Cinetocoros , Proteína Centromérica A/metabolismo , Segregação de Cromossomos , Animais , Galinhas
2.
Genome Biol ; 25(1): 52, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38378611

RESUMO

BACKGROUND: Centromeres are essential for faithful chromosome segregation during mitosis and meiosis. However, the organization of satellite DNA and chromatin at mouse centromeres and pericentromeres is poorly understood due to the challenges of assembling repetitive genomic regions. RESULTS: Using recently available PacBio long-read sequencing data from the C57BL/6 strain, we find that contrary to the previous reports of their homogeneous nature, both centromeric minor satellites and pericentromeric major satellites exhibit a high degree of variation in sequence and organization within and between arrays. While most arrays are continuous, a significant fraction is interspersed with non-satellite sequences, including transposable elements. Using chromatin immunoprecipitation sequencing (ChIP-seq), we find that the occupancy of CENP-A and H3K9me3 chromatin at centromeric and pericentric regions, respectively, is associated with increased sequence enrichment and homogeneity at these regions. The transposable elements at centromeric regions are not part of functional centromeres as they lack significant CENP-A enrichment. Furthermore, both CENP-A and H3K9me3 nucleosomes occupy minor and major satellites spanning centromeric-pericentric junctions and a low yet significant amount of CENP-A spreads locally at centromere junctions on both pericentric and telocentric sides. Finally, while H3K9me3 nucleosomes display a well-phased organization on major satellite arrays, CENP-A nucleosomes on minor satellite arrays are poorly phased. Interestingly, the homogeneous class of major satellites also phase CENP-A and H3K27me3 nucleosomes, indicating that the nucleosome phasing is an inherent property of homogeneous major satellites. CONCLUSIONS: Our findings reveal that mouse centromeres and pericentromeres display a high diversity in satellite sequence, organization, and chromatin structure.


Assuntos
Elementos de DNA Transponíveis , Nucleossomos , Camundongos , Animais , Proteína Centromérica A/genética , Camundongos Endogâmicos C57BL , Centrômero , Cromatina , DNA Satélite , Autoantígenos
3.
Mol Biol Cell ; 35(4): ar51, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38381564

RESUMO

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC-FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.


Assuntos
Proteínas Cromossômicas não Histona , Transferência Ressonante de Energia de Fluorescência , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Ciclo Celular , Centrômero/metabolismo , Proteína Centromérica A/metabolismo
4.
Cell Signal ; 117: 111110, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38382691

RESUMO

Glutamine addiction is a significant hallmark of metabolic reprogramming in tumors and is crucial to the progression of cancer. Nevertheless, the regulatory mechanisms of glutamine metabolism in endometrial cancer (EC) remains elusive. In this research, we found that elevated expression of CENPA and solute carrier family 38 member 1 (SLC38A1) were firmly associated with worse clinical stage and unfavorable outcomes in EC patients. In addition, ectopic overexpression or silencing of CENPA could either enhance or diminish glutamine metabolism and tumor progression in EC. Mechanistically, CENPA directly regulated the transcriptional activity of the target gene, SLC38A1, leading to enhanced glutamine uptake and metabolism, thereby promoting EC progression. Notably, a prognostic model utilizing the expression levels of CENPA and SLC38A1 genes independently emerged as a prognostic factor for EC. More importantly, CENPA and SLC38A1 were significantly elevated and positively correlated, as well as indicative of poor prognosis in multiple cancers. In brief, our study confirmed that CENPA is a critical transcription factor involved in glutamine metabolism and tumor progression through modulating SLC38A1. This revelation suggests that targeting CENPA could be an appealing therapeutic approach to address pan-cancer glutamine addiction.


Assuntos
Sistema A de Transporte de Aminoácidos , Proteína Centromérica A , Neoplasias do Endométrio , Glutamina , Feminino , Humanos , Sistema A de Transporte de Aminoácidos/genética , Sistema A de Transporte de Aminoácidos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Glutamina/metabolismo , Histonas , Fatores de Transcrição/metabolismo , Proteína Centromérica A/metabolismo
5.
mBio ; 15(3): e0318523, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38380929

RESUMO

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteína Centromérica A/genética , Filogenia , Proteínas Cromossômicas não Histona/genética , Centrômero/metabolismo , Schizosaccharomyces/genética , DNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Saccharomyces cerevisiae/genética
6.
Nat Cell Biol ; 26(1): 45-56, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38168769

RESUMO

To faithfully segregate chromosomes during vertebrate mitosis, kinetochore-microtubule interactions must be restricted to a single site on each chromosome. Prior work on pair-wise kinetochore protein interactions has been unable to identify the mechanisms that prevent outer kinetochore formation in regions with a low density of CENP-A nucleosomes. To investigate the impact of higher-order assembly on kinetochore formation, we generated oligomers of the inner kinetochore protein CENP-T using two distinct, genetically engineered systems in human cells. Although individual CENP-T molecules interact poorly with outer kinetochore proteins, oligomers that mimic centromeric CENP-T density trigger the robust formation of functional, cytoplasmic kinetochore-like particles. Both in cells and in vitro, each molecule of oligomerized CENP-T recruits substantially higher levels of outer kinetochore components than monomeric CENP-T molecules. Our work suggests that the density dependence of CENP-T restricts outer kinetochore recruitment to centromeres, where densely packed CENP-A recruits a high local concentration of inner kinetochore proteins.


Assuntos
Proteínas Cromossômicas não Histona , Cinetocoros , Humanos , Proteína Centromérica A/genética , Cinetocoros/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/genética , Centrômero/metabolismo , Nucleossomos , Mitose
7.
Oncogene ; 43(11): 804-820, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38279062

RESUMO

HJURP is overexpressed in several cancer types and strongly correlates with patient survival. However, the mechanistic basis underlying the association of HJURP with cancer aggressiveness is not well understood. HJURP promotes the loading of the histone H3 variant, CENP-A, at the centromeric chromatin, epigenetically defining the centromeres and supporting proper chromosome segregation. In addition, HJURP is associated with DNA repair but its function in this process is still scarcely explored. Here, we demonstrate that HJURP is recruited to DSBs through a mechanism requiring chromatin PARylation and promotes epigenetic alterations that favor the execution of DNA repair. Incorporation of HJURP at DSBs promotes turnover of H3K9me3 and HP1, facilitating DNA damage signaling and DSB repair. Moreover, HJURP overexpression in glioma cell lines also affected global structure of heterochromatin independently of DNA damage induction, promoting genome-wide reorganization and assisting DNA damage response. HJURP overexpression therefore extensively alters DNA damage signaling and DSB repair, and also increases radioresistance of glioma cells. Importantly, HJURP expression levels in tumors are also associated with poor response of patients to radiation. Thus, our results enlarge the understanding of HJURP involvement in DNA repair and highlight it as a promising target for the development of adjuvant therapies that sensitize tumor cells to irradiation.


Assuntos
Cromatina , Glioma , Humanos , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glioma/genética
8.
Cell Mol Life Sci ; 81(1): 27, 2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38212546

RESUMO

BACKGROUND: Breast cancer is a lethal malignancy affecting females worldwide. It has been reported that upregulated centromere protein A (CENPA) expression might indicate unfortunate prognosis and can function as a prognostic biomarker in breast cancer. This study aimed to investigate the accurate roles and downstream mechanisms of CENPA in breast cancer progression. METHODS: CENPA protein levels in breast cancer tissues and cell lines were analyzed by Western blot and immunohistochemistry assays. We used gain/loss-of-function experiments to determine the potential effects of CENPA and phospholipase A2 receptor (PLA2R1) on breast cancer cell proliferation, migration, and apoptosis. Co-IP assay was employed to validate the possible interaction between CENPA and DNA methyltransferase 1 (DNMT1), as well as PLA2R1 and hematopoietically expressed homeobox (HHEX). PLA2R1 promoter methylation was determined using methylation-specific PCR assay. The biological capabilities of CENPA/PLA2R1/HHEX axis in breast cancer cells was determined by rescue experiments. In addition, CENPA-silenced MCF-7 cells were injected into mice, followed by measurement of tumor growth. RESULTS: CENPA level was prominently elevated in breast cancer tissues and cell lines. Interestingly, CENPA knockdown and PLA2R1 overexpression both restrained breast cancer cell proliferation and migration, and enhanced apoptosis. On the contrary, CENPA overexpression displayed the opposite results. Moreover, CENPA reduced PLA2R1 expression through promoting DNMT1-mediated PLA2R1 promoter methylation. PLA2R1 overexpression could effectively abrogate CENPA overexpression-mediated augment of breast cancer cell progression. Furthermore, PLA2R1 interacted with HHEX and promoted HHEX expression. PLA2R1 knockdown increased the rate of breast cancer cell proliferation and migration but restrained apoptosis, which was abrogated by HHEX overexpression. In addition, CENPA silencing suppressed tumor growth in vivo. CONCLUSION: CENPA knockdown restrained breast cancer cell proliferation and migration and attenuated tumor growth in vivo through reducing PLA2R1 promoter methylation and increasing PLA2R1 and HHEX expression. We may provide a promising prognostic biomarker and novel therapeutic target for breast cancer.


Assuntos
Neoplasias , Receptores da Fosfolipase A2 , Feminino , Animais , Camundongos , Proteína Centromérica A/metabolismo , Receptores da Fosfolipase A2/genética , Receptores da Fosfolipase A2/metabolismo , Genes Homeobox , Linhagem Celular Tumoral , Metilação de DNA/genética , Biomarcadores/metabolismo , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética
9.
Nucleic Acids Res ; 52(2): 643-659, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38038247

RESUMO

Mislocalization of overexpressed CENP-A (Cse4 in budding yeast, Cnp1 in fission yeast, CID in flies) contributes to chromosomal instability (CIN) in yeasts, flies, and human cells. Mislocalization of CENP-A is observed in many cancers and this correlates with poor prognosis. Structural mechanisms that contribute to mislocalization of CENP-A are poorly defined. Here, we show that interaction of histone H4 with Cse4 facilitates an in vivo conformational change in Cse4 promoting its mislocalization in budding yeast. We determined that Cse4 Y193A mutant exhibits reduced sumoylation, mislocalization, interaction with histone H4, and lethality in psh1Δ and cdc48-3 strains; all these phenotypes are suppressed by increased gene dosage of histone H4. We developed a new in vivo approach, antibody accessibility (AA) assay, to examine the conformation of Cse4. AA assay showed that wild-type Cse4 with histone H4 is in an 'open' state, while Cse4 Y193A predominantly exhibits a 'closed' state. Increased gene dosage of histone H4 contributes to a shift of Cse4 Y193A to an 'open' state with enhanced sumoylation and mislocalization. We provide molecular insights into how Cse4-H4 interaction changes the conformational state of Cse4 in vivo. These studies advance our understanding for mechanisms that promote mislocalization of CENP-A in human cancers.


Assuntos
Proteínas Cromossômicas não Histona , Histonas , Proteínas de Saccharomyces cerevisiae , Humanos , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Neoplasias/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilação
10.
Genetics ; 226(2)2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-37950911

RESUMO

Chromosome segregation is crucial for the faithful inheritance of DNA to the daughter cells after DNA replication. For this, the kinetochore, a megadalton protein complex, assembles on centromeric chromatin containing the histone H3 variant CENP-A, and provides a physical connection to the microtubules. Here, we report an unanticipated role for enzymes required for ß-1,6- and ß-1,3-glucan biosynthesis in regulating kinetochore function in Saccharomyces cerevisiae. These carbohydrates are the major constituents of the yeast cell wall. We found that the deletion of KRE6, which encodes a glycosylhydrolase/ transglycosidase required for ß-1,6-glucan synthesis, suppressed the centromeric defect of mutations in components of the kinetochore, foremost the NDC80 components Spc24, Spc25, the MIND component Nsl1, and Okp1, a constitutive centromere-associated network protein. Similarly, the absence of Fks1, a ß-1,3-glucan synthase, and Kre11/Trs65, a TRAPPII component, suppressed a mutation in SPC25. Genetic analysis indicates that the reduction of intracellular ß-1,6- and ß-1,3-glucans, rather than the cell wall glucan content, regulates kinetochore function. Furthermore, we found a physical interaction between Kre6 and CENP-A/Cse4 in yeast, suggesting a potential function for Kre6 in glycosylating CENP-A/Cse4 or another kinetochore protein. This work shows a moonlighting function for selected cell wall synthesis proteins in regulating kinetochore assembly, which may provide a mechanism to connect the nutritional status of the cell to cell-cycle progression and chromosome segregation.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , beta-Glucanas , Saccharomyces cerevisiae/genética , Cinetocoros/metabolismo , Proteína Centromérica A/genética , Glucanos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Centrômero/metabolismo , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/genética
11.
Nucleic Acids Res ; 52(4): 1688-1701, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38084929

RESUMO

Centromeric chromatin plays a crucial role in kinetochore assembly and chromosome segregation. Centromeres are specified through the loading of the histone H3 variant CENP-A by the conserved chaperone Scm3/HJURP. The N-terminus of Scm3/HJURP interacts with CENP-A, while the C-terminus facilitates centromere localization by interacting with the Mis18 holocomplex via a small domain, called the Mis16-binding domain (Mis16-BD) in fission yeast. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) of unknown function. Here, we find that CYS binds zinc in vitro and is essential for the localization and function of fission yeast Scm3. Disrupting CYS by deletion or introduction of point mutations within its zinc-binding motif prevents Scm3 centromere localization and compromises kinetochore integrity. Interestingly, CYS alone can localize to the centromere, albeit weakly, but its targeting is greatly enhanced when combined with Mis16-BD. Expressing a truncated protein containing both Mis16-BD and CYS, but lacking the CENP-A binding domain, causes toxicity and is accompanied by considerable chromosome missegregation and kinetochore loss. These effects can be mitigated by mutating the CYS zinc-binding motif. Collectively, our findings establish the essential role of the cysteine-rich domain in fungal Scm3 proteins and provide valuable insights into the mechanism of Scm3 centromere targeting.


Assuntos
Proteínas de Transporte , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Transporte/genética , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cisteína/metabolismo , Cinetocoros/metabolismo , Chaperonas Moleculares/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Zinco/metabolismo
12.
Nat Commun ; 14(1): 7947, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-38040722

RESUMO

The centromere components cohesin, CENP-A, and centromeric DNA are essential for biorientation of sister chromatids on the mitotic spindle and accurate sister chromatid segregation. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We use ChIP-seq and super-resolution microscopy with single particle averaging to examine the geometry of essential centromeric components on human chromosomes. Both modalities suggest cohesin is enriched at pericentromeric DNA. CENP-A localizes to a subset of the α-satellite DNA, with clusters separated by ~562 nm and a perpendicular intervening ~190 nM wide axis of cohesin in metaphase chromosomes. Differently sized α-satellite arrays achieve a similar core structure. Here we present a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes, α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation to add components of the chromosome segregation machinery.


Assuntos
Centrômero , DNA Satélite , Humanos , DNA Satélite/genética , Proteína Centromérica A/genética , Centrômero/metabolismo , Mitose , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fuso Acromático/metabolismo , Cromátides/metabolismo , Segregação de Cromossomos
13.
Nat Commun ; 14(1): 8227, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38086807

RESUMO

Centromeres are epigenetically defined via the presence of the histone H3 variant CENP-A. Contacting CENP-A nucleosomes, the constitutive centromere associated network (CCAN) and the kinetochore assemble, connecting the centromere to spindle microtubules during cell division. The DNA-binding centromeric protein CENP-B is involved in maintaining centromere stability and, together with CENP-A, shapes the centromeric chromatin state. The nanoscale organization of centromeric chromatin is not well understood. Here, we use single-molecule fluorescence and cryoelectron microscopy (cryoEM) to show that CENP-A incorporation establishes a dynamic and open chromatin state. The increased dynamics of CENP-A chromatin create an opening for CENP-B DNA access. In turn, bound CENP-B further opens the chromatin fiber structure and induces nucleosomal DNA unwrapping. Finally, removal of CENP-A increases CENP-B mobility in cells. Together, our studies show that the two centromere-specific proteins collaborate to reshape chromatin structure, enabling the binding of centromeric factors and establishing a centromeric chromatin state.


Assuntos
Cromatina , Proteínas Cromossômicas não Histona , Proteína Centromérica A/metabolismo , Microscopia Crioeletrônica , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/metabolismo , Nucleossomos , DNA/metabolismo , Autoantígenos/metabolismo
14.
Int J Biol Sci ; 19(16): 5218-5232, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37928273

RESUMO

The centromere proteins (CENPs), a critical mitosis-related protein complexes, are involved in the kinetochore assembly and chromosome segregation. In this study, we identified that CENPA was significantly up-regulated in HCC and highly expressed CENPA correlated with poor prognosis for HCC patients. Knockdown of CENPA inhibited HCC cell proliferation and tumor growth in vitro and in vivo. Mechanistically, CENPA transcriptionally activated and cooperated with YY1 to drive the expression of cyclin D1 (CCND1) and neuropilin 2 (NRP2). Moreover, we identified that CENPA can be lactylated at lysine 124 (K124). The lactylation of CENPA at K124 promotes CENPA activation, leading to enhanced expression of its target genes. In summary, CENPA function as a transcriptional regulator to promote HCC via cooperating with YY1. Targeting the CENPA-YY1-CCND1/NRP2 axis may provide candidate therapeutic targets for HCC.


Assuntos
Carcinoma Hepatocelular , Proteína Centromérica A , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Histonas , Neoplasias Hepáticas/metabolismo , Fator de Transcrição YY1/genética , Proteína Centromérica A/metabolismo
15.
Int J Mol Sci ; 24(21)2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37958853

RESUMO

Centromeric chromatin is thought to play a critical role in ensuring the faithful segregation of chromosomes during mitosis. However, our understanding of this role is presently limited by our poor understanding of the structure and composition of this unique chromatin. The nucleosomal variant, CENP-A, localizes to narrow regions within the centromere, where it plays a major role in centromeric function, effectively serving as a platform on which the kinetochore is assembled. Previous work found that, within a given cell, the number of microtubules within kinetochores is essentially unchanged between CENP-A-localized regions of different physical sizes. However, it is unknown if the amount of CENP-A is also unchanged between these regions of different sizes, which would reflect a strict structural correspondence between these two key characteristics of the centromere/kinetochore assembly. Here, we used super-resolution optical microscopy to image and quantify the amount of CENP-A and DNA within human centromere chromatin. We found that the amount of CENP-A within CENP-A domains of different physical sizes is indeed the same. Further, our measurements suggest that the ratio of CENP-A- to H3-containing nucleosomes within these domains is between 8:1 and 11:1. Thus, our results not only identify an unexpectedly strict relationship between CENP-A and microtubules stoichiometries but also that the CENP-A centromeric domain is almost exclusively composed of CENP-A nucleosomes.


Assuntos
Microscopia , Nucleossomos , Humanos , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/metabolismo , Cromatina , Cinetocoros/metabolismo , Autoantígenos/química
16.
G3 (Bethesda) ; 14(1)2023 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-37962556

RESUMO

Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast), marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful; however, co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast; however, this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell cycle progression and kinetochore function in wild-type (WT) cells. Suppressors of CENP-A toxicity included gene deletions of subunits of 3 conserved chromatin remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation.


Assuntos
Cinetocoros , Proteínas de Saccharomyces cerevisiae , Humanos , Cinetocoros/metabolismo , Histonas/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Centrômero/genética , Centrômero/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
17.
EMBO Rep ; 24(12): e57702, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-37983946

RESUMO

Successful mitosis depends on the timely establishment of correct chromosomal attachments to microtubules. The kinetochore, a modular multiprotein complex, mediates this connection by recognizing specialized chromatin containing a histone H3 variant called Cse4 in budding yeast and CENP-A in vertebrates. Structural features of the kinetochore that enable discrimination between Cse4/CENP-A and H3 have been identified in several species. How and when these contribute to centromere recognition and how they relate to the overall structure of the inner kinetochore are unsettled questions. More generally, this molecular recognition ensures that only one kinetochore is built on each chromatid and that this happens at the right place on the chromatin fiber. We have determined the crystal structure of a Cse4 peptide bound to the essential inner kinetochore Okp1-Ame1 heterodimer from budding yeast. The structure and related experiments show in detail an essential point of Cse4 contact and provide information about the arrangement of the inner kinetochore.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/metabolismo
18.
Sci Adv ; 9(46): eadi5764, 2023 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-37967185

RESUMO

Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying centromere protein-A (CENP-A) nucleosomes at the nexus of a satellite repeat that we identified and termed π-satellite (π-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring ~6 mega-base pairs of a homogenized π-sat-related repeat, π-satB, that contains >20,000 functional CENP-B boxes. There, CENP-B abundance promotes accumulation of microtubule-binding components of the kinetochore and a microtubule-destabilizing kinesin of the inner centromere. We propose that the balance of pro- and anti-microtubule binding by the new centromere is what permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition.


Assuntos
Autoantígenos , Proteínas Cromossômicas não Histona , Camundongos , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Nucleossomos , Mamíferos/genética
19.
PLoS Genet ; 19(11): e1011066, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38019881

RESUMO

The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila. In Drosophila, CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. These two functions, however, happen at different times in the cell cycle. Furthermore, in Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. We have investigated the dynamics of function of CENP-C during the extended meiotic prophase of Drosophila oocytes and found that maintaining high levels of CENP-C for metaphase I requires expression during prophase. In contrast, CID is relatively stable and does not need to be expressed during prophase to remain at high levels in metaphase I of meiosis. Expression of CID during prophase can even be deleterious, causing ectopic localization to non-centromeric chromatin, abnormal meiosis and sterility. CENP-C prophase loading is required for multiple meiotic functions. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. CENP-C is one of the few proteins identified in which expression during prophase is required for meiotic chromosome segregation. An implication of these results is that the failure to maintain recruitment of CENP-C during the extended prophase in oocytes would result in chromosome segregation errors in oocytes.


Assuntos
Proteínas de Drosophila , Meiose , Animais , Meiose/genética , Segregação de Cromossomos/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Prófase/genética , Centrômero/genética , Centrômero/metabolismo , Drosophila/genética , Drosophila/metabolismo , Mitose , Cinetocoros/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo
20.
Cell Rep ; 42(10): 113178, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37742188

RESUMO

Centromeres are crucial for chromosome segregation, but their underlying sequences evolve rapidly, imposing strong selection for compensatory changes in centromere-associated kinetochore proteins to assure the stability of genome transmission. While this co-evolution is well documented between species, it remains unknown whether population-level centromere diversity leads to functional differences in kinetochore protein association. Mice (Mus musculus) exhibit remarkable variation in centromere size and sequence, but the amino acid sequence of the kinetochore protein CENP-A is conserved. Here, we apply k-mer-based analyses to CENP-A chromatin profiling data from diverse inbred mouse strains to investigate the interplay between centromere variation and kinetochore protein sequence association. We show that centromere sequence diversity is associated with strain-level differences in both CENP-A positioning and sequence preference along the mouse core centromere satellite. Our findings reveal intraspecies sequence-dependent differences in CENP-A/centromere association and open additional perspectives for understanding centromere-mediated variation in genome stability.


Assuntos
Autoantígenos , Proteínas Cromossômicas não Histona , Animais , Camundongos , Autoantígenos/genética , Autoantígenos/metabolismo , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Camundongos Endogâmicos
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