Distinct functions of POT1 proteins contribute to the regulation of telomerase recruitment to telomeres

ARTICLE https://doi.org/10.1038/s41467-021-25799-7 OPEN Distinct functions of POT1 proteins contribute to the regulation of telomerase recruitment to telomeres 1234567890():,; Peili Gu1, Shuting Jia2, Taylor Takasugi Sandy Chang 1,5,6 ✉ 1, Valerie M. Tesmer3, Jayakrishnan Nandakumar 3, Yong Chen 4& Human shelterin components POT1 and TPP1 form a stable heterodimer that protects telomere ends from ATR-dependent DNA damage responses and regulates telomerasedependent telomere extension. Mice possess two functionally distinct POT1 proteins. POT1a represses ATR/CHK1 DNA damage responses and the alternative non-homologous end-joining DNA repair pathway while POT1b regulates C-strand resection and recruits the CTC1-STN1-TEN1 (CST) complex to telomeres to mediate C-strand fill-in synthesis. Whether POT1a and POT1b are involved in regulating the length of the telomeric G-strand is unclear. Here we demonstrate that POT1b, independent of its CST function, enhances recruitment of telomerase to telomeres through three amino acids in its TPP1 interacting C-terminus. POT1b thus coordinates the synthesis of both telomeric G- and C-strands. In contrast, POT1a negatively regulates telomere length by inhibiting telomerase recruitment to telomeres. The identification of unique amino acids between POT1a and POT1b helps us understand mechanistically how human POT1 switches between end protective functions and promoting telomerase recruitment. 1 Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06520, USA. 2 Laboratory of Molecular Genetics of Aging and Tumor, School of Medicine, Kunming University Science and Technology, Kunming 650500, China. 3 Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA. 4 State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China. 5 Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA. 6 Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA. ✉email: NATURE COMMUNICATIONS | (2021)12:5514 | https://doi.org/10.1038/s41467-021-25799-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25799-7 T elomeres are TTAGGG repetitive DNA-ribonucleoprotein complexes that cap the ends of all eukaryotic chromosomes1,2. Telomeres terminate in G-rich singlestranded (ss) DNA overhangs and thus have the potential to initiate inappropriate DNA repair reactions. However, ss G-overhangs are also required for telomere elongation by telomerase. A major question in the telomere field is how telomeres a G3 Pot1b-/- TelG/DAPI G3 Pot1b+/- > > are able to protect chromosome ends from being recognized as damaged DNA while still functioning as substrates for telomerase-mediated telomere elongation. A potential solution to this problem resides in a complex of telomere-specific binding proteins, termed shelterin, which protects telomeres from inappropriately activating DNA damage checkpoints1,3. Three sequence-specific DNA-binding proteins are recruited to b c 4 > > > telomere > Relative frequency > 3 G3 Pot1b+/- 6081 G3 Pot1b-/- 2024 p<0.0001 2 1 0 0 POT1bWT GFP 5000 10000 15000 f > Pot1b-/- g p=0.9846 p=0.9600 p=0.5592 p=0.2177 > p=0.9786 p=0.0056 telomere 10 p=0.0047 p=0.0017 p=0.1575 8 6 POT1bF62A POT1aWT 20 5 0 0 Relative frequency Day 0 GFP POT1bWT POT1bF62A POT1aWT Day 0 GFP POT1bWT POT1bF62A POT1aWT Day 0 GFP POT1bWT POT1bF62A POT1aWT PO GF T P PO 1b W T T1 b F6 PO 2A T1 a WT telomere h GFP Median:2234 p=0.7961 10 2 e p=0.9996 p=0.6060 15 4 TelG/DAPI 20000 arbitrary telomere intensity PO GF T P PO 1b W T T1 PO b F62A T1 a WT TelG/DAPI d POT1bWT Median: 4042 POT1bF62A Median: 2527 POT1aWT Median: 2183 23 kb * 9.41 kb EtBr staining 2 6.56 kb 1.05 1.00 1.93 0.85 0.94 arbitrary telomere intensity arbitrary telomere intensity 1.00 1.00 0.25 1.05 0.95 Relative frequency arbitrary telomere intensity arbitrary telomere intensity native denature NATURE COMMUNICATIONS | (2021)12:5514 | https://doi.org/10.1038/s41467-021-25799-7 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25799-7 ARTICLE Fig. 1 POT1b promotes telomere elongation. a PNA-telomere FISH shows telomeric signals present in G3 Pot1b+/− and G3 Pot1b−/− sarcoma cells. TelG: PNA probe Cy3-OO-(CCCTAA)4. Red arrows point to telomere-free chromosome fusions and white arrows point to telomere-free chromosome ends. Scale bar: 5 µm. b Distribution of telomere lengths, designated as arbitary telomere intensity, in G3 Pot1b+/− and G3 Pot1b−/− sarcomas determined by quantitative (Q) telomere-FISH. Telomeres from a minimum of 30 metaphases were scored in three independent experiments. Numbers indicate median telomere lengths. c Quantification of the indicated types of fusions per chromosome in Pot1b−/− sarcomas of the indicated generations. Data show the mean ± standard deviation (s.d.) from three independent experiments. At least 30 metaphases were analyzed for each sample. d PNA-FISH of G3 Pot1b−/− sarcoma expressing GFP, POT1aWT, POT1bWT or POT1bF62A constructs. TelG: PNA probe Cy3-OO-(CCCTAA)4. Red arrows point to telomere-free chromosome fusions; white arrows point to telomere-free chromosome ends. Scale bar: 5 µm. e Distribution of telomere intensities by Q-FISH from metaphases in (d). A minimum of 40 metaphases were scored per cell type in three independent experiments. f Quantification of telomere-free ends in (d). Data show the mean ± s.d. from two independent experiments. At least 30 metaphases were analyzed per construct. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. g Quantification of telomere-free chromosome fusions in (d). Data show the mean ± s.d. from two independent experiments. At least 30 metaphases were analyzed per construct. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. h Detection of G-overhangs (native gel) and total telomere lengths (denature gel) by TRF Southern in G3 Pot1b−/− sarcomas expressing the indicated constructs. Ethidium bromide (EtBr) image shows that all samples contained equal amounts of genomic DNA. Molecular weight markers are indicted. *: DNA band used for quantification. Numbers indicate relative G-overhang and total telomere signals, with telomere signals set to 1.0 for cells expressing GFP. chromosomal ends: the duplex telomere binding proteins TRF1 and TRF2 and the telomere ssDNA binding protein Protection of Telomeres 1 (POT1). POT1 forms a functional heterodimer with TPP1, and in turn, TPP1 tethers POT1 to telomeres by interacting with the TIN2-TRF1 and TIN2-TRF2 complexes4. POT (...truncated)