Telomere integrity is essential for cell survival and proliferation. Accordingly, dysfunctional telomeres can initiate genomic instability, cellular senescence, and organismal aging. Telomere structure is provided by binding of shelterin to telomeric DNA repeats forming a nucleoprotein complex. Similar to the human shelterin, in fission yeast, Schizosaccharomyces pombe, shelterin is composed of telomeric sequence-specific double-stranded and single-stranded DNA binding proteins, Taz1 and Pot1, respectively, accompanied by their protein interaction partners, Rap1, Poz1, and Tpz1, which form a "protein bridge" connecting Taz1 and Pot1. Shelterin protects telomere integrity as well as regulates telomerase. With the parent grant support, we biochemically identified Tpz1 mutants that can individually but specifically disrupt its interactions with Poz1, Ccq1, or Pot1. Using these separation-of-function mutants of Tpz1, we found that the complete linkage between telomere dsDNA and ssDNA binding proteins within the shelterin complex is required for defining the telomerase-nonextendible state of telomeres. Moreover, epistasis analyses revealed that Tpz1 also participates in the activation of telomeres to the extendible state via its interaction with Ccq1. Our results suggest critical regulatory roles of Tpz1 in the telomere ON/OFF binary switch. Here, we request supplemental funds to use emerging technologies and interdisciplinary teams to further advance our mechanistic understanding of shelterin to a new level. The two aims are: (1) Identify shelterin interactomes and post-translational modifications on shelterin components using chemical cross-linking coupled with mass spectrometry analysis (CXMS);(2) Reveal the functional roles of Tpz1 in coordinating shelterin components for telomerase-mediated and recombination-based telomere elongation via analyzing telomere sequences using single molecule, real-time (SMRT) next- generation sequencing technology.

Public Health Relevance

Telomeres are closely involved in stem cell differentiation and cancer cell proliferation. The results of this study will lead to new mechanistc models for telomere length regulation and chromosome end protection. Therefore, valuable targets for mechanism- driven design of cancer and aging therapeutics may be identified through this research.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM098943-04S1
Application #
8738357
Study Section
Special Emphasis Panel (ZGM1-TRN-0 (MI))
Program Officer
Carter, Anthony D
Project Start
2012-05-01
Project End
2017-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
4
Fiscal Year
2014
Total Cost
$67,594
Indirect Cost
$23,844
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Tadeo, Xavier; Wang, Jiyong; Kallgren, Scott P et al. (2013) Elimination of shelterin components bypasses RNAi for pericentric heterochromatin assembly. Genes Dev 27:2489-99
Jun, Hyun-Ik; Liu, Jinqiang; Jeong, Heetae et al. (2013) Tpz1 controls a telomerase-nonextendible telomeric state and coordinates switching to an extendible state via Ccq1. Genes Dev 27:1917-31