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Team Publications
Molecular Mechanisms of Chromosome Dynamics
Year of publication 2013
Daniele Fachinetti, H Diego Folco, Yael Nechemia-Arbely, Luis P Valente, Kristen Nguyen, Alex J
Wong, Quan Zhu, Andrew J Holland, Arshad Desai, Lars E T Jansen, Don W Cleveland (2013 Feb
7)
A two-step mechanism for epigenetic specification of centromere identity and
function.
Nature cell biology : 1056-66 : DOI : 10.1038/ncb2805
Summary
The basic determinant of chromosome inheritance, the centromere, is specified in many
eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast,
chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to
be the epigenetic mark that acts through a two-step mechanism to identify, maintain and
propagate centromere function indefinitely. Initially, centromere position is replicated and
maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENPA substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATDcontaining chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A
for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human
centromeres or direct recruitment of CENP-C, respectively.
Year of publication 2012
Andrew J Holland, Daniele Fachinetti, Quan Zhu, Manuel Bauer, Inder M Verma, Erich A Nigg, Don
W Cleveland (2012 Dec 20)
The autoregulated instability of Polo-like kinase 4 limits centrosome duplication
to once per cell cycle.
Genes & development : 2684-9 : DOI : 10.1101/gad.207027.112
Summary
Centrioles organize the centrosome, and accurate control of their number is critical for the
maintenance of genomic integrity. Centriole duplication occurs once per cell cycle and is
controlled by Polo-like kinase 4 (Plk4). We showed previously that Plk4 phosphorylates itself
to promote its degradation by the proteasome. Here we demonstrate that this autoregulated
instability controls the abundance of endogenous Plk4. Preventing Plk4 autoregulation
causes centrosome amplification, stabilization of p53, and loss of cell proliferation;
moreover, suppression of p53 allows growth of cells carrying amplified centrosomes. Plk4
autoregulation thus guards against genome instability by limiting centrosome duplication to
once per cell cycle.
Andrew J Holland, Daniele Fachinetti, Joo Seok Han, Don W Cleveland (2012 Nov 12)
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1
Team Publications
Molecular Mechanisms of Chromosome Dynamics
Inducible, reversible system for the rapid and complete degradation of proteins
in mammalian cells.
Proceedings of the National Academy of Sciences of the United States of America : E3350-7 : DOI
: 10.1073/pnas.1216880109
Summary
Inducible degradation is a powerful approach for identifying the function of a specific protein
or protein complex. Recently, a plant auxin-inducible degron (AID) system has been shown to
degrade AID-tagged target proteins in nonplant cells. Here, we demonstrate that an AIDtagged protein can functionally replace an endogenous protein depleted by RNAi, leading to
an inducible null phenotype rapidly after auxin addition. The AID system is shown to be
capable of controlling the stability of AID-tagged proteins that are in either nuclear or
cytoplasmic compartments and even when incorporated into protein complexes. Induced
degradation occurs rapidly after addition of auxin with protein half-life reduced to as little as
9 min and proceeding to completion with first-order kinetics. AID-mediated instability is
demonstrated to be rapidly reversible. Induced degradation is shown to initiate and continue
in all cell cycle phases, including mitosis, making this system especially useful for identifying
the function(s) of proteins of interest during specific points in the mammalian cell cycle.
Andrew J Holland, Daniele Fachinetti, Sandrine Da Cruz, Quan Zhu, Benjamin Vitre, Mariana
Lince-Faria, Denaly Chen, Nicole Parish, Inder M Verma, Monica Bettencourt-Dias, Don W
Cleveland (2012 Mar 30)
Polo-like kinase 4 controls centriole duplication but does not directly regulate
cytokinesis.
Molecular biology of the cell : 1838-45 : DOI : 10.1091/mbc.E11-12-1043
Summary
Centrioles organize the centrosome, and accurate control of their number is critical for the
maintenance of genomic integrity. Centrioles duplicate once per cell cycle, and duplication is
coordinated by Polo-like kinase 4 (Plk4). We previously demonstrated that Plk4 accumulation
is autoregulated by its own kinase activity. However, loss of heterozygosity of Plk4 in mouse
embryonic fibroblasts has been proposed to cause cytokinesis failure as a primary event,
leading to centrosome amplification and gross chromosomal abnormalities. Using targeted
gene disruption, we show that human epithelial cells with one inactivated Plk4 allele undergo
neither cytokinesis failure nor increase in centrosome amplification. Plk4 is shown to localize
exclusively at the centrosome, with none in the spindle midbody. Substantial depletion of
Plk4 by small interfering RNA leads to loss of centrioles and subsequent spindle defects that
lead to a modest increase in the rate of cytokinesis failure. Therefore, Plk4 is a centriolelocalized kinase that does not directly regulate cytokinesis.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2