The ciliate Tetrahymena thermophila excises approximately 6000 specific DNA segments from its somatic nucleus during development. This project aims to understand the regulation of this massive genome reorganization and ultimately learn fundamental principles governing chromosome structure and stability. Understanding how particular DNA segments, called internal eliminated sequences (IES), are selectively excised is challenged by the fact that they share little common structure. Currently, we have limited knowledge of the protein machinery that recognizes and excises these 6000 IES. While four proteins have been linked to this process, their exact roles have yet to be fully elucidated. We have identified five genes encoding novel proteins putatively involved in DNA rearrangement. These were identified using a Green Fluorescence Protein (GFP)-based screening strategy designed to find developmentally expressed proteins that localize specifically to the differentiating nuclei where and when DNA rearrangement occurs. To demonstrate whether these five candidates are required for rearrangement, we will knock out their genes and analyze the phenotype of the resulting transgenic strains. Furthermore, we will test whether these proteins interact with previously identified DNA rearrangement proteins and/or associate with the IES during their excision. Initial characterization of one of these genes, LIA1, indicates that its protein very likely participates in DNA rearrangement. Characterization of additional components of the DNA rearrangement machinery will greatly enhance our understanding of this process. This biological phenomenon provides a unique system with which to discover mechanisms cells use to recognize individual chromosomal segments throughout the genome. Such mechanisms are likely critical for ensuring chromosome stability. Given that cancer cells commonly exhibit aberrant DNA rearrangements, identification of the cellular mechanisms that ensure faithful chromosome maintenance is an important step leading to an understanding of the molecular basis of disease associated with genetic instability.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069593-02
Application #
6917002
Study Section
Genetics Study Section (GEN)
Program Officer
Carter, Anthony D
Project Start
2004-08-01
Project End
2009-07-31
Budget Start
2005-08-01
Budget End
2006-07-31
Support Year
2
Fiscal Year
2005
Total Cost
$244,800
Indirect Cost
Name
Washington University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Wiley, Emily A; Horrell, Scott; Yoshino, Alyssa et al. (2018) Diversification of HP1-like Chromo Domain Proteins in Tetrahymena thermophila. J Eukaryot Microbiol 65:104-116
Carle, Christine M; Zaher, Hani S; Chalker, Douglas L (2016) A Parallel G Quadruplex-Binding Protein Regulates the Boundaries of DNA Elimination Events of Tetrahymena thermophila. PLoS Genet 12:e1005842
Horrell, Scott A; Chalker, Douglas L (2014) LIA4 encodes a chromoshadow domain protein required for genomewide DNA rearrangements in Tetrahymena thermophila. Eukaryot Cell 13:1300-11
Schwope, Rachel M; Chalker, Douglas L (2014) Mutations in Pdd1 reveal distinct requirements for its chromodomain and chromoshadow domain in directing histone methylation and heterochromatin elimination. Eukaryot Cell 13:190-201
Shieh, Annie Wan Yi; Chalker, Douglas L (2013) LIA5 is required for nuclear reorganization and programmed DNA rearrangements occurring during tetrahymena macronuclear differentiation. PLoS One 8:e75337
Motl, Jason A; Chalker, Douglas L (2009) Subtraction by addition: domesticated transposases in programmed DNA elimination. Genes Dev 23:2455-60
Chalker, Douglas L (2009) Transposons that clean up after themselves. Genome Biol 10:224
Chalker, Douglas L (2008) Dynamic nuclear reorganization during genome remodeling of Tetrahymena. Biochim Biophys Acta 1783:2130-6
Yao, Meng-Chao; Yao, Ching-Ho; Halasz, Lia M et al. (2007) Identification of novel chromatin-associated proteins involved in programmed genome rearrangements in Tetrahymena. J Cell Sci 120:1978-89
Rexer, Charles H; Chalker, Douglas L (2007) Lia1p, a novel protein required during nuclear differentiation for genome-wide DNA rearrangements in Tetrahymena thermophila. Eukaryot Cell 6:1320-9

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