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1.
J Cell Biol ; 218(1): 134-149, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30396998

RESUMO

The centromere is an important genomic locus for chromosomal segregation. Although the centromere is specified by sequence-independent epigenetic mechanisms in most organisms, it is usually composed of highly repetitive sequences, which associate with heterochromatin. We have previously generated various chicken DT40 cell lines containing differently positioned neocentromeres, which do not contain repetitive sequences and do not associate with heterochromatin. In this study, we performed systematic 4C analysis using three cell lines containing differently positioned neocentromeres to identify neocentromere-associated regions at the 3D level. This analysis reveals that these neocentromeres commonly associate with specific heterochromatin-rich regions, which were distantly located from neocentromeres. In addition, we demonstrate that centromeric chromatin adopts a compact structure, and centromere clustering also occurs in vertebrate interphase nuclei. Interestingly, the occurrence of centromere-heterochromatin associations depend on CENP-H, but not CENP-C. Our analyses provide an insight into understanding the 3D architecture of the genome, including the centromeres.


Assuntos
Centrômero/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Epigênese Genética , Genoma , Heterocromatina/ultraestrutura , Animais , Linhagem Celular Tumoral , Centrômero/efeitos dos fármacos , Centrômero/metabolismo , Galinhas , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , Citometria de Fluxo , Heterocromatina/efeitos dos fármacos , Heterocromatina/metabolismo , Ácidos Indolacéticos/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/ultraestrutura , Metiltransferases/genética , Metiltransferases/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
2.
Biomed Res ; 39(2): 75-85, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29669986

RESUMO

Separation of sister chromatids is a drastic and irreversible step in the cell cycle. The key biochemistry behind this event is the proteolysis mediated by the ubiquitin ligase called the anaphase promoting complex, or APC/C. Securin and cyclin B1 are the two established substrates for APC/C whose degradation releases separase and inactivates cyclin B1-dependent kinase 1 (cdk1), respectively, at the metaphase-to-anaphase transition. In this study, we have combined biochemical quantifications with mathematical simulations to characterize the kinetic regulation of securin and cyclin B1, in the cytoplasmic and chromosomal compartments, and found that they are differentially distributed and degraded with different rates. Modeling their interaction with separase predicted that activation timing of separase well coincides with the decline of securin-separase concentration in the cytoplasm. Notably, it also coincides with the peak of cyclin B1-separase level on chromosomes, which appeared crucial to coordinate the timing for separase activation and cdk1 inhibition. We have also conducted phosphoproteomic analysis and identified Ki67 as a chromosomal cdk1 substrate whose dephosphorylation is facilitated by cyclin B1-separase interaction in anaphase.


Assuntos
Anáfase/fisiologia , Ciclina B1/metabolismo , Metáfase/fisiologia , Securina/metabolismo , Algoritmos , Citocinese , Células HeLa , Humanos , Mitose/fisiologia , Modelos Biológicos , Fosforilação , Transporte Proteico , Separase/metabolismo
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