Defects in the equal partitioning of chromosomes at cell division causes aneuploidy, a genetic catastrophe that results in spontaneous abortion or birth defects if it arises in the gametes and that is a major contributor to gene dosage imbalances in almost all human cancers. The centromere is the locus on each chromosome that directs accurate chromosome segregation at cell division in healthy cells. The focus of this project is to address two related major questions regarding centromeres: what is the nature of the epigenetic mark that specifies human centromeres and how is this epigenetic mark propagated through cell divisions? Centromeres are the chromosomal loci that confer genetic stability at cell division, but the DNA sequences typically found at the loci are neither necessary nor sufficient for centromere function. Rather, centromeres are specified in metazoans and many other eukaryotes through an epigenetic process. Key to epigenetic centromere specification is a histone H3 variant, CENP-A, and in this proposal we now build on the major findings we made in the previous funding cycle regarding its structure/dynamics, cell cycled-coupled chromatin assembly, and the organization and recruitment of centromere components it directs to human centromeres and neocentromeres. We have identified three critical areas of investigation that are now ripe for eliciting major insight at centromeres: 1) identifying the majo form of the histone/DNA particle into which it assembles at functional human centromeres-representing the fundamental unit of centromeric chromatin and the prime candidate to epigenetically specify centromere location, 2) determining the molecular basis for the cell cycle-coupled program of centromere inheritance, and 3) investigating the stability of CENP-A nucleosomes at individual centromeres and the role therein of other centromere constituents. We will pursue these topics using biochemical, genomic, proteomic, molecular genetic and cell biological approaches, and together these studies have the promise to generate valuable insight into the epigenetic mechanisms that specify and maintain centromere location on the chromosome.

Public Health Relevance

Chromosomal inheritance must be flawless every time the cell divides or else unequal chromosome partitioning in the daughter cells, along with the imbalanced dosage of the genes that they carry, will lead to major medical problems such as spontaneous abortion of embryos and fetuses, birth defects in newborns, and tumor formation and progression in adults. Genome partitioning is controlled by proteins, rather than by a particular DNA sequence on the chromosome. The work proposed here promises to advance our knowledge of how this critical process is performed without error in healthy cells and which molecules may be to blame when catastrophic loss or gain of a chromosome occurs in disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM082989-06
Application #
8576976
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2008-07-01
Project End
2017-06-30
Budget Start
2013-07-16
Budget End
2014-06-30
Support Year
6
Fiscal Year
2013
Total Cost
$315,985
Indirect Cost
$115,985
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Falk, Samantha J; Lee, Jaehyoun; Sekulic, Nikolina et al. (2016) CENP-C directs a structural transition of CENP-A nucleosomes mainly through sliding of DNA gyres. Nat Struct Mol Biol 23:204-8
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Bailey, Aaron O; Panchenko, Tanya; Shabanowitz, Jeffrey et al. (2016) Identification of the Post-translational Modifications Present in Centromeric Chromatin. Mol Cell Proteomics 15:918-31
Logsdon, Glennis A; Barrey, Evelyne J; Bassett, Emily A et al. (2015) Both tails and the centromere targeting domain of CENP-A are required for centromere establishment. J Cell Biol 208:521-31
McKinley, Kara L; Sekulic, Nikolina; Guo, Lucie Y et al. (2015) The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface. Mol Cell 60:886-98
Falk, Samantha J; Guo, Lucie Y; Sekulic, Nikolina et al. (2015) Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere. Science 348:699-703
Wei, Sijie; Falk, Samantha J; Black, Ben E et al. (2015) A novel hybrid single molecule approach reveals spontaneous DNA motion in the nucleosome. Nucleic Acids Res 43:e111
Geiss, Christian P; Keramisanou, Dimitra; Sekulic, Nikolina et al. (2014) CENP-A arrays are more condensed than canonical arrays at low ionic strength. Biophys J 106:875-82
DeNizio, Jamie E; Elsässer, Simon J; Black, Ben E (2014) DAXX co-folds with H3.3/H4 using high local stability conferred by the H3.3 variant recognition residues. Nucleic Acids Res 42:4318-31
Bodor, Dani L; Mata, João F; Sergeev, Mikhail et al. (2014) The quantitative architecture of centromeric chromatin. Elife 3:e02137

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