In all eukaryotes, the large ribosomal RNAs are transcribed from repeated ribosomal DNA (rDNA) genes. These rDNA repeats form nucleoli, which are specialized, non-membrane-bound sub-nuclear organelles that are the sites of ribosome assembly. Additionally, nucleoli are dynamic hubs through which numerous proteins shuttle. Less well investigated is the role of nucleoli in organizing the three dimensional structure of mammalian genomes. Long-range chromosome interactions are of great interest because they can regulate the developmental timing or the variegation of gene expression in mammalian cells. Deep sequencing analyses of DNA associated with isolated nucleoli from human somatic cell lines have shown that specific loci, termed nucleolar-associated domains (NADs), form frequent three-dimensional associations with nucleoli. NADs are dynamic, being redistributed to the nuclear periphery or to pericentric heterochromatin foci upon nucleolar alteration via inhibition of rDNA transcription. The human cell lines in which NADs have been studied to date are not suited for answering broad questions about the role of NADs in mammalian development. Early development is a critical period to study NAD biological function, not just because of the fundamental biological events that occur, but also because interactions between pericentromeric chromatin and perinucleolar regions are particularly dynamic during mammalian preimplantation embryonic development. We therefore propose that the biological importance of the 3D genome associations maintained by nucleoli should be explored in a system that allows analysis of mammalian developmental processes; that is, a system in which the functionality of these interactions can be explored in four dimensions. Therefore, we propose for the first time to map of the nucleolar-associated domains (NADs) in the mouse genome, determine how these associations are altered during embryonic stem cell (ESC) differentiation, and develop tools for study of these higher-order chromosome interaction in fixed and live single cells. In keeping with the goals of the NIH Initiative, we intend to produe databases and tools for understanding the 4D regulation of mammalian genome structure and function via NAD interactions, as a comprehensive foundation for the mammalian developmental biology community. Furthermore, we will determine how these associations are altered upon differentiation into each of the three germ layers, and how they are correlated with the global genome reorganization that occurs in post-implantation epiblasts. In addition to these population measurements, we will generate tools for the visualization of the repeat-rich DNAs associated with nucleoli in live, single cells via CRISPR-based targeting. In this manner, our project will be the first to analyze the dynamics of NAD-mediated genome organization during mammalian cell differentiation. In sum, the comprehensive database created by this project will constitute a major new tool for the mammalian developmental biology and genomics communities.

Public Health Relevance

The three-dimensional organization of chromosomes is regulated in part by associations with the largest sub- nuclear organelle, the nucleolus. However, nucleolus-chromosome associations have not been previously explored during cellular differentiation. Therefore, we plan to map these associations in unprecedented detail during mouse embryonic stem cell differentiation, and to develop tools to visualize these associations in live cells. These studies will provide invaluable tools for studying the structure and function of the genome during landmark events in mammalian embryology.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Research Project--Cooperative Agreements (U01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Satterlee, John S
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Massachusetts Medical School Worcester
Anatomy/Cell Biology
Schools of Medicine
United States
Zip Code
Ma, Hanhui; Tu, Li-Chun; Naseri, Ardalan et al. (2018) CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods 15:928-931
Sun, Xiaoming; Kaufman, Paul D (2018) Ki-67: more than a proliferation marker. Chromosoma 127:175-186
Matheson, Timothy D; Kaufman, Paul D (2017) The p150N domain of chromatin assembly factor-1 regulates Ki-67 accumulation on the mitotic perichromosomal layer. Mol Biol Cell 28:21-29
Sun, Xiaoming; Bizhanova, Aizhan; Matheson, Timothy D et al. (2017) Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells. Mol Cell Biol 37:
Belaghzal, Houda; Dekker, Job; Gibcus, Johan H (2017) Hi-C 2.0: An optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation. Methods 123:56-65
Oomen, Marlies E; Dekker, Job (2017) Epigenetic characteristics of the mitotic chromosome in 1D and 3D. Crit Rev Biochem Mol Biol 52:185-204
Nora, Elphège P; Goloborodko, Anton; Valton, Anne-Laure et al. (2017) Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization. Cell 169:930-944.e22
Ma, Hanhui; Tu, Li-Chun; Naseri, Ardalan et al. (2016) CRISPR-Cas9 nuclear dynamics and target recognition in living cells. J Cell Biol 214:529-37
Valton, Anne-Laure; Dekker, Job (2016) TAD disruption as oncogenic driver. Curr Opin Genet Dev 36:34-40
Dekker, Job; Mirny, Leonid (2016) The 3D Genome as Moderator of Chromosomal Communication. Cell 164:1110-1121

Showing the most recent 10 out of 11 publications