Telomerase is an RNA-protein complex that replicates the very ends (telomeres) of the linear chromosomes of humans and other eukaryotes. Telomere maintenance is required for continuous proliferation of cells, and in most human cancers this is achieved by reactivating the gene for the catalytic subunit of telomerase (telomerase reverse transcriptase or TERT). The trigger for reactivation remained mysterious until 2013, when two research groups reported mutations in the promoter of the TERT gene present in multiple tumor types but not in adjacent healthy tissue. Recently we reported that these TERT promoter mutations were indeed associated with higher production of TERT protein, higher telomerase enzyme activity, and longer telomeres in urothelial cancer (UC), and associated with poor patient survival. However, very little is known about the molecular mechanisms by which the promoter mutations lead to TERT activation, which has impeded progress both in basic science and in clinical applications. Now we propose to unravel the mechanisms of TERT activation in UC and hepatocellular carcinoma (HCC) cell lines. Many of these cell lines are heterozygous for the TERT promoter mutation ? i.e., only one of the two TERT genes in the cell has the mutation. Thus, we will compare the two alleles to test for differences in RNA polymerase II occupancy, marks of epigenetic silencing, and production of mRNA, allowing a specific hypothesis about TERT gene silencing and reactivation to be tested. To move beyond association and test for causation, we will use CRISPR-Cas9 genome editing to insert or erase TERT promoter mutations and assess whether this is sufficient to switch TERT production. If the promoter mutation is not sufficient, we will inactivate factors such as the histone methyltransferase PRC2 (which adds repressive chromatin marks) either by knockdown or by adding a validated small-molecule inhibitor and test for reactivation. Furthermore, bioinformatics (gene expression data) reveals candidates for other components involved in gene reactivation that will be tested. The long-term goal of this project is to understand the state of the repressed and activated TERT genes and the steps involved in telomerase reactivation in cancer. This basic science study will provide information about cancer biology and is likely to give information that is clinically relevant.

Public Health Relevance

Telomerase extends human chromosome ends, which is required for continuous cell proliferation. Reactivation of telomerase, often by an activating mutation in a gene regulatory region, drives many cancers. We propose experiments to understand the molecular mechanisms of telomerase activation, which will have implications for diagnosis and treatment of multiple cancers.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM099705-06
Application #
9220833
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Willis, Kristine Amalee
Project Start
2012-05-01
Project End
2020-01-31
Budget Start
2017-02-01
Budget End
2018-01-31
Support Year
6
Fiscal Year
2017
Total Cost
$192,064
Indirect Cost
$57,064
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80303
Stern, Josh Lewis; Paucek, Richard D; Huang, Franklin W et al. (2017) Allele-Specific DNA Methylation and Its Interplay with Repressive Histone Marks at Promoter-Mutant TERT Genes. Cell Rep 21:3700-3707
Lim, Ci Ji; Zaug, Arthur J; Kim, Hee Jin et al. (2017) Reconstitution of human shelterin complexes reveals unexpected stoichiometry and dual pathways to enhance telomerase processivity. Nat Commun 8:1075
Long, Yicheng; Wang, Xueyin; Youmans, Daniel T et al. (2017) How do lncRNAs regulate transcription? Sci Adv 3:eaao2110
Schmidt, Jens C; Zaug, Arthur J; Cech, Thomas R (2016) Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres. Cell 166:1188-1197.e9
Shukla, Siddharth; Schmidt, Jens C; Goldfarb, Katherine C et al. (2016) Inhibition of telomerase RNA decay rescues telomerase deficiency caused by dyskerin or PARN defects. Nat Struct Mol Biol 23:286-92
Stern, Josh Lewis; Theodorescu, Dan; Vogelstein, Bert et al. (2015) Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERT expression in multiple cancers. Genes Dev 29:2219-24
Dalby, Andrew B; Hofr, Ctirad; Cech, Thomas R (2015) Contributions of the TEL-patch amino acid cluster on TPP1 to telomeric DNA synthesis by human telomerase. J Mol Biol 427:1291-303
Xi, Linghe; Schmidt, Jens C; Zaug, Arthur J et al. (2015) A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression. Genome Biol 16:231
Borah, Sumit; Xi, Linghe; Zaug, Arthur J et al. (2015) Cancer. TERT promoter mutations and telomerase reactivation in urothelial cancer. Science 347:1006-10
Schmidt, Jens C; Cech, Thomas R (2015) Human telomerase: biogenesis, trafficking, recruitment, and activation. Genes Dev 29:1095-105

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