@article{pan_kaur_barnes_detwiler_sanford_liu_xu_mahn_tang_hao_et al._2021, title={Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA}, volume={297}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2021.101080}, abstractNote={TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. Telomeres are nucleoprotein structures that prevent the degradation or fusion of the ends of linear chromosomes, which are threatened by at least seven distinct DNA damage response (DDR) pathways (1Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1344) Google Scholar, 2Muraki K. Nyhan K. Han L. Murnane J.P. Mechanisms of telomere loss and their consequences for chromosome instability.Front. Oncol. 2012; 2: 135Crossref PubMed Google Scholar, 3de Lange T. Shelterin-mediated telomere protection.Annu. Rev. Genet. 2018; 52: 223-247Crossref PubMed Scopus (280) Google Scholar). 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A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar). Consistent with its key role in telomere maintenance, germline inactivation of TIN2 in mice is embryonic lethal (41Chiang Y.J. Kim S.H. Tessarollo L. Campisi J. Hodes R.J. Telomere-associated protein TIN2 is essential for early embryonic development through a telomerase-independent pathway.Mol. Cell. Biol. 2004; 24: 6631-6634Crossref PubMed Scopus (61) Google Scholar). Removal of TIN2 leads to the formation of telomere dysfunction-induced foci (TIFs). Importantly, clinical studies further highlight the biological significance of TIN2 in telomere protection (42Savage S.A. Giri N. Baerlocher G.M. Orr N. Lansdorp P.M. Alter B.P. TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita.Am. J. Hum. 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However, since TIN2 itself does not directly bind to DNA and instead serves as a "mediator/enhancer" for shelterin and telomerase activities, defining TIN2's distinct function at the molecular level has been challenging. The bottleneck for studying TIN2 lies in the fact that results from bulk biochemical assays do not fully reveal the heterogeneity and dynamics of the protein–protein and protein–DNA interactions. Furthermore, cell-based assays only provide information on the outcomes from downstream effectors after the knocking down of TIN2 that also removes TRF1 and TRF2 from telomeres. These approaches do not allow us to investigate the molecular structures and dynamics in which TIN2 directly participates. In vivo, the amount of TIN2 is sufficient for binding every TRF1 and TRF2 molecule (44Takai K.K. Hooper S. Blackwood S. Gandhi R. de Lange T. In vivo stoichiometry of shelterin components.J. Biol. Chem. 2010; 285: 1457-1467Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), while TPP1 and POT1 are ~10-fold less than TRF1 and TIN2. Thus, it is important to study the DNA-binding properties of TRF1-TIN2 complexes. To fill this important knowledge gap, we applied complementary single-molecule imaging platforms, including atomic force microscopy (AFM) (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 46Wang H. Nora G.J. Ghodke H. Opresko P.L. Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding.J. Biol. Chem. 2011; 286: 7479-7489Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar), total internal reflection fluorescence microscopy (TIRFM) (48Erie D.A. Weninger K.R. Single molecule studies of DNA mismatch repair.DNA Repair. 2014; 20: 71-81Crossref PubMed Scopus (46) Google Scholar), and the DNA tightrope assay to monitor TRF1-TIN2-mediated DNA compaction and DNA-DNA bridging (49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar, 50Countryman P. Fan Y. Gorthi A. Pan H. Strickland J. Kaur P. Wang X. Lin J. Lei X. White C. You C. Wirth N. Tessmer I. Piehler J. 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Importantly, our results demonstrate that TIN2 protects the disassembly of TRF1-TIN2-mediated DNA-DNA bridging by Tankyrase 1. In addition, the N-terminal domain of TPP1 inhibits TRF1-TIN2-mediated DNA-DNA bridging. In summary, this study uncovered the unique biophysical function of TIN2 as a telomeric architectural protein, acting together with TRF1 to mediate interactions between distant telomeric sequences. Tankyrase 1 and TPP1 regulate TRF1-TIN2-mediated DNA-DNA bridging. Furthermore, this work establishes a unique combination of single-molecule imaging platforms for future examination of TIN2 disease variants and provides a new direction for investigating molecular mechanisms underlying diverse TIN2 functions. A previous study suggested that TIN2 modulates the bridging of telomeric DNA by TRF1 (31Kim S.H. Han S. You Y.H. Chen D.J. Campisi J. The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro.EMBO Rep. 2003; 4: 685-691Crossref PubMed Scopus (42) Google Scholar). However, the bulk biochemical assays using short telomeric DNA (six telomeric repeats) did not provide information regarding the structure and dynamics of the TRF1-TIN2-DNA complex. To investigate the molecular function of TIN2, we applied AFM imaging to investigate how TIN2 affects the telomeric DNA-DNA pairing mediated by TRF1 at the single-molecule level on longer telomeric DNA substrates (270 TTAGGG repeats). We purified TRF1 (Fig. S1A) and obtained TIN2S (1–354 amino acids, 39.4 kDa) and TIN2L (1–451 amino acids, 50.0 kDa) proteins purified from insect cells (Fig. 1A and Fig. S1D). Previously, we established an AFM imaging-based calibration method to investigate the oligomeric states and protein–protein interactions by correlating AFM volumes of proteins and their molecular weights (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar, 52Wang H. Yang Y. Erie D.A. Characterization of protein-protein interactions using atomic force microscopy.in: Schuck P. Protein Interactions Biophysical approaches for the Study of Complex Reversible Systems. Springer Science+Business Media, LLC, Berlin, Germany2007: 39-78Crossref Google Scholar). AFM volumes of TRF1 alone in solution showed two distinct peaks, which were consistent with TRF1 monomers (51 KDa) and dimers (102 KDa, Fig. S1B). In addition, based on the population of TRF1 under the monomer and dimer peaks (53Wang H. DellaVecchia M.J. Skorvaga M. Croteau D.L. Erie D.A. Van Houten B. UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.J. Biol. Chem. 2006; 281: 15227-15237Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), the estimated TRF1 dimer equilibrium dissociation constant (Kd) is 18.4 nM (Fig. S1C). Meanwhile, AFM volumes of purified TIN2S at 41.3 nm3 (±28.3 nm3) and TIN2L at 41.9 nm3 (±12.8 nm3) were consistent with the notion that TIN2 does not interact with itself (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), and TIN2 exists in a monomeric state in solution (Fig. S1D). Furthermore, we conducted size-exclusive chromatography using TRF1 and TIN2S and confirmed the presence of TRF1 dimers, TIN2 monomers, as well as the interaction between TRF1 and TIN2S in solution (Fig. S2). To further validate the activities of TIN2, we used electrophoresis mobility shift assays (EMSAs) to verify the interaction of TIN2 with TRF1 on a double-stranded telomeric DNA substrate (48 bp containing three TTAGGG repeats, Fig. S3, A–C). Consistent with previous studies (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), EMSA experiments showed that TIN2S and TIN2L did not directly bind to telomeric dsDNA (Fig. S3A). Both TRF1-TIN2S and TRF1-TIN2L induced a clear supershift of the telomeric DNA substrate compared with TRF1 alone (Complex III in Fig. S3, B and C), indicating the formation of stable TRF1-TIN2-telomeric DNA complexes. Next, to study TRF1-TIN2 DNA binding at the single-molecule level, we used the linear DNA substrate (5.4 kb) that contains 1.6 kb (270 TTAGGG) telomeric repeats in the middle region that is 35%–50% from DNA ends (T270 DNA, Experimental procedures, Fig. 1A) (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar). Previously, AFM and electron microscopy imaging–based studies established that TRF1 specifically binds to the telomeric region and mediates DNA-DNA pairing (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar, 22Bianchi A. Stansel R.M. Fairall L. Griffith J.D. Rhodes D. de Lange T. TRF1 binds a bipartite telomeric site with extreme spatial flexibility.EMBO J. 1999; 18: 5735-5744Crossref PubMed Scopus (163) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar). To study the function of TIN2, we preincubated TRF1 without or with TIN2 (either TIN2S or TIN2L), followed by the addition}, number={3}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Pan, Hai and Kaur, Parminder and Barnes, Ryan and Detwiler, Ariana C. and Sanford, Samantha Lynn and Liu, Ming and Xu, Pengning and Mahn, Chelsea and Tang, Qingyu and Hao, Pengyu and et al.}, year={2021}, month={Sep} }
 @article{chakraborty_pan_tang_woolard_xu_2018, title={The Extracellular Domain of Pollen Receptor Kinase 3 is structurally similar to the SERK family of co-receptors}, volume={8}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-018-21218-y}, abstractNote={Abstract}, journal={SCIENTIFIC REPORTS}, author={Chakraborty, Sayan and Pan, Haiyun and Tang, Qingyu and Woolard, Colin and Xu, Guozhou}, year={2018}, month={Feb} }