Genome-wide YFP Fluorescence Complementation Screen Identifies New Regulators for Telomere Signaling in Human Cells
2010; Elsevier BV; Volume: 10; Issue: 2 Linguagem: Inglês
10.1074/mcp.m110.001628
ISSN1535-9484
AutoresOk-Hee Lee, Hyeung Kim, Quanyuan He, Hwa Jin Baek, Dong Yang, Liuh‐Yow Chen, Jiancong Liang, Heekyung Kate Chae, Amin Safari, Dan Liu, Zhou Songyang,
Tópico(s)Retinal Development and Disorders
ResumoDetection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks. To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Maintenance of vertebrate telomeres requires the concerted action of members of the Telomere Interactome, built upon the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. Of the ∼12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric proteins. The majority of the identified proteins have not been previously linked to telomere biology, including regulators of post-translational modifications such as protein kinases and ubiquitin E3 ligases. Results from this study shed light on the molecular niche that is fundamental to telomere regulation in humans, and provide a valuable tool to investigate signaling pathways in mammalian cells. Detection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks. To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Maintenance of vertebrate telomeres requires the concerted action of members of the Telomere Interactome, built upon the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. Of the ∼12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric proteins. The majority of the identified proteins have not been previously linked to telomere biology, including regulators of post-translational modifications such as protein kinases and ubiquitin E3 ligases. Results from this study shed light on the molecular niche that is fundamental to telomere regulation in humans, and provide a valuable tool to investigate signaling pathways in mammalian cells. During mammalian DNA replication, linear chromosomal ends will gradually erode because of the inability of the DNA replication machinery to replicate the extreme 5′ terminus of a linear DNA sequence (1.Watson J.D. The regulation of DNA synthesis in eukaryotes.Adv. Cell Biol. 1971; 2: 1-46PubMed Google Scholar, 2.Olovnikov A.M. A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon.J. Theor. Biol. 1973; 41: 181-190Crossref PubMed Scopus (1423) Google Scholar). This inherent "end replication problem" is circumvented through specialized chromosomal end structures (telomeres) and the action of the RNA-containing DNA polymerase - telomerase (3.Autexier C. Greider C.W. Telomerase and cancer: revisiting the telomere hypothesis.Trends Biochem. Sci. 1996; 21: 387-391Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 4.McEachern M.J. Krauskopf A. Blackburn E.H. Telomeres and their control.Annu. Rev. Genet. 2000; 34: 331-358Crossref PubMed Scopus (608) Google Scholar, 5.Cech T.R. Life at the End of the Chromosome: Telomeres and Telomerase.Angew Chem. Int Ed. Engl. 2000; 39: 34-43Crossref PubMed Google Scholar, 6.Harley C.B. Telomerase is not an oncogene.Oncogene. 2002; 21: 494-502Crossref PubMed Google Scholar, 7.Cong Y.S. Wright W.E. Shay J.W. Human telomerase and its regulation.Microbiol. Mol. Biol. Rev. 2002; 66 (table of contents): 407-425Crossref PubMed Scopus (675) Google Scholar, 8.de Lange T. Protection of mammalian telomeres.Oncogene. 2002; 21: 532-540Crossref PubMed Google Scholar, 9.Wong J.M. Collins K. Telomere maintenance and disease.Lancet. 2003; 362: 983-988Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Telomere homeostasis is essential for genome stability, cell survival, and growth. Telomeres and telomerase help to ensure genome integrity in eukaryotes by enabling complete replication of the ends of linear DNA molecules, and preventing chromosomal rearrangements or fusion. For dividing cells such as stem cells and the majority of cancer cells, the telomerase is an essential positive regulator of their telomere length and ultimately determines the proliferative potential of these cells. Mammalian telomeres consist of a series of (TTAGGG)n sequence repeats and terminate in 3′ single-stranded DNA overhangs that are extendable by the telomerase (10.Cech T.R. Nakamura T.M. Lingner J. Telomerase is a true reverse transcriptase. A review.Biochemistry. 1997; 62: 1202-1205PubMed Google Scholar). Exposed linear chromosome ends or naturally occurring double-stranded breaks pose additional risks including activation of DNA damage responses. The ends of telomeres in mammalian cells appear to fold back in a T-loop structure, with the 3′ G-rich single-stranded overhang invading into the double-stranded telomere regions to form the d-loop (11.Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. de Lange T. Mammalian telomeres end in a large duplex loop.Cell. 1999; 97: 503-514Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar). The structure of the telomeres, coupled with the coordinated action of a collection of proteins that protect the ends of chromosomes (12.Blackburn E.H. Switching and signaling at the telomere.Cell. 2001; 106: 661-673Abstract Full Text Full Text PDF PubMed Scopus (1761) Google Scholar, 13.Maser R.S. DePinho R.A. Connecting chromosomes, crisis, and cancer.Science. 2002; 297: 565-569Crossref PubMed Scopus (485) Google Scholar, 14.Smogorzewska A. de Lange T. Regulation of telomerase by telomeric proteins.Annu. Rev. Biochem. 2004; 73: 177-208Crossref PubMed Scopus (660) Google Scholar, 15.Wright W.E. Shay J.W. Telomere-binding factors and general DNA repair.Nat. Genet. 2005; 37: 116-118Crossref PubMed Scopus (38) Google Scholar), contributes to the maintenance of telomere integrity, genome stability, and proper cell cycle progression. In mammals, the most widely studied telomere-associated proteins include the double-stranded DNA binding proteins TRF1 and TRF2 (16.Bianchi A. Smith S. Chong L. Elias P. de Lange T. TRF1 is a dimer and bends telomeric DNA.EMBO J. 1997; 16: 1785-1794Crossref PubMed Scopus (269) Google Scholar, 17.Broccoli D. Smogorzewska A. Chong L. de Lange T. Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2.Nat. Genet. 1997; 17: 231-235Crossref PubMed Scopus (755) Google Scholar), the single-stranded telomeric DNA binding protein POT1 (18.Baumann P. Cech T.R. Pot1, the putative telomere end-binding protein in fission yeast and humans.Science. 2001; 292: 1171-1175Crossref PubMed Scopus (801) Google Scholar), and three associated factors (RAP1, TIN2, and TPP1) (19.Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (425) Google Scholar, 20.Li B. Oestreich S. de Lange T. Identification of human Rap1: implications for telomere evolution.Cell. 2000; 101: 471-483Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 21.Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. PTOP interacts with POT1 and regulates its localization to telomeres.Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (333) Google Scholar, 22.Ye J.Z. Hockemeyer D. Krutchinsky A.N. Loayza D. Hooper S.M. Chait B.T. de Lange T. POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex.Genes Dev. 2004; 18 (Epub 2004 Jul 1): 1649-1654Crossref PubMed Scopus (346) Google Scholar, 23.Houghtaling B.R. Cuttonaro L. Chang W. Smith S. A dynamic molecular link between the telomere length regulator TRF1 and the chromosome end protector TRF2.Curr. Biol. 2004; 14: 1621-1631Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Work from our lab and others suggest that TPP1, along with POT1, TIN2, TRF1, TRF2, and RAP1, form a higher order complex (the telosome/shelterin) at the telomeres (24.Liu D. O'Connor M.S. Qin J. Songyang Z. Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins.J. Biol. Chem. 2004; 279: 51338-51342Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 25.Ye J.Z. Donigian J.R. Van Overbeek M. Loayza D. Luo Y. Krutchinsky A.N. Chait B.T. De Lange T. TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.J. Biol. Chem. 2004; 16: 16Google Scholar, 26.Kim S.H. Beausejour C. Davalos A.R. Kaminker P. Heo S.J. Campisi J. TIN2 mediates functions of TRF2 at human telomeres.J. Biol. Chem. 2004; 279: 43799-43804Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 27.de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres.Genes Dev. 2005; 19: 2100-2110Crossref PubMed Scopus (2271) Google Scholar). Information regarding the state of the telomere ends can be transmitted from TRF1 and TRF2 to POT1, through TPP1 and the other subunits (28.Xin H. Liu D. Songyang Z. The telosome/shelterin complex and its functions.Genome Biol. 2008; 9: 232Crossref PubMed Scopus (152) Google Scholar). Furthermore, TRF1 and TRF2 function as bona fide protein hubs and interact with a diverse array of factors/complexes that are involved in cell cycle, DNA repair, and recombination to maintain telomere structure and length (12.Blackburn E.H. Switching and signaling at the telomere.Cell. 2001; 106: 661-673Abstract Full Text Full Text PDF PubMed Scopus (1761) Google Scholar, 13.Maser R.S. DePinho R.A. Connecting chromosomes, crisis, and cancer.Science. 2002; 297: 565-569Crossref PubMed Scopus (485) Google Scholar, 27.de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres.Genes Dev. 2005; 19: 2100-2110Crossref PubMed Scopus (2271) Google Scholar, 29.Wright W.E. Shay J.W. Cellular senescence as a tumor-protection mechanism: the essential role of counting.Curr. Opin. Genet. Dev. 2001; 11: 98-103Crossref PubMed Scopus (261) Google Scholar, 30.Kim S.H. Kaminker P. Campisi J. Telomeres, aging and cancer: in search of a happy ending.Oncogene. 2002; 21: 503-511Crossref PubMed Google Scholar, 31.Baumann P. Are mouse telomeres going to pot?.Cell. 2006; 126: 33-36Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 32.Blasco M.A. The epigenetic regulation of mammalian telomeres.Nat. Rev. Genet. 2007; 8: 299-309Crossref PubMed Scopus (542) Google Scholar, 33.Verdun R.E. Karlseder J. Replication and protection of telomeres.Nature. 2007; 447: 924-931Crossref PubMed Scopus (379) Google Scholar, 34.Longhese M.P. DNA damage response at functional and dysfunctional telomeres.Genes Dev. 2008; 22: 125-140Crossref PubMed Scopus (143) Google Scholar). Consistent with the end protection function of this complex, many factors that are known to participate in DNA damage responses are recruited to the telomeres, such as the Mre11/Rad50/NBS1 complex, PARP-1, Ku70/80, DNA helicases BLM and WRN, Rad51D, nucleotide excision repair protein ERCC1/XPF, DNA nuclease Apollo, and the BRCT domain-containing protein MCPH1 (3.Autexier C. Greider C.W. Telomerase and cancer: revisiting the telomere hypothesis.Trends Biochem. Sci. 1996; 21: 387-391Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 4.McEachern M.J. Krauskopf A. Blackburn E.H. Telomeres and their control.Annu. Rev. Genet. 2000; 34: 331-358Crossref PubMed Scopus (608) Google Scholar, 10.Cech T.R. Nakamura T.M. Lingner J. Telomerase is a true reverse transcriptase. A review.Biochemistry. 1997; 62: 1202-1205PubMed Google Scholar, 20.Li B. Oestreich S. de Lange T. Identification of human Rap1: implications for telomere evolution.Cell. 2000; 101: 471-483Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 35.Song K. Jung D. Jung Y. Lee S.G. Lee I. Interaction of human Ku70 with TRF2.FEBS Lett. 2000; 481: 81-85Crossref PubMed Scopus (148) Google Scholar, 36.Li B. Comai L. Functional interaction between Ku and the werner syndrome protein in DNA end processing.J. Biol. Chem. 2000; 275: 28349-28352Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 37.Zhu X.D. Küster B. Mann M. Petrini J.H. de, Lange T. Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres.Nat. Genet. 2000; 25: 347-352Crossref PubMed Scopus (509) Google Scholar, 38.Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases.J. Biol. Chem. 2002; 277 (Epub 42002 Aug 41113): 41110-41119Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 39.Takai H. Smogorzewska A. de Lange T. DNA damage foci at dysfunctional telomeres.Curr. Biol. 2003; 13: 1549-1556Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, 40.Du X. Shen J. Kugan N. Furth E.E. Lombard D.B. Cheung C. Pak S. Luo G. Pignolo R.J. DePinho R.A. Guarente L. Johnson F.B. Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes.Mol. Cell. Biol. 2004; 24: 8437-8446Crossref PubMed Scopus (186) Google Scholar, 41.Chang S. Multani A.S. Cabrera N.G. Naylor M.L. Laud P. Lombard D. Pathak S. Guarente L. DePinho R.A. Essential role of limiting telomeres in the pathogenesis of Werner syndrome.Nat. Genet. 2004; 36 (Epub 2004 Jul 2004): 877-882Crossref PubMed Scopus (381) Google Scholar, 42.Li B. Navarro S. Kasahara N. Comai L. Identification and biochemical characterization of a Werner's syndrome protein complex with Ku70/80 and poly(ADP-ribose) polymerase-1.J. Biol. Chem. 2004; 279 (Epub 12004 Jan 13620): 13659-13667Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 43.Tarsounas M. Muñoz P. Claas A. Smiraldo P.G. Pittman D.L. Blasco M.A. West S.C. Telomere maintenance requires the RAD51D recombination/repair protein.Cell. 2004; 117: 337-347Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 44.d'Adda di Fagagna F. Teo S.H. Jackson S.P. Functional links between telomeres and proteins of the DNA-damage response.Genes Dev. 2004; 18: 1781-1799Crossref PubMed Scopus (224) Google Scholar, 45.Stavropoulos D.J. Bradshaw P.S. Li X. Pasic I. Truong K. Ikura M. Ungrin M. Meyn M.S. The Bloom syndrome helicase BLM interacts with TRF2 in ALT cells and promotes telomeric DNA synthesis.Hum. Mol. Genet. 2002; 11: 3135-3144Crossref PubMed Scopus (160) Google Scholar, 46.Zhu X.D. Niedernhofer L. Kuster B. Mann M. Hoeijmakers J.H. de Lange T. ERCC1/XPF removes the 3′ overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes.Mol. Cell. 2003; 12: 1489-1498Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 47.O'Connor M.S. Safari A. Liu D. Qin J. Songyang Z. The human Rap1 protein complex and modulation of telomere length.J. Biol. Chem. 2004; 279 (Epub 22004 Apr 28520): 28585-28591Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 48.Lillard-Wetherell K. Machwe A. Langland G.T. Combs K.A. Behbehani G.K. Schonberg S.A. German J. Turchi J.J. Orren D.K. Groden J. Association and regulation of the BLM helicase by the telomere proteins TRF1 and TRF2.Hum. Mol. Genet. 2004; 13 (Epub 2004 Jun 30): 1919-1932Crossref PubMed Scopus (124) Google Scholar, 49.Opresko P.L. Otterlei M. Graakjaer J. Bruheim P. Dawut L. Kølvraa S. May A. Seidman M.M. Bohr V.A. The Werner syndrome helicase and exonuclease cooperate to resolve telomeric D loops in a manner regulated by TRF1 and TRF2.Mol. Cell. 2004; 14: 763-774Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 50.Collins K. Mitchell J.R. Telomerase in the human organism.Oncogene. 2002; 21: 564-579Crossref PubMed Google Scholar, 51.Granger M.P. Wright W.E. Shay J.W. Telomerase in cancer and aging.Crit. Rev. Oncol. Hematol. 2002; 41: 29-40Crossref PubMed Scopus (139) Google Scholar, 52.Kim H. Lee O.H. Xin H. Chen L.Y. Qin J. Chae H.K. Lin S.Y. Safari A. Liu D. Songyang Z. TRF2 functions as a protein hub and regulates telomere maintenance by recognizing specific peptide motifs.Nat. Struct. Mol. Biol. 2009; 16: 372-379Crossref PubMed Scopus (103) Google Scholar, 53.Peng G. Lin S.Y. BRIT1/MCPH1 is a multifunctional DNA damage responsive protein mediating DNA repair-associated chromatin remodeling.Cell Cycle. 2009; 8: 3071-3072Crossref PubMed Scopus (11) Google Scholar). To date, much has been learned regarding the core telomere binding components, factors that constitutively associate with the telomeres. However, much remains unknown regarding the factors that are recruited to the telomeres upon damage or other signaling events, as well as the signaling cascades that must take place on or near the telomeres. In other words, the micro-environment—the complex regulatory network of protein-protein interactions—within which telomere homeostasis is achieved remains to be elucidated. Signaling regulators are often of low abundance, and their association with the targets may be transient or weak. Although conventional proteomic methods such as immunoprecipitation (IP) and mass spectrometry have been particularly informative in identifying core interacting proteins, regulatory components may be below the detection threshold. We have developed a high-throughput protein-protein interaction screening strategy based on the principle of the yellow fluorescent protein (YFP) 1The abbreviations used are:YFPyellow fluorescence proteinPCAprotein complementation assayBiFCbimolecular fluorescence complementationIPimmunoprecipitationORFopen reading frameWPRweighted positive ratiosGSTglutathione S-transferase. 1The abbreviations used are:YFPyellow fluorescence proteinPCAprotein complementation assayBiFCbimolecular fluorescence complementationIPimmunoprecipitationORFopen reading frameWPRweighted positive ratiosGSTglutathione S-transferase.-based protein complementation assay (PCA/bimolecular fluorescent complementation (BiFC)) (52.Kim H. Lee O.H. Xin H. Chen L.Y. Qin J. Chae H.K. Lin S.Y. Safari A. Liu D. Songyang Z. TRF2 functions as a protein hub and regulates telomere maintenance by recognizing specific peptide motifs.Nat. Struct. Mol. Biol. 2009; 16: 372-379Crossref PubMed Scopus (103) Google Scholar, 54.Ghosh I. Hamilton A.D. Regan L. antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein.J. Am. Chem. Soc. 2000; 122: 5658Crossref Scopus (428) Google Scholar, 55.Hu C.D. Chinenov Y. Kerppola T.K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.Mol. Cell. 2002; 9: 789-798Abstract Full Text Full Text PDF PubMed Scopus (1206) Google Scholar, 56.Chen L.Y. Liu D. Songyang Z. Telomere maintenance through spatial control of telomeric proteins.Mol. Cell. Biol. 2007; 27: 5898-5909Crossref PubMed Scopus (90) Google Scholar). In the PCA/BiFC assay, protein-protein interactions bring the two fragments (YFPn and YFPc) of YFP (tagged to two separate proteins) to close proximity and allow for their cofolding into a functional fluorescent protein (57.Wilson C.G. Magliery T.J. Regan L. Detecting protein-protein interactions with GFP-fragment reassembly.Nat. Methods. 2004; 1: 255-262Crossref PubMed Scopus (110) Google Scholar, 58.Hu C.D. Kerppola T.K. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis.Nat. Biotechnol. 2003; 21 (Epub 2003 Apr 14): 539-545Crossref PubMed Scopus (639) Google Scholar). PCA/BiFC enables the examination of interactions in live cells, providing spatial information about protein-protein interactions. Here we report the identification of over 300 telosome/shelterin associating proteins that mediate diverse signaling pathways. Many of these proteins regulate post-translational modifications including protein phosphorylation and ubiquitination. Our findings provide a high-resolution map of the telomere interactome (19.Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (425) Google Scholar, 59.O'Connor M.S. Safari A. Xin H. Liu D. Songyang Z. A critical role for TPP1 and TIN2 interaction in high-order telomeric complex assembly.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11874-11879Crossref PubMed Scopus (184) Google Scholar, 60.Songyang Z. Liu D. Inside the mammalian telomere interactome: regulation and regulatory activities of telomeres.Crit. Rev. Eukaryot Gene Expr. 2006; 16: 103-118Crossref PubMed Scopus (42) Google Scholar), and should greatly facilitate further studies of telomere signaling. yellow fluorescence protein protein complementation assay bimolecular fluorescence complementation immunoprecipitation open reading frame weighted positive ratios glutathione S-transferase. yellow fluorescence protein protein complementation assay bimolecular fluorescence complementation immunoprecipitation open reading frame weighted positive ratios glutathione S-transferase. pDONR223 vectors encoding SOX2, TRF1, RAP1, TIN2, and POT1 came from human ORFeome v3.1 (Open Biosystems). Sequences encoding human TRF2 and TPP1 were PCR amplified and cloned into pENTR/d-TOPO vector (Invitrogen). Through Gateway recombination, the six telomere open reading frames (ORFs) were transferred individually into either pBabe-CMV-YFPn-DEST-neo or pBabe-CMV-DEST-YFPn-neo vectors (56.Chen L.Y. Liu D. Songyang Z. Telomere maintenance through spatial control of telomeric proteins.Mol. Cell. Biol. 2007; 27: 5898-5909Crossref PubMed Scopus (90) Google Scholar). The resulting mammalian expression constructs enable tagging of Venus YFPn fragments at either the N- or C terminus of a protein. The vectors were then transfected individually into Phoenix cells for retrovirus production and subsequent infection of HTC75 cells. Infected cells were selected with 300 μg/ml G418 for up to 10 days to obtain cells stably expressing YFPn-tagged bait proteins. For each bait, cells that expressed either N- or C-terminally tagged YFPn-fusion proteins were mixed (1:1) for subsequent screens. Individual Gateway recombination reactions (in 96-well plates) were performed for all 12,212 ORFs from hORFeome with a mixture of pCl-CMV-YFPc-DEST-puro and pCL-CMV-DEST-YFPc-puro vectors (1:1) (56.Chen L.Y. Liu D. Songyang Z. Telomere maintenance through spatial control of telomeric proteins.Mol. Cell. Biol. 2007; 27: 5898-5909Crossref PubMed Scopus (90) Google Scholar), to generate ORFs tagged with YFPc at either the N- or C terminus. The reaction products were subsequently used to transform DH5α and selected by ampicilin. Plasmid extractions were then carried out in 96-well plates for each pool of bacterial transformants using PureLink HQ 96 plasmid purification kit (Invitrogen) and Biomek FX Laboratory Automation Work station (Beckman Coulter). 11,880 ORFs were successfully cloned into the YFPc vectors. The YFPc-prey collections were then transfected into Phoenix cells to generate retroviruses for infection of bait cells. Retroviral supernatant was collected at 48 h or 72 h post-transfection, and stored in −80 °C before use. All transfection steps were done in 96-well formats using the Biomek 3000 Laboratory Automation Work station (Beckman Coulter). Cells from each bait cell line were seeded onto 96-well plates and infected with the arrayed YFPc-tagged prey library. At 2 days following the infection, cells were selected with 1 μg/ml of puromycin for 5–10 days. We were able to obtain 10058 SOX2, 11,685 TRF1, 11,006 TRF2, 11,724 RAP1, 11,398 TIN2, 11,385 TPP1, and 11,330 POT1 cell lines infected by the YFPc-tagged prey library. All work was performed using the Biomek3000 Laboratory Automation Work station. Cells were then harvested for high-throughput flow cytometric analysis using the LSRII flow cytometer equipped with a HTS sampler (BD Biosciences). CytoArray is a data analysis platform custom designed for processing the large amount of flow cytometry data from the arrayed screen. Data processing is roughly divided into four major steps: defining positive regions, calculating weighted positive ratios (WPR), determining statistically significant cutoff values, and removing common contaminants (Supplemental Fig. S2). Data from each well are plotted with green fluorescent protein (GFP) on the x axis and phycoerythrin on the y axis. First, any samples with <200 data points are automatically discarded. The remaining profiles are processed plate-by-plate for each bait. All the profiles within a plate are compiled to create a composite profile. It is assumed that the majority of these flow cytometry profiles would be negative for PCA/BiFC signals and closely resemble each other. Therefore, the composite profile can be used as an internal control to gate for negative versus positive regions when superimposed onto individual profiles within the same plate (Supplemental Fig. S3). For the composite profile, CytoArray determines the adjusted vertex and weight center of data point distribution, and uses the y value of the adjusted vertex and x value of the weight center to define the top and left boundary of the positive region. Starting from here, the remaining boundaries are defined by scanning across the distribution profile until this positive region contains 5% of data points. CytoArray calculates weighted positive ratios (WPR) rather than positive percentages (PP: positive cell number/total live cell number) to measure PCA/BiFC signals, because PP ignores signal intensity (ratio of YFP/phycoerythrin) and does not differentiate between marginal versus significant data points. WPR increases the signal to noise ratio and improves the sensitivity of detection (Supplemental Fig. S3). WPR ranks all positive data points (on an arbitrary scale of 1–5) according to their distance from the leftmost boundary of the positive region, with the data points furthest away from that boundary assigned the highest value. Next, CytoArray calculates the WPR cutoff. When the distribution of the WPR values from all the proteins that were defined as extracellular (based on cellular component annotation in Gene Ontology database) is compared with that of the WPR values from the entire screen for each bait, the trend is clear that the higher the WPR value, the fewer of these proteins can be found. Using these two distribution curves, CytoArray calculates the WPR ratios between them. For example, when the ratio is 90%, the corresponding WPR value is the threshold cutoff with a 90% positive ratio (Supplemental Fig. S4). Finally, common contaminants and nonspecific binding proteins are filtered out using data from similar screens of the unrelated bait SOX2. To validate the interactions between each bait and prey protein pair, coprecipitation bythe glutathione S-transferase (GST) pull-down was performed in 96-well format using 96-well filter plates from Invitrogen. Sequences encoding telomeric proteins and candidate prey proteins were cloned into pDEST-27 (Invitrogen) for tagging with GST and pCl-2xFLAG for tagging with FLAG, respectively. Each bait-prey pair was cotransfected into 293T cells. The transfected cells were harvested after 2 days, and lysed with 1×NETN buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.5% Nonidet P-40, and 1 mm EDTA). The whole cell extract was then transferred to a 96-well lysate clearing filter plate (25 μm, Phenix), and centrifuged at 500 × g for 2 min (4 °C). The cleared lysate was then transferred into a 96-well binding plate (Purelink Clarificaiton plate, Invitrogen) preloaded with 100 μl/well of glutathione Sepharose 4B beads (10% slurry) (GE Healthcare Bio-Sciences AB), and incubated for 2 h at 4 °C with gentle agitation. The binding plate was then washed three times with 1× NETN, and the bound proteins eluted with elution buffer (50 mm Tris-HCl, pH 8.0, 20% glycerol, 20 mm reduced glutathione), centrifuged at 500 × g for 2 min and blotted onto polyvinylidene fluoride membranes using the Bio-Dot apparatus (Bio-Rad). The membrane was then probed with anti-FLAG-HRP (Sigma) or anti-GST-HRP (GE Healthcare Bio-Sciences AB) antibodies. To investigate protein-protein interactions in human cells, we adopted and modified the YFP-based PCA/BiFC method (52.Kim H. Lee O.H. Xin H. Chen L.Y. Qin J. Chae H.K. Lin S.Y. Safari A. Liu D. Songyang Z. TRF2 functions as a protein hub and regulates telomere maintenance by recognizing specific peptide motifs.Nat. Struct. Mol. Biol. 2009; 16: 372-379Crossref PubMed Scopus (103) Google Scholar, 56.Chen L.Y. Liu D. Songyang Z. Telomere maintenance through spatial control of telomeric proteins.Mol. Cell. Biol. 2007; 27: 5898-5909Crossref PubMed Scopus (90) Google Scholar, 58.Hu C.D. Kerppola T.K. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis.Nat. Biotechnol. 2003; 21 (Epub 2003 Apr 14): 539-545Crossref PubMed Scopus (639) Google Scholar). The N-terminal fragment (residues 1–155) of Venus YFP (YFPn) or the C-terminal fragment (residues 156–239) of YFP (YFPc) were tethered to either N- or C-terminal ends of candidate proteins and expressed in human cells for fluorescence complementation (Figs. 1A and 1B). A flexible linker of ∼30 amino acids, which covers a distance of ∼10 nm, was engineered to maximize complementation. Tagging protein pairs on either end with the two YFP fragments would cover all possible interaction configurations. Testing all these combinations individually, however, would be impractical on a large scale. Here, we performed PCA/BiFC assays using pools of cells expressing N- or C-terminally tagged fusion proteins (Fig. 1B). For example, bait-expressing cells were a mixture of cells that individually expressed
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