Telomere-dependent and telomereindependent roles of RAP1 in regulating human stem cell homeostasis

Abstract

RAP1 is a well-known telomere-binding protein, but its functions in human stem cells have remained unclear. Here we generated RAP1-deficient human embryonic stem cells (hESCs) by using CRISPR/Cas9 technique and obtained RAP1-deficient human mesenchymal stem cells (hMSCs) and neural stem cells (hNSCs) via direc- ted differentiation. In both hMSCs and hNSCs, RAP1 not only negatively regulated telomere length but also acted as a transcriptional regulator of RELN by tuning the methylation status of its gene promoter. RAP1 defi- ciency enhanced self-renewal and delayed senescence in hMSCs, but not in hNSCs, suggesting complicated lineage-specific effects of RAP1 in adult stem cells. Altogether, these results demonstrate for the first time that RAP1 plays both telomeric and nontelomeric roles in regulating human stem cell homeostasis.

Keywords

RAP1 / stem cell / telomere / RELN / methylation

References

[1] Anders S, Pyl PT, Huber W (2015) HTSeq–a Python framework to work with high-throughput sequencing data. Nat Methods 31:166–169

[2] Arnoult N, Van Beneden A, Decottignies A (2012) Telomere length regulates TERRA levels through increased trimethylation of telomeric H3K9 and HP1alpha. Nat Struct Mol Biol 19:948–956

[3] Bandaria JN, Qin P, Berk V, Chu S, Yildiz A (2016) Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin. Cell 164:735–746

[4] Blackburn EH (2001) Switching and signaling at the telomere. Cell 106:661–673

[5] Blackburn EH, Epel ES, Lin J (2015) Human telomere biology: A contributory and interactive factor in aging, disease risks, and protection. Science 350:1193–1198

[6] Bourgeron T, Xu Z, Doumic M, Teixeira MT (2015) The asymmetry of telomere replication contributes to replicative senescence heterogeneity. Sci Rep 5:15326

[7] Calado RT, Dumitriu B (2013) Telomere dynamics in mice and humans. Semin Hematol 50:165–174

[8] Cawthon RM (2002) Telomere measurement by quantitative PCR. Nucleic Acids Res 30:e47

[9] Chen J, Bardes EE, Aronow BJ, Jegga AG (2009) ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic acids Res 37:W305–311

[10] Dahl JA, Collas P (2008) A rapid micro chromatin immunoprecipitation assay (microChIP). Nat Protoc 3:1032–1045

[11] Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O (2009) Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4:1798–1806

[12] Di Palma T, Filippone MG, Pierantoni GM, Fusco A, Soddu S, Zannini M (2013) Pax8 has a critical role in epithelial cell survival and proliferation. Cell Death Dis 4:e729

[13] Ding Y, Liang X, Zhang Y, Yi L, Shum HC, Chen Q, Chan BP, Fan H, Liu Z, Tergaonkar V (2018) Rap1 deficiency-provoked paracrine dysfunction impairs immunosuppressive potency of mesenchymal stem cells in allograft rejection of heart transplantation. Cell Death Dis 9:386

[14] Doerks T, Copley RR, Schultz J, Ponting CP, Bork P (2002) Systematic identification of novel protein domain families associated with nuclear functions. Genome Res 12:47–56

[15] Donato R, Sorci G, Giambanco I (2017) S100A6 protein: functional roles. Cell Mol Life Sci 74:2749–2760

[16] Duan S, Yuan G, Liu X, Ren R, Li J, Zhang W, Wu J, Xu X, Fu L, Li Y (2015) PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype. Nat Commun 6:10068

[17] Feretzaki M, Lingner J (2017) A practical qPCR approach to detect TERRA, the elusive telomeric repeat-containing RNA. Methods 114:39–45

[18] Ferrara-Romeo I, Martinez P, Blasco MA (2018) Mice lacking RAP1 show early onset and higher rates of DEN-induced hepatocellular carcinomas in female mice. PloS ONE 13:e0204909

[19] Fu L, Xu X, Ren R, Wu J, Zhang W, Yang J, Ren X, Wang S, Zhao Y, Sun L (2016) Modeling xeroderma pigmentosum associated neurological pathologies with patients-derived iPSCs. Protein Cell 7:210–221

[20] Geng L, Liu Z, Zhang W, Li W, Wu Z, Wang W, Ren R, Su Y, Wang P, Sun L (2018) Chemical screen identifies a geroprotective role of quercetin in premature aging. Protein Cell. https://doi.org/10.1007/s13238-018-0567-y

[21] Goodell MA, Rando TA (2015) Stem cells and healthy aging. Science 350:1199–1204

[22] Greider CW (1991) Telomeres. Curr Opin Cell Biol 3:444–451

[23] Ha G, Roth A, Lai D, Bashashati A, Ding J, Goya R, Giuliany R, Rosner J, Oloumi A, Shumansky K (2012) Integrative analysis of genome-wide loss of heterozygosity and monoallelic expression at nucleotide resolution reveals disrupted pathways in triple-negative breast cancer. Genome Res 22:1995–2007

[24] Hohensinner PJ, Kaun C, Buchberger E, Ebenbauer B, Demyanets S, Huk I, Eppel W, Maurer G, Huber K, Wojta J (2016) Age intrinsic loss of telomere protection via TRF1 reduction in endothelial cells. Biochim Biophys Acta 1863:360–367

[25] Ishii K, Kubo KI, Nakajima K (2016) Reelin and Neuropsychiatric Disorders. Front Cell Neurosci 10:229

[26] Kabir S, Hockemeyer D, de Lange T (2014) TALEN gene knockouts reveal no requirement for the conservedhuman shelterin protein Rap1 in telomere protection and length regulation. Cell Rep 9:1273–1280

[27] Kabir S, Sfeir A, de Lange T (2010) Taking apart Rap1: an adaptor protein with telomeric and non-telomeric functions. Cell cycle 9:4061–4067

[28] Kanoh J, Ishikawa F (2001) spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast. Curr Biol 11:1624–1630

[29] Khurana E, Fu Y, Chen J, Gerstein M (2013) Interpretation of genomic variants using a unified biological network approach. PLoS Comput Biol 9:e1002886

[30] Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360

[31] Kim H, Li F, He Q, Deng T, Xu J, Jin F, Coarfa C, Putluri N, Liu D, Songyang Z (2017) Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells. Cell Discov 3:17034

[32] Kubben N, Zhang W, Wang L, Voss TC, Yang J, Qu J, Liu GH, Misteli T (2016) Repression of the antioxidant NRF2 pathway in premature aging. Cell 165:1361–1374

[33] Kundakovic M, Chen Y, Costa E, Grayson DR (2007) DNA methyltransferase inhibitors coordinately induce expression of the human reelin and glutamic acid decarboxylase 67 genes. Mol Pharmacol 71:644–653

[34] Kundakovic M, Chen Y, Guidotti A, Grayson DR (2009) The reelin and GAD67 promoters are activated by epigenetic drugs that facilitate the disruption of local repressor complexes. Mol Pharmacol 75:342–354

[35] Kyrion G, Liu K, Liu C, Lustig AJ (1993) RAP1 and telomere structure regulate telomere position effects in Saccharomyces cerevisiae. Genes Dev 7:1146–1159

[36] Lai TP, Wright WE, Shay JW (2016) Generation of digoxigeninincorporated probes to enhance DNA detection sensitivity. BioTechniques 60:306–309

[37] Lancaster MA, Knoblich JA (2014) Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc 9:2329–2340

[38] Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME, Homfray T, Penninger JM, Jackson AP, Knoblich JA (2013) Cerebral organoids model human brain development and microcephaly. Nature 501:373–379

[39] Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359

[40] Lensch MW, Schlaeger TM, Zon LI, Daley GQ (2007) Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera. Cell Stem Cell 1:253–258

[41] Li B, de Lange T (2003) Rap1 affects the length and heterogeneity of human telomeres. Mol Biol Cell 14:5060–5068

[42] Li WP, Liu P, Pilcher BK, Anderson RG (2001) Cell-specific targeting of caveolin-1 to caveolae, secretory vesicles, cytoplasm or mitochondria. J Cell Sci 114:1397–1408

[43] Lintas C, Sacco R, Persico AM (2016) Differential methylation at the RELN gene promoter in temporal cortex from autistic and typically developing post-puberal subjects. J Neurodev Disord 8:18

[44] Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C (2011) Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature 472:221–225

[45] Liu GH, Suzuki K, Li M, Qu J, Montserrat N, Tarantino C, Gu Y, Yi F, Xu X, Zhang W (2014) Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs. Nat Commun 5:4330

[46] Londono-Vallejo JA (2004) Telomere length heterogeneity and chromosome instability. Cancer letters 212:135–144

[47] Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G (2013) The hallmarks of aging. Cell 153:1194–1217

[48] Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550

[49] Luo K, Vega-Palas MA, Grunstein M (2002) Rap1-Sir4 binding independent of other Sir, yKu, or histone interactions initiates the assembly of telomeric heterochromatin in yeast. Genes Dev 16:1528–1539

[50] Martinez P, Gomez-Lopez G, Garcia F, Mercken E, Mitchell S, Flores JM, de Cabo R, Blasco MA (2013) RAP1 protects from obesity through its extratelomeric role regulating gene expression. Cell reports 3:2059–2074

[51] Martinez P, Gomez-Lopez G, Pisano DG, Flores JM, Blasco MA (2016) A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance. Aging Cell 15(6):1113–1125

[52] Martinez P, Thanasoula M, Carlos AR, Gomez-Lopez G, Tejera AM, Schoeftner S, Dominguez O, Pisano DG, Tarsounas M, Blasco MA (2010) Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites. Nat Cell Biol 12:768–780

[53] Moretti P, Shore D (2001) Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Molecular and cellular biology 21:8082–8094

[54] Nabil Fikri RM, Norlelawati AT, Nour El-Huda AR, Hanisah MN, Kartini A, Norsidah K, Nor Zamzila A (2017) Reelin (RELN) DNA methylation in the peripheral blood of schizophrenia. J Psychiatr Res 88:28–37

[55] Nanavaty V, Sandhu R, Jehi SE, Pandya UM, Li B (2017) Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids. Nucleic Acids Res 45:5785–5796

[56] O’Connor MS, Safari A, Liu D, Qin J, Songyang Z (2004) The human Rap1 protein complex and modulation of telomere length. The Journal of biological chemistry 279:28585–28591

[57] Omura N, Li CP, Li A, Hong SM, Walter K, Jimeno A, Hidalgo M, Goggins M(2008) Genome-wide profiling of methylated promoters in pancreatic adenocarcinoma. Cancer Biol Ther 7:1146–1156

[58] Palm W, de Lange T (2008) How shelterin protects mammalian telomeres. Annu Rev Genet 42:301–334

[59] Pan H, Guan D, Liu X, Li J, Wang L, Wu J, Zhou J, Zhang W, Ren R, Zhang W (2016) SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2. Cell Res 26:190–205

[60] Proctor CJ, Kirkwood TB (2003) Modelling cellular senescence as a result of telomere state. Aging cell 2:151–157

[61] Reddel RR (2014) Telomere maintenance mechanisms in cancer: clinical implications. Curr Pharm Des 20:6361–6374

[62] Ren R, Deng L, Xue Y, Suzuki K, Zhang W, Yu Y, Wu J, Sun L, Gong X, Luan H (2017a) Visualization of aging-associated chromatin alterations with an engineered TALE system. Cell Res 27:483–504

[63] Ren R, Ocampo A, Liu GH, Izpisua Belmonte JC (2017b) Regulation of stem cell aging by metabolism and epigenetics. Cell Metab 26:460–474

[64] Sato N, Fukushima N, Chang R, Matsubayashi H, Goggins M (2006) Differential and epigenetic gene expression profiling identifies frequent disruption of the RELN pathway in pancreatic cancers. Gastroenterology 130:548–565

[65] Schmutz I, de Lange T (2016) Shelterin. Curr Biol 26:R397–399

[66] Schulze M, Violonchi C, Swoboda S, Welz T, Kerkhoff E, Hoja S, Bruggemann S, Simburger J, Reinders J, Riemenschneider MJ (2017) RELN signaling modulates glioblastoma growth and substrate-dependent migration. Brain Pathol 28(5):695–709

[67] Sekine K, Kubo K, Nakajima K (2014) How does Reelin control neuronal migration and layer formation in the developing mammalian neocortex? Neurosci Res 86:50–58

[68] Sfeir A, Kabir S, van Overbeek M, Celli GB, de Lange T (2010) Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal. Science 327:1657–1661

[69] Shore D, Nasmyth K (1987) Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements. Cell 51:721–732

[70] Smogorzewska A, de Lange T (2004) Regulation of telomerase by telomeric proteins. Annu Rev Biochem 73:177–208

[71] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872

[72] Vincent A, Omura N, Hong SM, Jaffe A, Eshleman J, Goggins M (2011) Genome-wide analysis of promoter methylation associated with gene expression profile in pancreatic adenocarcinoma. Clin Cancer Res 17:4341–4354

[73] Wagner KD, Ying Y, Leong W, Jiang J, Hu X, Chen Y, Michiels JF, Lu Y, Gilson E, Wagner N (2017) The differential spatiotemporal expression pattern of shelterin genes throughout lifespan. Aging 9:1219–1232

[74] Wang P, Liu Z, Zhang X, Li J, Sun L, Ju Z, Li J, Chan P, Liu GH, Zhang W (2018a) CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-kappaB/RelA in maintaining the homeostasis of human vascular cells. Protein Cell 9(11):945–965

[75] Wang S, Hu B, Ding Z, Dang Y, Wu J, Li D, Liu X, Xiao B, Zhang W, Ren R (2018b) ATF6 safeguards organelle homeostasis and cellular aging in human mesenchymal stem cells. Cell Discov 4:2

[76] Wu Z, Zhang W, Song M, Wang W, Wei G, Li W, Lei J, Huang Y, Sang Y, Chan P (2018) Differential stem cell aging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome. Protein Cell 9:333–350

[77] Xiao L, Lan X, Shi X, Zhao K, Wang D, Wang X, Li F, Huang H, Liu J (2017) Cytoplasmic RAP1 mediates cisplatin resistance of nonsmall cell lung cancer. Cell Death Dis 8:e2803

[78] Xin H, Liu D, Songyang Z (2008) The telosome/shelterin complex and its functions. Genome Biol 9:232

[79] Xu Z, Duc KD, Holcman D, Teixeira MT (2013) The length of the shortest telomere as the major determinant of the onset of replicative senescence. Genetics 194:847–857

[80] Yang D, Xiong Y, Kim H, He Q, Li Y, Chen R, Songyang Z (2011) Human telomeric proteins occupy selective interstitial sites. Cell Res 21:1013–1027

[81] Yang J, Li J, Suzuki K, Liu X, Wu J, Zhang W, Ren R, Zhang W, Chan P, Izpisua Belmonte JC (2017) Genetic enhancement in cultured human adult stem cells conferred by a single nucleotide recoding. Cell Res 27:1178–1181

[82] Yang X, Figueiredo LM, Espinal A, Okubo E, Li B (2009) RAP1 is essential for silencing telomeric variant surface glycoprotein genes in Trypanosoma brucei. Cell 137:99–109

[83] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920

[84] Zha Y, Gan P, Yao Q, Ran FM, Tan J (2014) Downregulation of Rap1 promotes 5-fluorouracil-induced apoptosis in hepatocellular carcinoma cell line HepG2. Oncol Rep 31:1691–1698

[85] Zhang W, Li J, Suzuki K, Qu J, Wang P, Zhou J, Liu X, Ren R, Xu X, Ocampo A (2015) Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging. Science 348:1160–1163

[86] Zhang W, Wan H, Feng G, Qu J,Wang J, Jing Y, Ren R, Liu Z, Zhang L, Chen Z (2018) SIRT6 deficiency results in developmental retardation in cynomolgus monkeys. Nature 560:661–665

[87] Zhu Y, Liu X, Ding X, Wang F, Geng X (2018) Telomere and its role in the aging pathways: telomere shortening, cell senescence and mitochondria dysfunction. Biogerontology 20:1–16

RIGHTS & PERMISSIONS

2019 The Author(s)