1. Distinct TERB1 Domains Regulate Different Protein Interactions in Meiotic Telomere Movement 
Jingjing Zhang, Zhaowei Tu, Yoshinori Watanabe, Hiroki Shibuya 
Cell Reports 
Volume 21, Issue 7, Pages (November 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 1715-1726DOI: (10.1016/j.celrep.2017.10.061)
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3. Figure 1
TRF1 Is Indispensable for the Recruitment of TERB1-TERB2-MAJIN to Meiotic Telomeres
(A) Schematic of the Trf1-conditional KO allele. Rectangle, exon; triangle, flippase recognition site. Exon 1 of Trf1 was excised by Cre recombinase.
(B) Schematic of the crossing strategy to get inducible Trf1 KO mice.
(C) Schematic of the inducible KO strategy for Trf1F/−; Vasa-CreERT mice in early spermatocytes. Tamoxifen (TAM) was injected into testes undergoing the first wave of spermatogenesis at postnatal day (P)10 (when the majority of spermatocytes are at the leptotene [Lep] stage). Cells were collected, fixed, and stained at P15 (when the majority of spermatocytes reach the pachytene [Pac] stage). Dip, diplotene; Sg, spermatogonia; Zyg, zygotene.
(D) Spermatocytes from Trf1F/−; Vasa-CreERT mice after TAM injection and staining with the indicated antibodies. Among the SYCP3-positive cell population, 79% showed residual TRF1 signals that represent cells that escaped from the inducible KO and 21% showed almost undetectable TRF1 signals that represent successful KO cells (TRF1 null; n = 360 spermatocytes).
(E) Frequencies of the meiotic stages in control (Trf1F/−) or Trf1-null (Trf1F/−; Vasa-CreERT2) testes after TAM injection. SYCP3-positive spermatocytes are classified into the following substages: Lep (no SYCE3); Zyg (partially assembled SYCE3); and Pac (fully assembled SYCE3). The mean values of two independent experiments are shown, and error bars are ±SD.
(F) Equator images of zygotene spermatocytes of WT or Trf1F/− and Trf1F/−; Vasa-CreERT (null) after TAM injection and staining with the indicated antibodies. An internal TRF2 focus at the end of chromosomal axis is magnified. Bottom graph shows the number of internal TRF2 foci (telomeres that were not attached to the NE) per zygotene nuclei of indicated genotypes. The mean value is shown with a red bar.
(G) Zygotene spermatocytes of Trf1F/−; Vasa-CreERT (null) and Trf1F/− (control) after TAM injection and staining with the indicated antibodies.
More than 15 cells were observed for each condition. Two-tailed t test; ∗∗p < 0.01; ∗∗∗∗p < Scale bars are 5 μm (unless otherwise indicated). See also Figure S1.
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4. Figure 2 The TERB1 C Terminus Is Sufficient to Recruit TERB2-MAJIN
(A) The SUN1 binding domain (aas 1–371), TRFB domain (aas 523–699), and MYB domain (aas 706–759) of TERB1 are shown. The TRFB domain is responsible for both TRF1 and TERB2 binding. TERB1-C (aas 523–767) was used in the following experiments.
(B and C) Terb1−/− zygotene spermatocytes expressing GFP-TERB1 (full length), GFP-TERB1-C, or no-GFP stained with SYCP3, GFP, and TERB2 (B) or MAJIN (C). More than 10 cells were observed for each condition.
(D) The number of internal TRF1 foci (telomeres that were not attached to the NE) in Terb1−/− spermatocytes expressing the indicated GFP fusion proteins. The median value is shown with a red bar. (Right) Equator images of zygotene Terb1−/− spermatocytes expressing the indicated GFP fusion proteins are shown.
(E) The number of TRF1 foci in Terb1−/− spermatocytes expressing the indicated GFP fusion proteins. The median value is shown as a red bar. Murine cells have 40 chromosomes (2n), and thus the number of TRF1 foci ranges from 80 (fully unpaired) to 40 (fully paired). (Right) Equator images of zygotene Terb1−/− spermatocytes expressing the indicated GFP fusion proteins are shown.
Two-tailed t test; ∗∗∗p < The scale bars represent 5 μm.
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5. Figure 3
Identification and Characterization of the TERB2 Binding Domain of TERB1
(A) The domain conformation of TERB1 showing the TRFB truncation series. The SUN1 binding domain (aas 1–371), TRFB domain (aas 523–699), and MYB domain (aas 706–759) are shown. The positive or negative Y2H interactions with TRF1 and TERB2 are summarized on the right. The ΔT2B mutant is highlighted in red.
(B) Y2H interaction results of the TRFB truncations summarized in (A). The p53-T antigen pair served as the positive control.
(C and D) Terb1−/− zygotene spermatocytes expressing GFP-TERB1 (full length), GFP-TERB1ΔT2B, or no-GFP stained with SYCP3, GFP, and TERB2 (C) or TERB2 (D). More than 10 cells were observed for each condition.
(E) The number of internal TRF1 foci in Terb1−/− spermatocytes expressing the indicated GFP fusion proteins. The median value is shown with a red bar. (Right) Equator images of zygotene Terb1−/− spermatocytes expressing the indicated GFP fusion proteins are shown.
(F) The number of TRF1 foci in Terb1−/− spermatocytes expressing the indicated GFP fusion proteins. The median value is shown as a red bar. (Right) Equator images of zygotene Terb1−/− spermatocytes expressing the indicated GFP fusion proteins are shown.
Two-tailed t test; N.S., not significant; ∗∗∗p < The scale bars represent 5 μm. See also Figure S2.
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6. Figure 4
Maintenance of Telomere Rigidity Is a Distinct Pathway Regulated by TERB1, but Not Downstream TERB2, MAJIN, or SUN1
(A) The domain conformation of TERB1. The SUN1 binding domain (aas 1–371), TRFB domain (aas 523–699), and MYB domain (aas 706–759) are shown. The TRFB domain binds to TRF1 and TERB2 through different surfaces, and the MYB domain binds to cohesin complexes.
(B) Fold enrichment of SMC3 immunostaining signal at telomeres in zygotene spermatocytes of the indicated genotypes normalized to WT (+/+) controls. All WT (+/+) and knockout (−/−) pairs were littermate mice that were simultaneously prepared, stained, and quantified. We quantified 10 telomeres from each cell (n = 10 cells) for each mutant. The error bars are ±SD. Representative images are shown in Figure S3.
(C) The mean number of defective telomeres. Structural defects in zygotene spermatocytes (Zyg) are classified into split (abnormally elongated TRF1 signal without an interconnection between the different chromosome axes) and bridge (abnormally elongated TRF1 signal interconnected between more than two chromosome axes). The structural defect in leptotene (Lep) spermatocytes is classified into a single category, split/bridge, because the chromosome axis is not yet formed at this stage. Representative images are shown in Figure S4A.
(D) Zygotene spermatocytes of the indicated genotypes stained with the indicated antibodies. Monotone and magnified images show the telomere structural defects that are frequently and specifically observed in Terb1−/− spermatocytes. Representative images in leptotene spermatocytes are shown in Figure S4B.
(E) (Left) Zygotene spermatocytes of the indicated genotypes stained with the indicated antibodies. Telomeric DNA is visualized by FITC-labeled FISH-probe (TEL-FISH) (green and monotone pictures). (Right) Quantification of the TEL-FISH signal intensity is shown with the mean value shown as a red bar. All WT (+/+) and knockout (−/−) pairs were littermates that were simultaneously prepared, stained, and quantified. The average values are normalized to that of WT (+/+). We quantified 20 un-synapsed telomeres from 10 cells (n = 200 telomeres) for each mutant.
Two-tailed t test; ∗p < 0.05; ∗∗∗p < The scale bars represent 5 μm (unless otherwise indicated). See also Figures S3 and S4.
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7. Figure 5 The TERB1 MYB Domain Is Required for Rapid Telomere Movement
(A) Time-lapse images (15-s intervals) of live zygotene-stage spermatocytes from Terb1−/− testes 72 hr after expressing the GFP-TERB1 constructs. GFP-negative cells (top) and GFP-TERB1-expressing cells (middle) and GFP-TERB1ΔMYB-expressing cells (bottom) are shown. GFP is shown in green, and DNA is visualized with Hoechst in blue (or in the bottom monotone panels). Asterisks indicate the identical heterochromatin. See Movies S1, S2, S3, S4, and S5 for whole images and other examples.
(B) Quantification of telomere (GFP-TERB1 or GFP-TERB1ΔMYB) velocity in Terb1−/− zygotene spermatocytes with the mean value shown as a red bar. Five telomeres are traced for 11 sequential time points (15-s intervals between each time point) from 12 cells (n = 60 telomeres). The separated graphs showing telomere velocity in 12 individual cells are shown in Figure S5A.
(C) Terb1−/− spermatocytes expressing GFP-TERB1, GFP-TERB1ΔMYB, or no GFP (−) stained with the indicated antibodies. The staining was performed after the time-lapse imaging shown in (A).
(D) Quantification of the split length of the TRF1 signal in Terb1−/− spermatocytes expressing the indicated GFP constructs or no GFP. The mean value is shown as a red bar. The representative images are shown in the most right panels in (C). More than 20 cells were quantified for each category.
(E) Schematic of the sequential assembly process of the meiotic telomere complex. TRF1 binds to the TRFB domain of TERB1 and recruits TERB1 onto telomeres. TERB1 sequentially recruits TERB2 and MAJIN through its T2B domain in order to facilitate telomere attachment. SUN1/KASH5 accumulation requires all three upstream factors TERB1, TERB2, and MAJIN to drive telomere movement. The TERB1 MYB domain recruits the cohesin complex independent from the TERB2 pathway and contributes to the formation of rigid telomere structures and telomere lengthening. All of these interactions culminate in rapid telomere and chromosome movements.
Two-tailed t test; ∗∗∗p < The scale bars represent 5 μm (unless otherwise indicated). See also Figure S5 and Movies S1, S2, S3, S4, and S5.
Cell Reports  , DOI: ( /j.celrep )
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