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.
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.
|Fachinetti, Daniele; Folco, H Diego; Nechemia-Arbely, Yael et al. (2013) A two-step mechanism for epigenetic specification of centromere identity and function. Nat Cell Biol 15:1056-66|
|Earnshaw, William C; Cleveland, Don W (2013) CENP-A and the CENP nomenclature: response to Talbert and Henikoff. Trends Genet 29:500-2|
|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|
|Black, Ben E; Cleveland, Don W (2011) Epigenetic centromere propagation and the nature of CENP-a nucleosomes. Cell 144:471-9|
|Lan, Weijie; Cleveland, Don W (2010) Multiclassifier proteomics to define complexes yields new chromosomal proteins. Dev Cell 19:356-9|
|Zeitlin, Samantha G; Baker, Norman M; Chapados, Brian R et al. (2009) Double-strand DNA breaks recruit the centromeric histone CENP-A. Proc Natl Acad Sci U S A 106:15762-7|
|Samoshkin, Alexander; Arnaoutov, Alexei; Jansen, Lars E T et al. (2009) Human condensin function is essential for centromeric chromatin assembly and proper sister kinetochore orientation. PLoS One 4:e6831|
|Foltz, Daniel R; Jansen, Lars E T; Bailey, Aaron O et al. (2009) Centromere-specific assembly of CENP-a nucleosomes is mediated by HJURP. Cell 137:472-84|
|Black, Ben E; Brock, Melissa A; Bedard, Sabrina et al. (2007) An epigenetic mark generated by the incorporation of CENP-A into centromeric nucleosomes. Proc Natl Acad Sci U S A 104:5008-13|
|Jansen, Lars E T; Black, Ben E; Foltz, Daniel R et al. (2007) Propagation of centromeric chromatin requires exit from mitosis. J Cell Biol 176:795-805|
Showing the most recent 10 out of 12 publications