Telomeric protein function and regulation Project Summary/Abstract Continued cell division mandates the maintenance of chromosome ends, which undergo shortening due to the inability of the replicative DNA polymerases to completely synthesize them. Telomerase is a unique ribonuclear protein enzyme that can bind chromosomes ends to extend them with repetitive sequences called telomeric DNA. Using this mechanism, telomerase can help reduce the erosion of chromosome ends and sustain the continued proliferation of actively dividing cells such as somatic stem cells, and cells that make up ~90% of human cancers. Whereas aberrant activation of telomerase in non-dividing somatic cells predisposes them to cancer, mutations in genes that reduce telomerase function in stem cells result in one of many premature aging diseases, including dyskeratosis congenita. Therefore understanding how telomerase function is tightly regulated in cells bears major implications for the biology of aging, and for diseases such as cancer. Telomeric DNA in the cells is not exposed, because this would result in its recognition and repair by the DNA damage response/repair machineries in the cell, leading to illicit end-to-end DNA-joining events at chromosome ends. Our cells therefore have in place a six-protein complex named shelterin that specifically binds telomeric DNA to protect it from illicit DNA repair events. TPP1 is a unique shelterin protein that is central to both telomerase function and chromosome end protection. However, how a single TPP1 gene facilitates the solutions of such distinct biological problems remains unknown. Using a multi-disciplinary approach that includes biochemistry, cell biology, single-molecule microscopy, X-ray crystallography, and bioinformatics, this proposal aims to understand how TPP1 upholds chromosome end protection and end replication.
Aim 1 of the proposal will reveal at a molecular level how TPP1 helps protect the single-stranded regions of telomeric DNA with the help of the POT1 protein.
Aim 2 of this proposal will involve a novel selective-knockout strategy using CRISPR- Cas9 technology, to ask how natural isoforms of TPP1 facilitate different aspects of end protection and end replication.
Aim 3 will explore the mechanism-of-action and pervasiveness of novel RNAs that regulate TPP1 expression in human cells using RNA biochemistry, cell biology and bioinformatics approaches. These studies will reveal the mechanistic basis for how a single TPP1 gene can orchestrate both chromosome end protection and end replication, and discover previously unanticipated mechanisms by which noncoding RNAs regulate TPP1 abundance and function in human cells.

Public Health Relevance

Telomeric protein function and regulation Relevance Telomeres and telomerase are complexes found at chromosome ends that are essential for proper cell function, and misregulation of which result in a wide range of diseases including cancer and inherited stem cell deficiency disorders. This project aims to reveal how a gene coding for the TPP1 protein facilitates telomere and telomerase function. These studies will help design inhibitors that impede telomerase function in cancer cells, and endorse the development of therapeutic strategies to ameliorate telomerase function in patients suffering from telomerase-deficiency diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM120094-01
Application #
9156409
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
2016-08-04
Project End
2021-07-31
Budget Start
2016-08-04
Budget End
2017-07-31
Support Year
1
Fiscal Year
2016
Total Cost
$305,170
Indirect Cost
$105,170
Name
University of Michigan Ann Arbor
Department
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Grill, Sherilyn; Tesmer, Valerie M; Nandakumar, Jayakrishnan (2018) The N Terminus of the OB Domain of Telomere Protein TPP1 Is Critical for Telomerase Action. Cell Rep 22:1132-1140
Gu, Peili; Jia, Shuting; Takasugi, Taylor et al. (2018) CTC1-STN1 coordinates G- and C-strand synthesis to regulate telomere length. Aging Cell :e12783
Bisht, Kamlesh; Grill, Sherilyn; Graniel, Jacqueline et al. (2017) A lentivirus-free inducible CRISPR-Cas9 system for efficient targeting of human genes. Anal Biochem 530:40-49
Pendlebury, Devon F; Fujiwara, Yasuhiro; Tesmer, Valerie M et al. (2017) Dissecting the telomere-inner nuclear membrane interface formed in meiosis. Nat Struct Mol Biol 24:1064-1072
Bisht, Kamlesh; Smith, Eric M; Tesmer, Valerie M et al. (2016) Structural and functional consequences of a disease mutation in the telomere protein TPP1. Proc Natl Acad Sci U S A 113:13021-13026