Abstract
Key message
Duplicate POT1 genes must rapidly diverge or be inactivated.
Abstract
Protection of telomeres 1 (POT1) encodes a conserved telomere binding protein implicated in both chromosome end protection and telomere length maintenance. Most organisms harbor a single POT1 gene, but in the few lineages where the POT1 family has expanded, the duplicate genes have diversified. Arabidopsis thaliana bears three POT1-like loci, POT1a, POT1b and POT1c. POT1a retains the ancestral function of telomerase regulation, while POT1b is implicated in chromosome end protection. Here we examine the function and evolution of the third POT1 paralog, POT1c. POT1c is a new gene, unique to A. thaliana, and was derived from a duplication event involving the POT1a locus and a neighboring gene encoding ribosomal protein S17. The duplicate S17 locus (dS17) is highly conserved across A. thaliana accessions, while POT1c is highly divergent, harboring multiple deletions within the gene body and two transposable elements within the promoter. The POT1c locus is transcribed at very low to non-detectable levels under standard growth conditions. In addition, no discernable molecular or developmental defects are associated with plants bearing a CRISPR mutation in the POT1c locus. However, forced expression of POT1c leads to decreased telomerase enzyme activity and shortened telomeres. Evolutionary reconstruction indicates that transposons invaded the POT1c promoter soon after the locus was formed, permanently silencing the gene. Altogether, these findings argue that POT1 dosage is critically important for viability and duplicate gene copies are retained only upon functional divergence.
Similar content being viewed by others
References
Arora A, Beilstein MA, Shippen DE (2016) Evolution of Arabidopsis protection of telomeres 1 alters nucleic acid recognition and telomerase regulation. Nucleic Acids Res 44:9821–98307. https://doi.org/10.1093/nar/gkw807
Baumann P, Cech TR (2001) Pot1, the putative telomere end-binding protein in fission yeast and humans. Science 292:1171–1175. https://doi.org/10.1126/science.1060036
Beilstein MA, Nagalingum NS, Clements MD, Manchester SR, Mathews S (2010) Dated molecular phylogenies indicate a Miocene origin for Arabidopsis thaliana. Proc Natl Acad Sci 107:18724–18728. https://doi.org/10.1073/pnas.0909766107
Beilstein MA, Renfrew KB, Song X, Shakirov EV, Zanis MJ, Shippen DE (2015) Evolution of the telomere-associated protein POT1a in Arabidopsis thaliana is characterized by positive selection to reinforce protein–protein interaction. Mol Biol Evol 32:1329–1341. https://doi.org/10.1093/molbev/msv025
Bewick AJ, Ji L, Niederhuth CE, Willing EM, Hofmeister BT, Shi X et al (2016) On the origin and evolutionary consequences of gene body DNA methylation. Proc Natl Acad Sci 113:9111–9116. https://doi.org/10.1073/pnas.1604666113
Birchler J, Veitia R (2007) The gene balance hypothesis: from classical genetics to modern genomics. Plant Cell 19:395–402. https://doi.org/10.1105/tpc.106.049338
Blanc G, Wolfe KH (2004) Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution. Plant Cell 16:1679–1691. https://doi.org/10.1105/tpc.021410
Bremer K (2002) Gondwanan evolution of the grass alliance of families (Poales). Evolution 56:1374–1387. https://doi.org/10.1111/j.0014-3820.2002.tb01451.x
Bunch JT, Bae NS, Leonardi J, Baumann P (2005) Distinct requirements for Pot1 in limiting telomere length and maintaining chromosome stability. Mol Cell Biol 25:5567–5578. https://doi.org/10.1128/MCB.25.13.5567-5578.2005
Cao J, Schneeberger K, Ossowski S, Gunther T, Bender S, Fitz J et al (2011) Whole-genome sequencing of multiple Arabidopsis thaliana populations. Nat Genet 43:956–965. https://doi.org/10.1038/ng.911
Cheng C, Shtessel L, Brady MM, Ahmed S (2012) Caenorhabditis elegans POT-2 telomere protein represses a mode of alternative lengthening of telomeres with normal telomere lengths. Proc Natl Acad Sci 109:7805–7810. https://doi.org/10.1073/pnas.1119191109
Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD et al (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452:215–219. https://doi.org/10.1038/nature06745
Cranert S, Heyse S, Linger BR, Lescasse R, Price C (2014) Tetrahymena Pot2 is a developmentally regulated paralog of Pot1 that localizes to chromosome breakage sites but not to telomeres. Eukaryot Cell 13:1519–1529. https://doi.org/10.1128/EC.00204-14
De Bodt S, Maere S, Van de Peer Y (2005) Genome duplication and the origin of angiosperms. Trends Ecol Evol. https://doi.org/10.1016/j.tree.2005.07.008
Denchi EL, De Lange T (2007) Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. Lett Nat 448:1068–1071. https://doi.org/10.1038/nature06065
Fitzgerald MS, Riha K, Gao F, Ren S, McKnight TD, Shippen DE (1999) Disruption of the telomerase catalytic subunit gene from Arabidopsis inactivates telomerase and leads to a slow loss of telomeric DNA. Proc Natl Acad Sci USA 96:14813–14818. https://doi.org/10.1073/pnas.96.26.14813
Gu Z, Steinmetz LM, Gu X, Scharfe C, Davis RW, Li WH (2003) Role of duplicate genes in genetic robustness against null mutations. Nature 421:63–66. https://doi.org/10.1038/nature01198
He H, Multani AS, Cosme-Blanco W, Tahara H, Ma J, Pathak S et al (2006) POT1b protects telomeres from end-to-end chromosomal fusions and aberrant homologous recombination. EMBO J 25:5180–5190. https://doi.org/10.1038/sj.emboj.7601294
Heacock M, Spangler E, Riha K, Puizina J, Shippen DE (2004) Molecular analysis of telomere fusions in Arabidopsis: multiple pathways for chromosome end-joining. EMBO J 23:2304–2313. https://doi.org/10.1038/sj.emboj.7600236
Henderson IR, Jacobsen SE (2007) Epigenetic inheritance in plants. Nature 447:418–424
Hockemeyer D, Sfeir AJ, Shay JW, Wright WE, de Lange T (2005) POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end. EMBO J 24:2667–2678. https://doi.org/10.1038/sj.emboj.7600733
Hockemeyer D, Daniels JP, Takai H, de Lange T (2006) Recent expansion of the telomeric complex in rodents: two distinct POT1 proteins protect mouse telomeres. Cell 126:63–77. https://doi.org/10.1016/j.cell.2006.04.044
Hori H, Higo K, Osawa S (1977) The rates of evolution in some ribosomal components. J Mol Evol 9:191–201
Horiguchi G, Van Lijsebettens M, Candela H, Micol JL, Tsukaya H (2012) Ribosomes and translation in plant developmental control. Plant Sci 191–192:24–34. https://doi.org/10.1016/j.plantsci.2012.04.008
Karamysheva ZN, Surivtseva Y, Vespa L, Shakirov E, Shippen DE (2004) A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis. JBC 279:47799–47807. https://doi.org/10.1074/jbc.M407938200
Kelleher C, Kurth I, Lingner J (2005) Human protection of telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro. Mol Cell Biol 25:808–818. https://doi.org/10.1128/MCB.25.2.808
Kellogg EA (1998) Relationships of cereal crops and other grasses. Proc Natl Acad Sci 95:2005–2010. https://doi.org/10.1073/pnas.95.5.2005
Kimura Y, Hawkins MTR, McDonough MM, Jacobs LL, Flynn LJ (2015) Corrected placement of Mus-Rattus fossil calibration forces precision in the molecular tree of rodents. Sci Rep 5:1–9. https://doi.org/10.1038/srep14444
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948
Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11:204–220. https://doi.org/10.1038/nrg2719
Lippman Z, Gendrel AV, Black M, Vaughn MW, Dedhia N, McCombie WR et al (2004) Role of transposable elements in heterochromatin and epigenetic control. Nature 430:471–476. https://doi.org/10.1038/nature02651
Lynch M, Conery JS (2000) The evolutionary fate and consequences of duplicate genes. Science 290:1151–1155. https://doi.org/10.1126/science.290.5494.1151
Lynch M, Conery JS (2003) The evolutionary demography of duplicate genes. J Struct Funct Genom 3:35–44
Lynch M, O’Hely M, Walsh B, Force A (2001) The probability of preservation of a newly arisen gene duplicate. Genetics 159:1789–1804. https://doi.org/10.1006/scdb.1996.0068
Meier B, Barber LJ, Liu Y, Shtessel L, Boulton SJ, Gartner A et al (2009) The MRT-1 nuclease is required for DNA crosslink repair and telomerase activity in vivo in Caenorhabditis elegans. EMBO J 28:3549–3563. https://doi.org/10.1038/emboj.2009.278
Molnar A, Melnyk C, Baulcombe DC (2011) Silencing signals in plants: a long journey for small RNAs. Genome Biol 12:1–8. https://doi.org/10.1186/gb-2010-11-12-219
Moore RC, Purugganan MD (2003) The early stages of duplicate gene evolution. Proc Natl Acad Sci 100:15682–15687. https://doi.org/10.1073/pnas.2535513100
Mosher RA, Schwach F, Studholme D, Baulcombe DC (2008) PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis. Proc Natl Acad Sci 105:3145–3150. https://doi.org/10.1073/pnas.0709632105
Naduparambil JK, Lescasse R, Linger BR, Price CM (2007) Tetrahymena POT1a regulates telomere length and prevents activation of a cell cycle checkpoint. Mol Cell Biol 27:1592–1601. https://doi.org/10.1128/MCB.01975-06
Nandakumar J, Podell ER, Cech TR (2010) How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA. Proc Natl Acad Sci 107:651–656. https://doi.org/10.1073/pnas.0911099107
Ohno S (ed) (1970) Duplication for the sake of producing more of the same. In: Evolution by gene duplication. Springer, Berlin, Heidelberg, pp 59–65
Palm W, Hockemeyer D, Kibe T, de Lange T (2009) Functional dissection of human and mouse POT1 proteins. Mol Cell Biol 29:471–482. https://doi.org/10.1128/MCB.01352-08
Paterson AH, Bowers JE, Chapman BA (2004) Ancient polyploidization predating divergence of the cereals, and its consequences for comparative genomics. Proc Natl Acad Sci 101:9903–9908. https://doi.org/10.1073/pnas.0307901101
Pignatta D, Erdmann RM, Scheer E, Picard CL, Bell GW, Gehring M (2014) Natural epigenetic polymorphisms lead to intraspecific variation in Arabidopsis gene imprinting. eLife 3:1–24. https://doi.org/10.7554/eLife.03198
Raices M, Verdun RE, Compton SA, Haggblom CI, Griffith JD, Dillin A et al (2008) C. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins. Cell 132:745–757. https://doi.org/10.1016/j.cell.2007.12.039
Renfrew KB, Song X, Lee JR, Arora A, Shippen DE (2014) POT1a and components of CST engage telomerase and regulate its activity in Arabidopsis. PLoS Genet 10:1–12. https://doi.org/10.1371/journal.pgen.1004738
Riha K, Fajkus J, Siroky J, Vyskot B (1998) Developmental control of telomere lengths and telomerase activity in plants. Plant Cell 10:1691–1698. https://doi.org/10.1105/tpc.10.10.1691
Rossignol P, Collier S, Bush M, Shaw P, Doonan JH (2007) Arabidopsis POT1A interacts with TERT-V (I8), and N-terminal splicing variant of telomerase. J Cell Sci 120:3678–3687. https://doi.org/10.1242/jcs.004119
Schrumpfová PP, Vychodilová I, Dvořáčková M, Majerska J, Dokladal L, Schorova S et al (2014) Telomere repeat binding proteins are functional components of Arabidopsis telomeres and interact with telomerase. Plant J 77:770–781. https://doi.org/10.1111/tpj.12428
Shakirov EV, Surovtseva YV, Osbun N, Shippen DE (2005) The Arabidopsis Pot1 and Pot2 proteins function in telomere length homeostasis and chromosome end protection. Mol Cell Biol 25:7725–7733. https://doi.org/10.1128/MCB.25.17.7725-7733.2005
Shakirov EV, Perroud P-F, Nelson AD, Quatrano RS, Shippen DE (2010) Protection of Telomeres 1 is required for telomere integrity in the moss Physcomitrella patens. Plant Cell 22:1838–1848. https://doi.org/10.1105/tpc.110.075846
Shtessel L, Lowden MR, Cheng C, Simon M, Ahmed S (2013) Caenorhabditis elegans POT-1 and POT-2 repress telomere maintenance pathways. Genetics 3:305–313. https://doi.org/10.1534/g3.112.004440
Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313
Steinert J, Schiml S, Fauser F, Puchta H (2015) Highly efficient heritable plant genome engineering using Cas9 ortholog from Streptococcus thermophilus and Staphylococcus aureus. Plant J 84:1295–1305
Stroud H, Hale CJ, Feng S, Caro E, Jacob Y, Michaels SD et al (2012) DNA methyltransferases are required to induce heterochromatic re-replication in Arabidopsis. PLoS Genet 8:1–7. https://doi.org/10.1371/journal.pgen.1002808
Stroud H, Greenberg M, Feng S (2013) Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152:352–364. https://doi.org/10.1016/j.cell.2012.10.054.Comprehensive
Surovtseva YV, Shakirov EV, Vespa L, Osbun N, Song X, Shippen DE (2007) Arabidopsis POT1 associates with the telomerase RNP and is required for telomere maintenance. EMBO J 26:3653–3661. https://doi.org/10.1038/sj.emboj.7601792
Theobald DL, Mitton-Fry RM, Wuttke DS (2003) Nucleic acid recognition by OB-fold proteins. Annu Rev Biophys Biomol Struct 32:115–133. https://doi.org/10.1016/j.neuron.2009.10.017.A
Tsugeki R, Kochieva EZ, Fedoroff NV (1996) A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation. Plant J 10:479–489. https://doi.org/10.1046/j.1365-313X.1996.10030479.x
Van Lijsebettens M, Vanderhaeghen R, De Block M, Bauw G, Villarroel R, Van Montagu M (1994) An S18 ribosomal protein gene copy at the Arabidopsis PFL locus affects plant development by its specific expression in meristems. EMBO J 13:3378–3388. https://doi.org/10.1111/j.0022-3646.1985.00072.x
Walsh JB (1995) How often do duplicated genes evolve new functions? Genetics 139:421–428
Wang Y, Wang X, Tang H, Tan X, Ficklin SP, Feltus AF et al (2011) Modes of gene duplication contribute differently to genetic novelty and redundancy, but show parallels across divergent angiosperms. PLoS ONE 6:1–17. https://doi.org/10.1371/journal.pone.0028150
Wang J, Tao F, Marowsky NC, Fan C (2016) Evolutionary fates and dynamic functionalization of young duplicate genes in Arabidopsis genomes. Plant Physiol 172:427–440. https://doi.org/10.1104/pp.16.01177
Wu L, Multani AS, He H, Cosme-Blanco W, Deng Y, Deng JM et al (2006) Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres. Cell 126:49–62. https://doi.org/10.1016/j.cell.2006.05.037
Wu DD, Wang X, Li Y, Zeng L, Irwin DM, Zhang Y (2014) “Out of pollen” hypothesis for origin of new genes in flowering plants: study from Arabidopsis thaliana. Genome Biol Evol 6:2822–2829. https://doi.org/10.1093/gbe/evu206
Xie X, Shippen DE (2018) DDM1 guards against telomere truncation in Arabidopsis. Plant Cell Rep 37:501–513. https://doi.org/10.1007/s00299-017-2245-6
Xue S, Barna M (2012) Specialized ribosomes: a new frontier in gene regulation and organismal biology. Nat Rev Mol Cell Biol 13:355–369. https://doi.org/10.1038/nrm3359
Yang L, Takuno S, Waters ER, Gaut BS (2011) Lowly expressed genes in Arabidopsis thaliana bear the signature of possible pseudogenization by promoter degradation. Mol Biol Evol 28:1193–1203. https://doi.org/10.1093/molbev/msq298
Yu Y, Tan R, Ren Q, Gao B, Sheng Z, Zhang J et al (2017) POT1 inhibits the efficiency but promotes the fidelity of nonhomologous end joining at non-telomeric DNA regions. Aging 9:2529–2543. https://doi.org/10.18632/aging.101339
Zhang X, Yazaki J, Sundaresan A, Cokus S, Chan SW, Chen H et al (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126:1189–1201. https://doi.org/10.1016/j.cell.2006.08.003
Zhang H, Tang K, Wang B, Duan CG, Lang Z, Zhu JK (2014) Protocol: a beginner’s guide to the analysis of RNA-directed DNA methylation in plants. Plant Methods 10:1–9. https://doi.org/10.1186/1746-4811-10-18
Zhiponova MK, Morohashi K, Vanhoutte I, Machemer-Noonan K, Revalska M, Van Montagu M et al (2014) Helix-loop-helix/basic helix-loop-helix transcription factor network represses cell elongation in Arabidopsis through an apparent incoherent feed-forward loop. Proc Natl Acad Sci 111:2824–2829. https://doi.org/10.1073/pnas.1400203111
Acknowledgements
The authors thank Holger Puchta for sharing plasmids and expertise on CRISPR, Dan Browne for assistance with computational analysis, and members of the Shippen Lab for critical feedback on the project.
Funding
This work was supported by a grant from the National Institutes of Health (GM065383 to D.E.S.).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by Attila Feher.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Fig. S1
Transcriptional analysis of POT1c. a Results from RT-PCR analysis of POT1c using nested PCR for flowers. Left: first PCR (40 cycles), and right: second (nested) PCR (40 cycles). b Results from RT-PCR analysis of POT1c using nested PCR for flowers, seedlings, and leaves. Individual bands were gel purified, and cloned into the pGEM-T vector for sequencing. The most prominent band (closed triangle) in each reaction contained a 717 nucleotide sequence corresponding to fully spliced POT1c (GenBank Accession MK585073). A few other bands (open triangles) displayed evidence of incomplete splicing or consisted of unrelated sequence, indicating they were non-specific amplification products. Fig. S2 Generation of a POT1c mutant. a Location of the deletion by CRISPR-Cas9 is indicated by the black triangle. Location of the internal ATG is indicated by the red arrow. b Chromatogram of WT, mutant (Mt), and heterozygous (Het) plants at the POT1c locus. Vertical line indicates the start of deletion. Heterozygous chromatograms containing forward sequence (Het Fw) and reverse sequence (Het Rv) are shown. Fig. S3 The pot1c-1 mutant does not exhibit a defect in telomere maintenance or telomerase activity. a Bulk telomere length of WT plants and plants deficient in either POT1a or POT1c. b Telomere length on the individual chromosome arms 1L or 3R in WT plants and plants deficient in either POT1a or POT1c. No difference in telomere length was observed. c Telomerase activity levels were compared for WT, pot1a (p = 0.0007), and pot1c (p = 0.81) mutants using quantitative TRAP. N = 4. *p < 0.05.Fig. S4 The POT1c transcript is not actively silenced. a Results for Chop PCR. No DNA (lane 1), untreated (lane 2), treated with DNA methylation-sensitive restriction enzymes SnaBI, HypCH4IV, and HhaI (lane 3), or treated with the restriction enzyme McrBC that cuts at sites of CG methylation (lane 4). Red asterisks indicate amplification of DNA from incomplete digestion by McrBC. b qPCR results for POT1c and POT1a transcripts in ddm1 and dcl2,3,4 mutants. POT1a, POT1c, and TAS1 transcripts were normalized to the GAPDH transcript level. The fold change in POT1c transcript was adjusted relative to POT1a. The mir173 target TAS1 was used as a control in the dcl2,3,4 mutant. c Results of RT-PCR in the ddm1. PCR data for WT and ddm1 were compared to the AtMu1 TSA2 transposable element to confirm the ddm1 mutation. Fig. S5 The POT1c locus lacks a functional promoter. a Positive control: GUS reporter under the control of the CYCB1 promoter in a pot1a mutant background. Negative control: untransformed WT. GUS reporter expressed from the POT1c promoter. SAM: shoot apical meristem. b PCR amplification of the POT1cp-GUS construct in three transformants. PCR amplification of the ATR locus is shown at the bottom for DNA quality. Fig. S6 Original figure of Fig. 6b. Forced expression of POT1c leads to perturbations in telomere length maintenance and decreased telomerase activity. Results of TRF analysis to measure bulk telomere length of wild type (WT) and POT1cOE plants. Table S1 PCR primer sequences. Table S2 Accessions used in Fig. 4b. (PDF 15211 kb)
Rights and permissions
About this article
Cite this article
Kobayashi, C.R., Castillo-González, C., Survotseva, Y. et al. Recent emergence and extinction of the protection of telomeres 1c gene in Arabidopsis thaliana. Plant Cell Rep 38, 1081–1097 (2019). https://doi.org/10.1007/s00299-019-02427-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-019-02427-9