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18488.Full.Pdf Single-cell imaging reveals unexpected heterogeneity of telomerase reverse transcriptase expression across human cancer cell lines Teisha J. Rowlanda,b,c,1, Gabrijela Dumbovica,b,1, Evan P. Hassa,b, John L. Rinna,b,c, and Thomas R. Cecha,b,c,2 aDepartment of Biochemistry, University of Colorado Boulder, Boulder, CO 80303; bBioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80303; and cHoward Hughes Medical Institute, University of Colorado Boulder, Boulder, CO 80303 Contributed by Thomas R. Cech, July 19, 2019 (sent for review May 16, 2019; reviewed by Steven Artandi and Karen L. Beemon) Telomerase is pathologically reactivated in most human cancers, and related pathological conditions (i.e., telomeropathies such where it maintains chromosomal telomeres and allows immortal- as dyskeratosis congenita, aplastic anemia, and pulmonary ization. Because telomerase reverse transcriptase (TERT) is usually fibrosis) (14). the limiting component for telomerase activation, numerous While many TERT expression studies have been published, studies have measured TERT mRNA levels in populations of cells these studies have been complicated by difficulties in detecting or in tissues. In comparison, little is known about TERT expression low levels of endogenously expressed TERT mRNA (15) and at the single-cell and single-molecule level. To address this, we have typically used methods that look at bulk expression levels analyzed TERT expression across 10 human cancer lines using within a cell population or tissue sample. Traditional cell pop- single-molecule RNA fluorescent in situ hybridization (FISH) and ulation studies provide little insight into cell-to-cell heteroge- made several unexpected findings. First, there was substantial cell- neity and spatial aspects of mRNA expression (16), which has to-cell variation in number of transcription sites and ratio of left unanswered—or provided only unclear answers to—many transcription sites to gene copies. Second, previous classification of lines as having monoallelic or biallelic TERT expression was intriguing questions about TERT expression. For example, while found to be inadequate for capturing the TERT gene expression population averages can be a good starting point for under- patterns. Finally, spliced TERT mRNA had primarily nuclear locali- standing overall expression levels of a given mRNA, these data zation in cancer cells and induced pluripotent stem cells (iPSCs), in cannot answer questions related to expression at the single-cell stark contrast to the expectation that spliced mRNA should be level, such as how many gene copies are active per cell and predominantly cytoplasmic. These data reveal unappreciated het- whether there is significant heterogeneity between cancer types erogeneity, complexity, and unconventionality in TERT expression or among cells in a given cell line, and can even be misleading across human cancer cells. when such heterogeneity is involved. Additionally, while cell fractionation can be done on a population of cells, studies of cancer | single-molecule imaging | telomerase | TERT | transcription population averages cannot provide insight into where molecules of TERT pre-mRNA and spliced (i.e., mature) mRNA reside elomeres, protective structures found at the ends of eukary- within single cells; an important consideration as subcellular Totic chromosomes, contain a highly repetitive DNA sequence and associated proteins; they are important for maintaining Significance chromosomal and genomic stability (1). In early human devel- opment, chromosomal telomere shortening that occurs due to Telomerase, which extends DNA at chromosome ends, is “ ” the end-replication problem during cell proliferation can be composed of an RNA template and a catalytic protein subunit, compensated by telomerase. Telomerase, first discovered in 1985 telomerase reverse transcriptase (TERT). TERT gene expression Tetrahymena in the ciliate (2), is a ribonucleoprotein enzyme that has been of great interest because it is required for pro- lengthens and maintains the telomeres. After development, liferation of most cancers, but expression investigations have telomerase is inactivated in most somatic cells, leading to pro- been limited due to low endogenous mRNA levels. Here, we gressive telomere shortening until a critical length halts cell di- employ single-molecule RNA fluorescent in situ hybridization vision and triggers cell senescence (the Hayflick limit). However, (FISH) in 10 human cancer cell lines and make findings that are ∼ telomerase is pathologically active in 80 to 90% of malignant unanticipated based upon bulk TERT mRNA measurements. For human cancers, which is considered an early cancer progression example, there is great cell-to-cell variation in the number of – event (3 5). transcription sites, and spliced TERT mRNA has greater nuclear Expression of the catalytic subunit of telomerase, telomerase localization than cytoplasmic. Overall, our findings provide in- reverse transcriptase (TERT) (6, 7), is required for telomerase sights regarding TERT expression, localization patterns, and activity. Introduction of TERT expression into normal human variability in cancer on a single-cell level. somatic cells leads to telomere elongation and cellular immor- talization, making its expression necessary, but not sufficient, for Author contributions: T.J.R., G.D., J.L.R., and T.R.C. designed research; T.J.R., G.D., and driving oncogenesis in most cancers (8, 9). Increased TERT ex- E.P.H. performed research; T.J.R., G.D., E.P.H., J.L.R., and T.R.C. analyzed data; and T.J.R., pression levels have also been found to be associated with poorer G.D., E.P.H., J.L.R., and T.R.C. wrote the paper. patient prognoses for several cancer types, including breast cancer, Reviewers: S.A., Stanford University; and K.L.B., Johns Hopkins University. urothelial bladder carcinomas, non–small-cell lung carcinomas, Conflict of interest statement: T.R.C. is on the board of directors of Merck, Inc., and a melanoma, and thyroid tumors (10–13), highlighting the impor- consultant for Storm Therapeutics, neither of which provided funding for this study. tance of understanding the role of TERT expression in cancer Published under the PNAS license. and its progression. 1T.J.R. and G.D. contributed equally to this work. It is thought that TERT expression, which is relatively low, 2To whom correspondence may be addressed. Email: [email protected]. must be tightly regulated to ensure normal telomere mainte- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nance. Even a small decrease in TERT activity, such as by 10 to 1073/pnas.1908275116/-/DCSupplemental. 20%, may potentially result in abnormal telomere maintenance Published online August 26, 2019. 18488–18497 | PNAS | September 10, 2019 | vol. 116 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1908275116 Downloaded by guest on September 25, 2021 localization can have profound effects on the function of RNA occurrence of TERT protein (visualized using an anti-FLAG an- molecules. tibody to the 3×FLAG-tagged TERT protein) and TERT mRNA Recent advancements in RNA imaging (15, 17) allow visuali- (visualized by TERT exon smFISH). TERT OE cells that were zation of mRNAs and actively transcribing alleles at the single- positive for anti-FLAG staining (i.e., expressing TERT protein) cell level even at low abundance. In the current study, we de- also showed clear TERT exon probe hybridization (Fig. 1 C, Top), termined single-cell TERT expression levels in several cancer while nontransfected cells had much lower levels of TERT mRNA cell lines that had been previously classified as having either and showed no anti-FLAG staining (Fig. 1 C, Bottom). These monoallelic or biallelic expression (MAE or BAE, respectively) experiments gave considerable confidence that the oligonucleo- of TERT by Huang et al. (18). They determined the MAE or tide probes specifically recognized TERT pre-mRNA and BAE status of these lines by quantifying allelic imbalances of spliced mRNA. heterozygous single-nucleotide polymorphisms (SNPs) in TERT As additional controls, we performed GAPDH exon and in- exons using whole-genome sequencing and RNA-sequencing tron smFISH on TERT OE and nontransfected cells (SI Ap- (RNA-seq) data from the Broad Institute’s Cancer Cell Line pendix, Fig. S1). GAPDH exon probes showed characteristic Encyclopedia (18). For reasons that remain unknown, MAE or signal in the cytoplasm, and nuclear exon signals colocalizing BAE of TERT was found to consistently associate with certain with intron signals marked active GAPDH transcription sites (SI cancer types; for example, melanoma and pancreatic cancer cell Appendix, Fig. S1B). Both nontransfected and TERT OE trans- lines had MAE of TERT, while breast and prostate lines had fected cells typically had 0 to 2 exon–intron colocalized nuclear BAE of TERT. Other cancer types were found to be composed “spots,” or signals. Some HEK293T cells had 3 exon–intron of a mixture of MAE and BAE lines. Overall, 44% (39/88) of cell colocalized spots per nucleus, which is not unexpected due to the lines investigated had MAE of TERT, while the other lines had abnormal karyotype of HEK293T cells (27). Altogether, these BAE of TERT. Nearly one-half (19/39) of MAE lines contained data are supportive of our TERT smFISH probes being specific a TERT promoter mutation known to reactivate TERT expres- for detecting and visualizing TERT RNA and TERT active sion via transcription factor recruitment (19–22),
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