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The TRF1-binding protein TERB1 promotes chromosome movement and telomere rigidity in meiosis

Nature Cell Biology volume 16, pages 145–156 (2014)Cite this article

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Abstract

During meiotic prophase, telomere-mediated chromosomal movement along the nuclear envelope is crucial for homologue pairing and synapsis. However, how telomeres are modified to mediate chromosome movement is largely elusive. Here we show that mammalian meiotic telomeres are fundamentally modified by a meiosis-specific Myb-domain protein, TERB1, that localizes at telomeres in mouse germ cells. TERB1 forms a heterocomplex with the canonical telomeric protein TRF1 and binds telomere repeat DNA. Disruption of Terb1 in mice abolishes meiotic chromosomal movement and impairs homologous pairing and synapsis, causing infertility in both sexes. TERB1 promotes telomere association with the nuclear envelope and deposition of the SUN–KASH complex, which recruits cytoplasmic motor complexes. TERB1 also binds and recruits cohesin to telomeres to develop structural rigidity, strikingly reminiscent of centromeres. Our study suggests that TERB1 acts as a central hub for the assembly of a conserved meiotic telomere complex required for chromosome movements.

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Figure 1: Identification of a telomere protein, TERB1.
Figure 2: Generation of Terb1-knockout mice.
Figure 3: TERB1 is required for homologue pairing/synapsis and recombination.
Figure 4: TERB1 is required for meiotic chromosome movement.
Figure 5: TERB1–TRF1 heterocomplex associates with telomeres.
Figure 6: TERB1 interacts with and enriches meiotic cohesin at telomeres.
Figure 7: Complementation analysis for Terb1−/− spermatocytes.
Figure 8: TERB1 assembles force-generating machineries at the nuclear envelope.

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Acknowledgements

We thank N. Takeda (University of Kumamoto) for assisting in the construction of knockout mice, and Y. Kishi, Y. Ito, M. Yoda and H. Sasaki (University of Tokyo) for technical advice. We also thank all of the members of our laboratory for their valuable support and discussion. This work was supported in part by a JSPS Research Fellowship (H.S.), a Grant-in-Aid for Scientific Research on Innovative and Priority Areas (K.I.), the Global COE Program (Integrative Life Science Based on the Study of Biosignalling Mechanisms), and a Grant-in-Aid for Specially Promoted Research (Y.W.) from MEXT.

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Authors and Affiliations

  1. Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Tokyo 113-0032, Japan

    Hiroki Shibuya, Kei-ichiro Ishiguro & Yoshinori Watanabe

  2. Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Tokyo 113-0032, Japan

    Hiroki Shibuya & Yoshinori Watanabe

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  1. Hiroki Shibuya
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H.S. designed and performed experiments, analysed data and wrote the paper; K-i.I. supervised the experiments; Y.W. supervised the experiment and project and wrote the paper.

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Correspondence to Yoshinori Watanabe.

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Integrated supplementary information

Supplementary Figure 1 Sequence alignment of TERB1 homologs in vertebrates.

M. musculus TERB1 was derived from our own sequencing of the cDNA (AB775896). R. norvegicus (XP_226205.5), H. sapiens (NP_001129977.1), P. troglodytes (XP_001159574.2), C. lupus (XP_536822.3), G. gallus(XP_001232198.2) and X. tropicalis (XP_002933719.1) data are from the NCBI protein database.

Supplementary Figure 2 3D tracing of individual telomeres and heterochromatin by live imaging.

Individual telomeres (arrowheads) and heterochromatin (asterisks) were traced for 3 min with 3D information (from sections 5 to 9) to evaluate the relative movements and distances shown in Fig. 2c. Bar, 5 μm.

Supplementary Figure 3 SUN1 but not SUN2 associations with TERB1.

a, Immunoprecipitates from mouse testis-chromatin extracts using TERB1 antibody or control IgG (same IP sample as Figure.6e) were analyzed by the indicated antibodies. SUN1, but not SUN2, associated with TERB1. b, Wild type testis suspension stained for SCP3 (red), SUN2 (green) and DNA (blue). SUN2 signals are detected at the NE in mitotic cells, but is absent in spermatocytes and haploid cells. Asterisks indicate haploid cells. Uncropped images of blots are shown in Supplementary Fig. 6. Bar, 15 μm.

Supplementary Figure 4 GFP-SUN1 and -TERB1 localizations at meiotic telomeres in wild spermatocytes.

The projected and equator images of wild type spermatocytes expressing GFP-SUN1 or -TERB1 stained for SCP3 (red), GFP (green) and TRF1 (blue). Note that overexpressed GFP-SUN1 associates with the NE with telomere accumulations, while GFP-TERB1 exclusively accumulates at telomeres. Bar, 5 μm.

Supplementary Figure 5 Characterization of the TERB1-TRF1 heterocomplex.

a, Purifications of MBP-TERB1, HIS-TRF1 and heterocomplex from E. coli extracts. Note, MBP-TERB1 form a heterocomplex with TRF1 when they are co-expressed in the same E. coli cells. b, Purifications of MBP-TERB1-TRFB domain and HIS-TRF1 from E. coli extracts. While full length MBP-TERB1 is degraded and the stoichiometry with HIS-TRF1 is difficult to be assessed (panel a), MBP-TERB1-TRFB doesn’t show any degradation band and clearly showed 1:1 stoichiometric binding with HIS-TRF1.

Supplementary Figure 6 Entire membranes of cropped immunoblots.

Blue labels on top indicate the antibody used for detection. Dashed red boxes show which region of the immunoblot were cropped for the individual figures.

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Live imaging of wild type spermatocytes.

Wild type spermatocytes co-expressing GFP-TRF1 and GFP-SCP3 were cultured in medium supplemented with Hoechst 33324. Images were collected using a DeltaVision microscopy system. Exposures of 0.15 sec (for GFP) and 0.025 sec (for Hoechst 33324) were acquired every 30 sec for 5 min. (AVI 3285 kb)

Live imaging of wild type spermatocytes in the presence of nocodazole.

Wild type spermatocytes co-expressing GFP-TRF1 and GFP-SCP3 were cultured in medium supplemented with Hoechst 33324 and nocodazole. Images were collected using a DeltaVision microscopy system. Exposures of 0.15 sec (for GFP) and 0.025 sec (for Hoechst 33324) were acquired every 30 sec for 5 min. (AVI 3285 kb)

Live imaging of TERB1−/− spermatocytes.

TERB1−/− spermatocytes co-expressing GFP-TRF1 and GFP-SCP3 were cultured in medium supplemented with Hoechst 33324. Images were collected using a DeltaVision microscopy system. Exposures of 0.15 sec (for GFP) and 0.025 sec (for Hoechst 33324) were acquired every 30 sec for 5 min. (AVI 3285 kb)

Live imaging of SUN1−/− spermatocytes.

SUN1−/− spermatocytes co-expressing GFP-TRF1 and GFP-SCP3 were cultured in medium supplemented with Hoechst 33324. Images were collected using a DeltaVision microscopy system. Exposures of 0.15 sec (for GFP) and 0.025 sec (for Hoechst 33324) were acquired every 30 sec for 5 min. (AVI 3285 kb)

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Shibuya, H., Ishiguro, Ki. & Watanabe, Y. The TRF1-binding protein TERB1 promotes chromosome movement and telomere rigidity in meiosis. Nat Cell Biol 16, 145–156 (2014). https://doi.org/10.1038/ncb2896

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