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Although originally thought to be silent chromosomal regions, centromeres are instead actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA- smFISH foci levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere-nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions. [ABSTRACT FROM AUTHOR]

Establishing the bipolar spindle in mammalian oocytes after their prolonged arrest is crucial for meiotic fidelity and subsequent development. In contrast to somatic cells, the first meiotic spindle assembles in the absence of centriolecontaining centrosomes. Ran-GTP can promote microtubule nucleation near chromatin, but additional unidentified factors are postulated for the activity of multiple acentriolar microtubule organizing centers in the oocyte. We now demonstrate that partially overlapping, nonredundant functions of Aurora A and Plk4 kinases contribute to initiate acentriolar meiosis I spindle formation. Loss of microtubule nucleation after simultaneous chemical inhibition of both kinases can be significantly rescued by drug-resistant Aurora A alone. Drug-resistant Plk4 can enhance Aurora A-mediated rescue, and, accordingly, Plk4 can phosphorylate and potentiate the activity of Aurora A in vitro. Both kinases function distinctly from Ran, which amplifies microtubule growth. We conclude that Aurora A and Plk4 are rate-limiting factors contributing to microtubule growth as the acentriolar oocyte resumes meiosis. [ABSTRACT FROM AUTHOR]

Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C. [ABSTRACT FROM AUTHOR]

Summary: During the first five rounds of cell division in the mouse embryo, spindles assemble in the absence of centrioles. Spindle formation initiates around chromosomes, but the microtubule nucleating process remains unclear. Here we demonstrate that Plk4, a protein kinase known as a master regulator of centriole formation, is also essential for spindle assembly in the absence of centrioles. Depletion of maternal Plk4 prevents nucleation and growth of microtubules and results in monopolar spindle formation. This leads to cytokinesis failure and, consequently, developmental arrest. We show that Plk4 function depends on its kinase activity and its partner protein, Cep152. Moreover, tethering Cep152 to cellular membranes sequesters Plk4 and is sufficient to trigger spindle assembly from ectopic membranous sites. Thus, the Plk4-Cep152 complex has an unexpected role in promoting microtubule nucleation in the vicinity of chromosomes to mediate bipolar spindle formation in the absence of centrioles. [Copyright &y& Elsevier]

As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1. • Mitotic centromere transcription requires cohesin • Prophase cohesin removal releases elongating Pol II and nascent RNA from chromatin • Chromatin release of Pol II/nascent RNA facilitates accurate chromosome segregation • The prophase pathway ensures G2/G1 gene expression reprograming across mitosis During mitosis, chromosomes condense and transcription is silenced to facilitate chromosome segregation. However, centromeres are transcribed during mitosis. Perea-Resa et al. find that mitotic cohesin regulation is important for mitotic transcriptional silencing, mitotic centromere transcription, and transcriptional restart in G1. Coupled cohesin/transcription dynamics ensures chromosome segregation and gene expression reprograming across mitosis. [ABSTRACT FROM AUTHOR]

Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant centromere protein A (CENP-A). Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential. • CENP-A nucleosomes are gradually incorporated in quiescent cells and oocytes • CENP-A deposition during quiescence is required for future chromosome segregation • RNA Polymerase transcription at centromeres promotes gradual CENP-A exchange • Terminally differentiated muscle cells fail to retain CENP-A nucleosomes Epigenetic marks must be retained during extended periods of quiescence to ensure the proper function of genomic loci. Although centromeric CENP-A nucleosomes were thought to be immobile, Swartz et al. identify gradual CENP-A deposition in quiescent cells and oocytes. CENP-A exchange is essential for faithful cell division following long arrests. [ABSTRACT FROM AUTHOR]