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Dissecting the telomere–inner nuclear membrane interface formed in meiosis

Nature Structural & Molecular Biology volume 24, pages 1064–1072 (2017)Cite this article

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Abstract

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere–INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1–TERB1 interface to reveal the structural basis for telomere–INM linkage. Disruption of this interface abrogates binding and compromises telomere–INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1–TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere–INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere–INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.

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Figure 1: Structural and biochemical dissection of the TRF1TRFH–TERB1TBM interface.
Figure 2: Weakening the TRF1–TERB1 interface compromises telomere–INM tethering.
Figure 3: Further strengthening of the TRF1–TERB1 interface disrupts cap exchange.
Figure 4: Defining protein complexes involved in telomere tethering versus chromosome end protection.

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Acknowledgements

We thank the Life Sciences Collaborative Access Team at Argonne National Laboratory beamline staff for help with X-ray data collection, M. Iyer for help with protein purifications, and S. Grill for excellent feedback on the manuscript. This work was funded by NIH Grants R00CA167644 (to J.N.), R01GM120094 (to J.N.), R01AG050509 (to J.N., co-investigator), American Cancer Society Research Scholar grant 130882-RSG-17-037-01-DMC (to J.N.), NIH Biology of Aging Training Grant (T32AG000114) awarded to the University of Michigan Geriatrics Center from the National Institute on Aging (fellowship to E.M.S.), and MEXT KAKENHI 25000014 (to Y.W.).

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Authors and Affiliations

  1. Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA

    Devon F Pendlebury, Valerie M Tesmer, Eric M Smith & Jayakrishnan Nandakumar

  2. Program in Chemical Biology, University of Michigan, Ann Arbor, Michigan, USA

    Devon F Pendlebury, Eric M Smith & Jayakrishnan Nandakumar

  3. University of Tokyo, Institute of Molecular and Cellular Biosciences, Laboratory of Chromosome Dynamics, Tokyo, Japan

    Yasuhiro Fujiwara & Yoshinori Watanabe

  4. Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden

    Hiroki Shibuya

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  1. Devon F Pendlebury
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Contributions

J.N., D.F.P., V.M.T., and Y.W. designed experiments; D.F.P., V.M.T., E.M.S., and J.N. conducted the crystallographic experiments; D.F.P. conducted cloning, protein purifications, and in vitro binding studies; Y.F. conducted in vivo cellular localization studies with guidance from H.S.; J.N., D.F.P., and Y.W. analyzed the data; J.N. and D.F.P. wrote the manuscript with input from all authors.

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Correspondence to Jayakrishnan Nandakumar.

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Integrated supplementary information

Supplementary Figure 1 The TRF1-TERB1 interface.

(a) Overlay of the two TRF1TRFH-TERB1TRFB complexes observed in the dimeric crystal structure. The two TRFH domains are shown in cartoon depiction (green and cyan), while the two TBM peptides are shown in stick representation (orange and pink). (b) 2Fo – Fc map of the core of the TRF1-TERB1 interface contoured at 1.0 σ.

Supplementary Figure 2 Mutations in the TRF1-TERB1 interface abrogate binding.

(a) GST pull down of His-Smt3-TERB1TBM WT and mutant proteins using GST-TRF1TRFH as bait. Quantitation at the bottom was performed by dividing the appropriate band intensities by the molecular weight of the species followed by normalization against the molecular weight-normalized GST-TRF1TRFH signal in that line. (b) GST pull down of TRF1TRFH (WT or F142A) using GST-TERB1TBM as bait.

Supplementary Figure 3 Competition data for TERB1 TBM mutants with TRF1TRFH.

(a) And (b) are independent experimental replicates of data shown in Fig. 1j and k, respectively. Mean of technical duplicate is plotted. Mean and s.e.m. of biological duplicates (of technical duplicates) are shown in Table 2. (c) Size-exclusion (Superdex 200) profile of the TERB1TRFB and TERB21-107 complex following Ni-affinity purification and removal of the His-Smt3 tag. (d) Coomassie-stained SDS-PAGE for WT and indicated TERB1 mutant TERB1TRFB - TERB21-107 complexes eluted by size-exclusion chromatography. Yellow boxes highlight fractions indicative of a 1:1 TERB1:TERB2 complex. (e) Molecular weights of WT and TERB1 mutant TERB1TRFB - TERB21-107 complexes were determined by size-exclusion chromatography (Superdex 75). The theoretical molecular weight of a homodimer of the TERB1TRFB - TERB21-107 heterodimer (in kDa) along with the experimentally determined molecular weights (in kDa) is shown in a tabular format. (f) Equal amounts (5 μg) of indicated, purified TERB1TRFB - TERB21-107 complexes were analyzed using coomassie-stained SDS-PAGE.

Supplementary Figure 4 Cap exchange is not prevented in WT spermatocytes expressing TERB1 mutants that disrupt the TRF1-TERB1 interface.

(a) Schematic depiction of TRF1 and TERB1 signal width and localization in early and late pachytene. While the TERB1 signal does not undergo a significant change in width throughout pachytene, the TRF1 signal becomes more diffuse resulting in an increase in its outer diameter. (b) WT late pachytene spermatocytes expressing GFP-TERB1 L647E, GFP-TERB1 T648D, GFP-TERB1 P649E, or GFP-TERB1 R651E-R652E (green) stained with TRF1 (red) and SYCP3 (blue) antibodies.

Supplementary Figure 5 Quantitation of anomalous TRF1 E93K and TERB1 I645F cap exchange.

(a) WT mouse spermatocytes expressing GFP-TRF1 E93K (green) stained with TRF1 (red) antibody in late pachytene. (b and c) Quantitation shows that GFP-TERB1 I645F distributes to the wider area compared to WT TERB1 protein. “n” and P-values indicated on graph in panel b.

Supplementary Figure 6 Composition and stoichiometry of the TRF1-TIN2 and TRF1-TERB1 complexes.

(a) Fluorescence-based competition experiments using Alexa Fluor 488-labeled GST-TIN2TBM pre-bound to biotin-tagged TRF1TRFH on streptavidin beads titrated with varying concentrations of unlabeled full-length TIN2 and TERB1TRFB – TERB21-107 proteins. Mean of technical duplicate is plotted. Error bars indicate s.e.m. (b) Size-exclusion (Superdex 200) profiles confirming intramolecular dimerization of TRFH-TRFH fusions (based on molecular weight determination). (c) Coomassie-stained SDS-PAGE for TRFH-TRFH intramolecular fusion proteins eluted by size-exclusion chromatography. (d) Replicate of experiment shown in Fig. 4c with quantitation shown at the bottom. (e) Mean and s.e.m. of duplicate experiments from panel d and Fig. 4c.

Supplementary Figure 7 Binding of TERB1 to TRF2 in vitro.

(a) Direct binding curves of biotin-tagged TRF1TRFH and TRF2TRFH with Alexa Fluor 488-labeled GST-TERB1TBM. Mean of technical duplicate is plotted. Mean and s.e.m. of duplicate measurements are indicated. (b) Competition experiments demonstrating similar affinity of TRF1 and TRF2 for TERB1TBM peptide. Mean of technical duplicate is plotted. Error bars indicate s.e.m. (c-d) Fluorescence-based competition experiments using Alexa Fluor 488-labeled GST-TERB1TBM pre-bound to biotin-tagged TRF2TRFH on streptavidin beads titrated with varying concentrations of indicated unlabeled TERB1TBM peptides (c) or TERB1TRFB – TERB21-107 complexes (d). For c and d, mean of technical duplicate is plotted and two biological replicates were performed. (e) Ternary pull down on amylose resin showing that TRF2 associates with MBP-tagged TERB1TRFB – TERB21-107 in the absence of Rap1 but not in its presence. (f) Ternary pull down on amylose resin showing that TRF2 associates with MBP-tagged TERB1TRFB – TERB21-107 in the presence or absence of TIN2.

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Pendlebury, D., Fujiwara, Y., Tesmer, V. et al. Dissecting the telomere–inner nuclear membrane interface formed in meiosis. Nat Struct Mol Biol 24, 1064–1072 (2017). https://doi.org/10.1038/nsmb.3493

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