Delivery of chromosomes, the basic units of inheritance, to each daughter cell during cell division is mediated by the centromere. Unlike typical genes for which the DNA sequence is crucial, in metazoans this central genetic element for insuring chromosome inheritance is determined epigenetically rather than by DNA sequence. Over the last 10 years, we have identified the epigenetic mark of centromere identity to be chromatin assembled with the centromere-selective histone variant CENP-A, identified its loading chaperone HJURP, and determined that centromeric chromatin is replicated only at exit from mitosis, half a cell cycle after centromere DNA replication. In the next five years, multiple directions will be undertaken for identifying how centromere identity is replicated and maintained epigenetically, including genome wide analyses to identify the molecular events that mediate an error correction mechanism we have identified which acts to maintain centromeric chromatin assembled with CENP-A, but strips CENP-A misloaded onto non-centromeric sites. Chromosome missegregation or errors in cytokinesis produce aneuploidy, a chromosome content other that a multiple of the haploid number. A major effort will focus on identifying the mechanisms underlying normal chromosome segregation and that act to prevent aneuploidy in the normal situation and testing the consequences of single chromosome missegregation or spindle pole amplification in driving tumorigenesis. We have previously identified the centromere-specific microtubule-dependent motor CENP-E, determined it to be a true microtubule tip tracking kinesin, and demonstrated that limiting amounts of it produce widespread, whole chromosomal aneuploidy in cells and in mice. We have used reconstruction with all purified components and gene targeting/silencing in cells and mice to identify key molecular mechanisms underlying the mitotic checkpoint (also known as the spindle assembly checkpoint), the primary guard against chromosome missegregation in mammals. In the upcoming 5 years, we propose to use gene replacement with CRISPR- Cas9 genome editing and auxin-inducible degron tags to identify key aspects of centromere replication, mitotic checkpoint activation and silencing function, including an initial focus on the joint action of the AAA+ ATPase TRIP13 in catalytic disassembly of mitotic checkpoint inhibitor(s) and/or initial mitotic checkpoint activation. The linkage of aneuploidy to tumorigenesis has long been recognized and aneuploidy is frequent in human cancers. The great German cytologist Theodor Boveri initially proposed related hypotheses that aneuploidy drives tumorigenesis from missegregation of individual chromosomes or an aberrant mitosis caused by centrosome amplification. Using mice that missegregate chromosomes at high frequency from reduced levels of the centromere motor protein CENP-E, we showed previously that whole chromosomal aneuploidy can facilitate tumorigenesis in some genetic contexts, but does not affect tumorigenesis caused by mutations in DNA repair, and delays tumorigenesis when combined with genetic lesions that also increase aneuploidy. We now will test how centrosome amplification affects tumorigenesis. Using a conditional mouse model we have produced in which extra centrosomes can be transiently induced, we will determine whether centrosome amplification promotes cellular transformation or the formation of spontaneous tumors, is capable of facilitating the development of carcinogen-induced tumors, and is able to accelerate the development (or increase the aggressiveness or metastatic potential) of tumors driven by the loss of a tumor suppressor gene. A related chromosomal abnormality linked to chromosome missegregation is chromothripsis (also known as chromoanagenesis), an event in which one (or two) chromosomes appear to have been shattered into tens to hundreds of small genomic fragments and religated back together in random order. Chromotriptic chromosomes were identified by sequencing and are now recognized to be present in a broad range of cancers. Efforts with human cells and genetic plant models have suggested that initial missegregation into micronuclei can trigger chromothripsis. We propose now to test mechanisms of chromothripsis using an approach to generate missegregation of a specific chromosome (the Y) into micronuclei at high efficiency. By exploiting a unique feature of the human Y centromere, we have produced cells in which we can produce selective, transient inactivation of the Y centromere, with the Y chromosome missegregated into micronuclei at high frequency. We will use this approach to determine whether sustained and/or transient centromere inactivation can produce stably heritable chromothripsis from chromosomes fragmented within micronuclei and to determine the repair mechanisms underlying reassembly of fragmented micronuclear chromosomes to generate chromothripsis. Related to this, new directions will be to identify the chromosome shattering and reassembly events that underlie gene amplification during acquired drug resistance, including generation of double minutes or homogenous staining regions.

Public Health Relevance

The essential function of mitosis is the delivery of a complete set of chromosomes to each daughter cell. Inheritance of each chromosome is mediated by a specialized region on it, called the centromere. Multiple directions will be undertaken for identifying how centromere identity is replicated and maintained. Errors in chromosome inheritance produce an abnormal chromosome number, called aneuploidy, and such errors have long been linked to human tumors. The effort here will identify key steps through which the major cell cycle mechanism in mitosis, the mitotic checkpoint, acts to prevent chromosome missegregation. Additionally, amplification of centrosomes, the microtubule organizing centers of mitotic spindles, has also long been associated with tumorigenesis. Using mice in which extra centrosome replication can be transiently induced, it will be determined whether centrosome amplification promotes cellular transformation, the formation of spontaneous tumors or accelerates the development of tumors that develop from loss of a tumor suppressor gene. Finally, sequencing efforts from human tumors have uncovered that a frequent event in a broad range of cancers is chromothripsis, a phenomenon in which one chromosome appears to have been shattered into tens to hundreds of small pieces and then stitched back together in random order. We propose now to test mechanisms of chromothripsis using an approach to generate missegregation of a specific chromosome (the Y) into micronuclei at high efficiency. We will also identify how chromosome fragmentation and religation produces the gene amplifications that occur in acquired drug resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
1R35GM122476-01
Application #
9275206
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Deatherage, James F
Project Start
2017-05-01
Project End
2022-02-28
Budget Start
2017-05-01
Budget End
2018-02-28
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Ludwig Institute for Cancer Research Ltd
Department
Type
DUNS #
627922248
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Nechemia-Arbely, Yael; Fachinetti, Daniele; Miga, Karen H et al. (2017) Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points. J Cell Biol 216:607-621
Ly, Peter; Teitz, Levi S; Kim, Dong H et al. (2017) Selective Y centromere inactivation triggers chromosome shattering in micronuclei and repair by non-homologous end joining. Nat Cell Biol 19:68-75
Ly, Peter; Cleveland, Don W (2017) Rebuilding Chromosomes After Catastrophe: Emerging Mechanisms of Chromothripsis. Trends Cell Biol 27:917-930
Ye, Qiaozhen; Kim, Dong Hyun; Dereli, Ihsan et al. (2017) The AAA+ ATPase TRIP13 remodels HORMA domains through N-terminal engagement and unfolding. EMBO J 36:2419-2434
Fachinetti, Daniele; Logsdon, Glennis A; Abdullah, Amira et al. (2017) CENP-A Modifications on Ser68 and Lys124 Are Dispensable for Establishment, Maintenance, and Long-Term Function of Human Centromeres. Dev Cell 40:104-113
Levine, Michelle S; Bakker, Bjorn; Boeckx, Bram et al. (2017) Centrosome Amplification Is Sufficient to Promote Spontaneous Tumorigenesis in Mammals. Dev Cell 40:313-322.e5