CENP-A Is Dispensable for Mitotic Centromere Function after Initial Centromere/Kinetochore Assembly

Hoffmann, Sebastian; Dumont, Marie; Barra, Viviana; Ly, Peter; Nechemia-Arbely, Yael; McMahon, Moira A.; Hervé, Solène; Cleveland, Don W.; Fachinetti, Daniele

doi:10.1016/j.celrep.2016.10.084

Cited by 93 publications

(136 citation statements)

References 45 publications

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“…components as well as the failure of the kinetochore to correctly assemble at the centromere and ensure faithful chromosome segregation (Régnier et al 2005;Fachinetti et al 2013;Hoffmann et al 2016). Remarkably, HJURP ablation in cell lines also causes mitotic defects due to the loss of CENP-A at centromeres (Dunleavy et al 2009;Foltz et al 2009).…”

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confidence: 99%

Essential role for centromeric factors following p53 loss and oncogenic transformation

et al. 2017

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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.

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Essential role for centromeric factors following p53 loss and oncogenic transformation

et al. 2017

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“…In our current manuscript, we now show that following rapid CENP-A depletion just before mitosis, CENP-B knock-out leads to loss of CENP-C/kinetochore centromeric signal concomitant with a high percentage of mitotic errors. 4 Our studies shed light on the puzzling role of CENP-B for centromere function. Altogether, we demonstrate a reciprocal interdependency of CENP-A chromatin and the underlying repetitive centromere DNA bound by CENP-B in the maintenance of human chromosome segregation 4 ( Fig.…”

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confidence: 82%

“…4 Our studies shed light on the puzzling role of CENP-B for centromere function. Altogether, we demonstrate a reciprocal interdependency of CENP-A chromatin and the underlying repetitive centromere DNA bound by CENP-B in the maintenance of human chromosome segregation 4 ( Fig. 1).…”

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confidence: 82%

“…Indeed, siRNA-mediated CENP-A knock-down is incomplete, and the protein remains detectable even 7 d post gene disruption. 2 In our current manuscript, 4 we now overcome these limitations by combining the use of genome editing with the Auxin-Inducible Degron (AID) tagging system. 5,6 Following tagging of both copies of endogenous CENP-A with AID tag, we achieve complete and rapid depletion of CENP-A after the addition of synthetic auxin (Indol-3-Acetic-Acid, IAA) in every stage of the cell cycle.…”

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confidence: 99%

“…5,6 Following tagging of both copies of endogenous CENP-A with AID tag, we achieve complete and rapid depletion of CENP-A after the addition of synthetic auxin (Indol-3-Acetic-Acid, IAA) in every stage of the cell cycle. 4 How CENP-A is evicted from the chromatin during these degradation processes and whether the entire nucleosome is disassembled and/or replaced are still questions to be answered.…”

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A time out for CENP-A

Hoffmann

Fachinetti

2017

Molecular & Cellular Oncology

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Proper chromosome segregation relies on a functional centromere-kinetochore interface. We showed that chromatin containing CENtromere Protein A (CENP-A) is essential for centromere assembly, but dispensable for chromosome segregation in the presence of CENP-B-bound DNA sequences. This demonstrates the existence of two contact points between the DNA and the kinetochore to mediate successful chromosome segregation.

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Real-Time De Novo Deposition of Centromeric Histone-Associated Proteins Using the Auxin-Inducible Degradation System

Hoffmann

Fachinetti

2018

Methods in Molecular Biology

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Measuring protein dynamics is essential to uncover protein function and to understand the formation of large protein complexes such as centromeres. Recently, genome engineering in human cells has improved our ability to study the function of endogenous proteins. By combining genome editing techniques with the auxin-inducible degradation (AID) system, we created a versatile tool to study protein dynamics. This system allows us to analyze both protein function and dynamics by enabling rapid protein depletion and reexpression in the same experimental setup. Here, we focus on the dynamics of the centromeric histone-associated protein CENP-C, responsible for the formation of the kinetochore complex. Following rapid removal and reactivation of a fluorescent version of CENP-C by auxin treatment and removal, we could follow CENP-C de novo deposition at centromeric regions during different stages of the cell cycle. In conclusion, the auxin degradation system is a powerful tool to assess and quantify protein dynamics in real time.

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CENP-A Is Dispensable for Mitotic Centromere Function after Initial Centromere/Kinetochore Assembly

Cited by 93 publications

References 45 publications

Essential role for centromeric factors following p53 loss and oncogenic transformation

Essential role for centromeric factors following p53 loss and oncogenic transformation

A time out for CENP-A

Real-Time De Novo Deposition of Centromeric Histone-Associated Proteins Using the Auxin-Inducible Degradation System

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