Delivery of chromosomes, the basic units of inheritance, to each daughter cell during cell division is mediated by the centromere. Unlike typical genes, in metazoans this central genetic element is not determined by DNA sequence. Rather, functional centromeres are determined epigenetically through stable acquisition of an unexplained, non-DNA """"""""mark"""""""". A prime candidate for a component of such an epigenetic mark is CENP-A, a histone H3 variant found exclusively at functional centromeres and which we have previously demonstrated to assemble into conformationally more constained, functionally divergent nucelsomes. We will now determine if the structural rigidity and compactness of nucleosomes targeted to the centromere is sufficient to maintain centromere identity and function in mammalian cells. Using an in vivo fluorescence pulse-chase tagging method we identified, we will determine the timing of loading of constitutive components required for CENP-A loading, as well as the DNA targets for centromre components CENP-T and CENP-W. To identify how centromeric chromatin is replicated, we will identify cell cycle-dependent covalent modification to CENP-A and its assembly chaperone HJURP, and will identify how timing and loading is altered in cells with reduced HJURP. Following our discovery that CENP-A and other components known for essential roles in centromere assembly are rapidly recruited to sites of DNA damage, we will assess the roles of CENP-A and its partners in DNA repair. This will include determination of the mechanism of transient CENP-A recruitment to sites of DNA damage and the extent of chromatin remodeling following DNA damage. Finally, methods will be developed for rapidly following de novo centromere assembly in mammalian cells after introduction of large arrays of centromeric alphoid DNA carried on bacterial or yeast artificial chromosomes. These will be used to identify factors critical for formation of new centromeres.

Public Health Relevance

Understanding how centromeres function and the genetic mechanisms that may generate failure of normal chromosome delivery have broad medical implications. Among these, errors of chromosome segregation lead to infertility. Moreover, many human tumors have highly abnormal numbers of chromosomes (that is, they are aneuploid), with initial chromosomal loss participating in the early steps of the transformation cascade in inherited cancers caused by heterozygous mutation in tumor suppressor genes and the more widespread aneuploidy characteristic of advance tumors thought to drive acquisition of malignant growth properties.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM074150-05
Application #
7785239
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Deatherage, James F
Project Start
2006-02-01
Project End
2014-01-31
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
5
Fiscal Year
2010
Total Cost
$353,470
Indirect Cost
Name
Ludwig Institute for Cancer Research Ltd
Department
Type
DUNS #
627922248
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Guo, Lucie Y; Allu, Praveen Kumar; Zandarashvili, Levani et al. (2017) Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition. Nat Commun 8:15775
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
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
Hoffmann, Sebastian; Dumont, Marie; Barra, Viviana et al. (2016) CENP-A Is Dispensable for Mitotic Centromere Function after Initial Centromere/Kinetochore Assembly. Cell Rep 17:2394-2404
Bertuzzi, Stefano; Cleveland, Don W (2015) The curious incident of the translational dog that didn't bark. Trends Cell Biol 25:187-9
Fachinetti, Daniele; Han, Joo Seok; McMahon, Moira A et al. (2015) DNA Sequence-Specific Binding of CENP-B Enhances the Fidelity of Human Centromere Function. Dev Cell 33:314-27
Rahdar, Meghdad; McMahon, Moira A; Prakash, Thazha P et al. (2015) Synthetic CRISPR RNA-Cas9-guided genome editing in human cells. Proc Natl Acad Sci U S A 112:E7110-7
Bodor, Dani L; Mata, João F; Sergeev, Mikhail et al. (2014) The quantitative architecture of centromeric chromatin. Elife 3:e02137
Earnshaw, W C; Allshire, R C; Black, B E et al. (2013) Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant. Chromosome Res 21:101-6

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