Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis

Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion... Telomeres integrity is indispensable for chromosomal stability by preventing chromosome erosion and end-to-end fusions. During meiosis, telomeres attach to the inner nuclear envelope and cluster into a highly crowded microenvironment at the bouquet stage, which requires specific mechanisms to protect the telomeres from fusion. Here, we demonstrate that germ cell-specific knockout of a shelterin complex subunit, Trf1, results in arrest of spermatocytes at two different stages. The obliterated telomere-nuclear envelope attachment in Trf1-deficient spermatocytes impairs homologue synapsis and recombination, resulting in a pachytene-like arrest, while the meiotic division arrest might stem from chromosome end-to- end fusion due to the failure of recruiting meiosis specific telomere associated proteins. Further investigations uncovered that TRF1 could directly interact with Speedy A, and Speedy A might work as a scaffold protein to further recruit Cdk2, thus protecting telomeres from fusion at this stage. Together, our results reveal a novel mechanism of TRF1, Speedy A, and Cdk2 in protecting telomere from fusion in a highly crowded microenvironment during meiosis. Introduction progressively shorter during mitotic cycles or due to environmental stresses. When telomeres are shortened and Telomeres are nucleoprotein structures located at the end become dysfunctional, cellular senescence is triggered and of chromosomes and telomere integrity is essential for tissue/organ aging might be accelerated. [2] Dysfunctional genome stability and cell survival. [1] Telomeres become telomeres also tend to form chromosomal end-to-end fusion, eventually causing genomic instability, cell cycle arrest, and finally cell death. [3] Protection of telomere fusion is mainly regulated by the telomerase complex and Edited by R.A. Knight the shelterin complex, which includes six subunits (TRF1, Lina Wang, Zhaowei Tu and Chao Liu contributed equally to this TRF2, RAP1, TIN2, TPP1, and POT1). [4–11] TRF1 and work. TRF2 directly bind to double-stranded telomeric DNA and Electronic supplementary material The online version of this article prevent the activation of both ATM- and ATR-dependent (https://doi.org/10.1038/s41418-017-0037-8) contains supplementary DNA damage signaling pathways. [12, 13] Mutations in material, which is available to authorized users. * Zijiang Chen Ministry of Education, Jinan, Shandong 250001, China chenzijiang@hotmail.com Institute of Molecular and Cell Biology (IMCB), Agency for * Wei Li Science, Technology, and Research (A*STAR), leways@ioz.ac.cn Singapore 138673, Republic of Singapore Department of Biochemistry, National University of Singapore, State Key Laboratory of Stem Cell and Reproductive Biology, Singapore 117599, Republic of Singapore Institute of Zoology, Chinese Academy of Sciences, University of the Chinese Academy of Sciences, Beijing 100049, Beijing 100101, China China Department of Chemistry and Molecular Biology, University of Key Laboratory for Major Obstetric Diseases of Guangdong Gothenburg, Gothenburg SE-405 30, Sweden Province, Key Laboratory of Reproduction and Genetics of Center for Reproductive Medicine, Shandong Provincial Hospital Guangdong Higher Education Institutes, The Third Affiliated Affiliated to Shandong University, Jinan, Shandong 250001, China Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, China The Key Laboratory for Reproductive Endocrinology of the 1234567890 Dual roles of TRF1 in tethering telomeres 1175 telomere related genes are associated with many diseases, crossing mice with a floxed Trf1 allele to Stra8-Cre mice, including Hoyeraal-Hreidarsson syndrome, dyskeratosis which express Cre recombinase during early-stage sperma- congenita, pulmonary fibrosis, and cancer. [14–16] togenesis beginning on day 3 after birth. [30] Germ cell- Telomeres are attached to the nuclear matrix and scattered specific Trf1 knockout mice will be referred to as Stra8- −/− −/− throughout the nucleus in somatic cells. [17–19]In contrast Trf1 . We found that the Stra8-Trf1 male mice were in germ cells, telomeres display an unprecedented behavior completely infertile. The size and weight of their testes were Flox/Flox during meiotic prophase I where they attach to the inner significantly reduced compared with that of Trf1 and +/− nuclear envelope (NE) and cluster to form a structure which Stra8-Trf1 mice (Fig. 1a-b) due to efficient Trf1 deletion resembles a bouquet of flowers, termed the bouquet stage. (Fig. 1c). Further histological examination revealed that Trf1- [17, 20, 21] This phenomenon is conserved in diverse deficient testes lacked post-meiotic cells (Fig. 1d) and the eukaryote species and required for homologous chromosome diameter of Trf1-deficient seminiferous tubules was decreased pairing, synapsis, and proper resolution of recombination significantly (Fig. 1e). Few spermatozoa could be detected in events. [22, 23] During the bouquet stage, telomeres are in the cauda epididymis and the total number of spermatozoa −/− close proximity to each other, thus creating a crowded was dramatically reduced in the Stra8-Trf1 mice compared microenvironment. Since the prerequisite for telomere fusion with that of the control group, indicating that the disruption of is that two telomeres are in close proximity to each other to Trf1 severely impairs spermatogenesis (Fig. 1f-g). The allow the reaction to occur, telomeres are vulnerable to amount of degenerated cells with highly condensed nuclei −/− unwanted end-to-end fusions at the bouquet stage. In this found in the seminiferous epithelium of Stra8-Trf1 mice crowded microenvironment, protection mechanisms must (Fig. 1d), which are resembling the characteristic of apoptotic have been evolved to protect them fusing with each other. cells, let us speculate that the reduction of post-meiotic cells in The attachment of telomeres to the NE is dependent on the Trf1-deficient testes is due to apoptosis. To verify this pos- transmembrane linker of nucleoskeleton and cytoskeleton sibility, we performed TUNEL assays and found that the (LINC)-complex, which contains SUN1, SUN2, KASH5, number of TUNEL positive cells per seminiferous tubule in −/− and adaptor proteins like TERB1, TERB2, MAJIN, Speedy the Stra8-Trf1 mice was significantly increased compared A, and TRF1. [24–29] Since TRF1 is a core subunit of the with that of the control group (Supplementary Figure 1), shelterin complex and also involved in anchoring telomeres suggesting that spermatocytes in Trf1-deficient testes are to the NE by interacting with TERB1, [28]weselected TRF1 eliminated through apoptosis. Altogether, these results indi- as the starting point to investigate the telomere fusion pro- cate that germ cell-specific disruption of Trf1 results in tection mechanism at the bouquet stage. We knocked out spermatogenetic failure and that TRF1 is essential for Flox/Flox Trf1 in germ cells by crossing Trf1 mice [13]with spermatogenesis. Stra8-Cre transgenic mice. [30] We found that Trf1 is essential for spermatogenesis since the knockout of Trf1 in male germ cells led to arrest at two stages: the pachytene-like The Trf1-deficient spermatocytes arrest at two different stages. and the meiotic division stages. It is the defective attachment of telomeres to NE that results in the pachytene-like arrest, Spermatogenesis is a process in the seminiferous epithelium whereas the meiotic division arrest stems from chromosome end-to-end fusions in Trf1 knockout spermatocytes. Further where various generations of germ cells undergo a series of developmental steps, including spermatogonial mitosis, investigations uncovered that Speedy A and cyclin- spermatocytic meiosis, and spermiogenesis. [31, 32] Sper- dependent kinase 2 (Cdk2) but not SUN1 were involved in protection from telomere fusion at the bouquet stage. Thus, in matogenesis can be subdivided into 12 stages in mouse testes using Periodic Acid Schiff (PAS) and hematoxylin addition to their roles in tethering telomeres to NE, our work defines novel functions for TRF1, Speedy A, and Cdk2 in staining of sections. [33] To identify which stages of sper- matogenesis were affected after Trf1 deletion, sections were protecting telomeres from fusion in a crowded micro- environment during meiosis. stained with PAS and hematoxylin and we determined that spermatogenesis of Trf1-deficient mice was blocked at stages IV and XII (Fig. 2a). Since early spermatocytes gradually develop into mid-pachytene spermatocytes at Results stage III–V and spermatocytes in either the first or the second meiotic division are present in stage XII, [33] our TRF1 is essential for spermatogenesis −/− results indicate that the Stra8-Trf1 spermatocytes might To study the molecular mechanism of protection from telo- be arrested at the pachytene and meiotic division stages. To confirm this hypothesis, we identified the various mere fusion at the bouquet stage, the core subunit of the shelterin complex, Trf1, was knocked out in germ cells by stages of meiotic prophase I by staining for a component of 1176 L. Wang et al. −/− Fig. 1 TRF1 is required for spermatogenesis. a The Stra8-Trf1 mice Arrows indicate apoptotic cells. e The diameter of the Flox/Flox +/− −/− testes were smaller than those of the Trf1 and Stra8-Trf1 seminiferous tubules in Stra8-Trf1 mice was smaller than that of Flox/Flox Flox/Flox mice (10-week-old, and the same to the below). b Quantification of the Trf1 mice. Trf1 (gray bar, 195.50 ± 4.49 μm), Flox/Flox +/− −/− testis weight of the Trf1 and Stra8-Trf1 mice. Testis weight: Stra8-Trf1 (white bar, 102.40 ± 3.62 μm). Data are presented as Flox/Flox +/− Trf1 (grey bar, 151.20 ± 1.42 mg), Stra8-Trf1 (black bar, mean ± SEM. ***P < 0.001. f Histological analysis of the caudal −/− Flox/Flox −/− 148.40 ± 1.96 mg), Stra8-Trf1 (white bar, 27.40 ± 1.69 mg). Data epididymides from 10-week-old Trf1 and Stra8-Trf1 mice. are presented as mean ± SEM. ***P < 0.001. c The TRF1 protein g The total number of sperm in the cauda epididymis was −/− −/− Flox/Flox levels were reduced in the testes of the Stra8-Trf1 mice. Histone significantly decreased in the Stra8-Trf1 mice. Trf1 (grey 6 −/− 6 H2A was used as the loading control. d PAS-hematoxylin analysis of bar, 19.03 ± 1.22 × 10 ), Stra8-Trf1 (white bar, 0.06 ± 0.11 × 10 ). Flox/Flox −/− the seminiferous tubules of Trf1 and Stra8-Trf1 mice. Data are presented as mean ± SEM. **P < 0.01. the synaptonemal complex, SYCP3. [34] All meiotic pro- TUNEL positive tubules and spermatocytes in stage IV phase I stages could be identified in the spermatocyte nuclei significantly higher than that of the control (Supplementary Flox/Flox of Trf1 testes, whereas only spermatocytes from the Figure 1d-e), suggesting that the deletion of Trf1 leads to a leptotene to the pachytene-like stages were observed in the pachytene-like arrest and results in spermatocyte death. −/− Stra8-Trf1 mice testes (Fig. 2b). Further quantification of Since germ cells of Trf1-deficient mice also displayed the meiotic prophase I stages in testes indicated that the stage XII arrest (Fig. 2a) and some diplotene and diakinesis −/− proportion of the zygotene cells was significantly increased spermatocytes could be identified in the Stra8-Trf1 and that of diplotene cells was decreased in the Trf1-defi- mouse testes, we speculated that some Trf1-deficient sper- cient testes (Fig. 2c). The percentage of spermatocytes in matocytes could bypass the pachytene checkpoint due to the the pachytene stage was not accumulated in Stra8- variation of TRF1 expression, TRF1 protein half-life, or −/− Trf1 mouse testes (Fig. 2c), most likely because the other reasons. We found that some of the germ cells were pachytene checkpoint-arrested spermatocytes undergoing then blocked at the meiotic division stage. To determine the apoptosis. [35] To further confirm this possibility, TUNEL fate of the meiotic division stage-arrested germ cells, we staining was performed, and TUNEL-positive signal was performed TUNEL staining and found that some sperma- detected in the pachytene-like spermatocytes in testes from tocytes undergoing meiotic division were TUNEL positive −/− Stra8-Trf1 mice (Fig. 2d), with the percentages of (Fig. 2d), with both the percentages of TUNEL positive Dual roles of TRF1 in tethering telomeres 1177 Fig. 2 Trf1-deficient spermatocytes arrest at two stages. a Seminiferous tubules Flox/Flox paraffinsections from Trf1 −/− and Stra8-Trf1 testis were stained with PAS-hematoxylin. A type A spermatogonia, In intermediate spermatogonia, B type B spermatogonia, PL preleptotene spermatocytes, L leptotene spermatocytes, Z zygotene spermatocytes, P pachytene spermatocytes, aP apoptotic pachytene spermatocytes, M meiotic divisions, aM abnormal meiotic divisions, sS secondary spermatocytes, rSt round spermatids, S spermatids. b Spermatocyte stages in pre- Flox/Flox metaphase in Trf1 and −/− Stra8-Trf1 spermatocytes. Flox/Flox −/− Trf1 and Stra8-Trf1 chromosome spreads of spermatocytes were immunostained with antibodies against SYCP3 (green) and DAPI (blue). c Meiotic stage Flox/Flox frequencies in Trf1 and −/− Stra8-Trf1 testes. Lep Flox/Flox (Leptotene): Trf1 (gray bar, 12.07 ± 0.94%), Stra8- −/− Trf1 (white bar, 20.85 ± 1.30%). Zyg (Zygotene): Flox/Flox Trf1 (gray bar, 6.71 ± −/− 1.34%), Stra8-Trf1 (white bar, 23.63 ± 6.42%). Pac (Pachytene): Flox/Flox Trf1 (gray bar, 55.31 ± −/− 2.50%), Stra8-Trf1 (white bar, 47.60 ± 2.75%). Dip (Diplotene): Flox/Flox Trf1 (gray bar, 23.00 ± −/− 2.71%), Stra8-Trf1 (white bar, 7.00 ± 0.89%). Dia (Diakinesis): Flox/Flox Trf1 (gray bar, 2.88 ± −/− 1.49%), Stra8-Trf1 (white bar, 0.89 ± 0.52%). Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001. d Representative Flox/Flox TUNEL results in Trf1 and −/− Stra8-Trf1 testes. Paraffin Flox/Flox sections from Trf1 and −/− Stra8-Trf1 testes were stained with TUNEL (green) and DAPI (blue) to determine apoptotic cells in stage IV and stage XII tubules together with pachytene and metaphase spermatocytes. tubules and spermatocytes in stage XII significantly higher pachytene-like stage but some of them bypass the pachytene than that of the control (Supplementary Figure 1f-g). Thus, checkpoint and are later blocked at the meiotic division Trf1 disruption leads to most spermatocytes arresting at the stage. 1178 L. Wang et al. Fig. 3 TRF1 is required for the attachment of telomeres to the nuclear control group. Zygotene to pachytene spermatocytes were counted for envelope in spermatocytes. a Increased detachment of telomeres from wild-type, whereas pachytene-like spermatocytes were counted for the −/− Flox/Flox −/− NE after Trf1 deletion. An increased number of telomeres were dis- Stra8-Trf1 spermatocytes. Trf1 , 1.17 ± 1.38; Stra8-Trf1 , −/− tributed at the inner site of nucleus in Stra8-Trf1 spermatocytes 9.56 ± 1.62. The numbers of internal telomere (FISH) foci are pre- Flox/Flox compared with the control group. Paraffin sections from Trf1 sented as mean ± SEM. **P < 0.01. c An increased number of telo- −/− and Stra8-Trf1 testes were immunostained with antibodies against meres were detached from NE in the Trf1-deficient spermatocytes. SYCP3 (red), Tel-FISH (green), and Lamin B (gray). An increased Electron micrographs showing telomeres (red arrowheads) and inner Flox/Flox −/− number of telomere (FISH) signals was observed at the inner site of the NE (white arrows) in the Trf1 pachytene and Stra8-Trf1 nucleus and did not co-localize with the NE (Lamin B) signals. White pachytene-like nuclei; asterisks indicate membrane vesicles. Schematic arrowheads indicate the inner telomeres. Zyg zygotene spermatocytes, illustrations represent the structures. AP attachment plate, CH chro- Pac pachytene spermatocytes. b The number of internal telomeres in matin, NE nuclear envelope, SC synaptonemal complex. −/− Stra8-Trf1 spermatocytes was increased compared with that of the Germ cell-specific Trf1 knockout impairs the pachytene stage, whereas several telomeres were still −/− attachment of telomeres to the nuclear membrane detached from the NE in the Stra8-Trf1 spermatocytes (Fig. 3a-b). Electron microscopy analysis indicated that the Since it has been reported that TERB1 is involved in telomeres either did not reach the NE, did not form an anchoring telomeres to the NE by interacting with TRF1, attachment plate (AP), or were bound to a membrane [28] we speculated that knocking out Trf1 might impair NE vesicle just before reaching the NE (Fig. 3c), which is attachment. To verify this hypothesis, we performed similar to the reported phenomenon in other telomere-NE immunofluorescence staining of the NE marker Lamin B attachment-deficient mice. [24–29, 36, 37] Thus, the dis- Flox/Flox and telomere FISH. In Trf1 spermatocytes, most of ruption of Trf1 seems to affect the telomere-NE attachment the telomeres were attached to the NE at the zygotene and during meiosis. Dual roles of TRF1 in tethering telomeres 1179 TRF1 is essential for homologue synapsis and Impaired telomere fusion protection in recombination Trf1-deficient spermatocytes leads to meiotic division arrest During meiotic prophase I, telomeres associate with the LINC complex to attach to the nuclear envelope and the It has been reported that the depletion of shelterin complex disruption of this attachment might affect homologous subunits, such as TRF1, leads to telomere de-protection and chromosome synapsis and recombination, thereby leading initiation of DNA damage response at the end of chromo- to pachytene-like arrest. [24, 26–29, 36, 37] To investigate somes with telomere fusions and cell cycle checkpoint homologous chromosomes synapsis, we stained chromo- activation in somatic cells. [5, 12, 15, 16, 44] Thus, it is some spreads with antibodies against SYCP3 and SYCP1, a possible that TRF1 depletion in germ cells could lead to key component of the synaptonemal complex [38] and chromosomal end-to-end fusions and result in meiotic found that the SYCP1 signals were not continuous with division arrest. We noticed fused chromosomes in Stra8- −/− SYCP3 and many regions of homologous chromosomes Trf1 pachytene-like spermatocytes (Figs. 2b, 4a-b). failed to synapse in the Trf1-deficient spermatocytes (Sup- Therefore, we performed telomeric FISH experiment toge- plementary Figure 2), suggesting the knockout of Trf1 ther with SYCP3 staining in chromosome spreads of sper- impairs homologue synapsis. matocyte nuclei. Indeed we found end-to-end fusions in the Chromosome synapsis and recombination are facilitated Trf1-deficient spermatocytes (Fig. 5a), which might origi- −/− by the introduction of DNA double-strand breaks (DSBs), nate from the bouquet stage in Stra8-Trf1 spermatocytes [39] which can be monitored by detecting γH2AX-positive (Supplementary Figure 3). In support of the above results, foci at the leptotene and zygotene stages and these are we uncovered that both the Tel-FISH intensity and area in decreased on autosomes after DSB repaired at the pachytene the Trf1-deficient spermatocytes were increased compared −/− stage (Fig. 4a). [40]Inthe Stra8-Trf1 spermatocytes, the to those in the control group (Fig. 5b-c), most likely due to γH2AX signal was detected at leptotene and zygotene stages, their elongation by telomere fusions (Fig. 5d). These results Flox/Flox which was similar to Trf1 spermatocytes, indicating suggest that germ cell-specific knockout of Trf1 may impair the normal production of DSBs (Fig. 4a). However, in the telomere integrity, leading to chromosomal instability with Trf1-deficient spermatocytes, the γH2AX signal was still telomere end-to-end fusions, finally resulting in a meiotic diffused on the autosomes at the pachytene-like stage, rather division arrest. than accumulated only on the XY bodies (Fig. 4a). Con- To investigate the protection mechanism of TRF1 in sistent with the γH2AX staining, ATR, which is integral to preventing telomere fusion during meiosis, we stained for the regulation of programmed DSB formation, [41, 42]was other shelterin complex subunits. Surprisingly, the protein −/− also detected on the autosomes of Stra8-Trf1 spermato- levels of the shelterin complex proteins TRF2, TIN2, and −/− cytes (Fig. 4b). These results indicate that the programmed POT1 were dramatically reduced in the Stra8-Trf1 DSBs are properly introduced, but DSB repair is impaired in mouse testes but TRF2 was still localized on telomeres, the Trf1-deficient spermatocytes. Since the DSB repair in even when fusion occurred (Fig. 5g and Supplementary meiosis is mainly dependent on homologous recombination, Figure 4a), which suggests that telomere might be protected we stained for the meiosis specific recombination related during meiosis through a meiosis-specific mechanism. −/− protein, DMC1 [39]in Stra8-Trf1 spermatocytes. The Recently, several LINC complex proteins and their adaptor number of DMC1 foci in the Trf1-deficient spermatocytes proteins have been identified, such as Speedy A, Cdk2, was dramatically increased compared with that of the TERB1, MAJIN, and SUN1. [24, 26, 28, 29, 36, 37] control group (Fig. 4c), indicating impaired homologous We screened potential effectors of telomere fusion at the recombination. The late-recombination marker MLH1 bouquet stage, and found that TRF2, RAP1, and SUN1 normally appeared at the designated crossing-over sites, were still localized on telomeres, but Speedy A and [43] and MLH1 immunofluorescence in Trf1-deficient Cdk2 were not recruited to the telomeres in Trf1-deficient spermatocytes indicated that the number of MLH1 foci spermatocytes (Fig. 5e-f and Supplementary Figures 4a-c). was significantly reduced compared with that of the control In addition, the protein levels of Speedy A and Cdk2 −/− group (Fig. 4d-e), suggesting the crossover formation were substantially decreased in the Stra8-Trf1 mouse was also impaired. Altogether, TRF1 is required for testes compared to those of the control groups (Fig. 5g). homologue synapsis and recombination, and the disruption These results indicate that the disruption of Trf1 impairs of Trf1 might affect the telomere-nuclear envelope attach- the localization of the meiotic telomere-related proteins, ment, lead to impaired homologue synapsis, recombination Speedy A and Cdk2, on telomeres, which might be and crossover formation, finally resulting in a pachytene-like indispensable for preventing telomere fusion during arrest. meiosis. 1180 L. Wang et al. Fig. 4 Impaired meiotic −/− recombination in Stra8-Trf1 Flox/Flox spermatocytes. a Trf1 −/− and Stra8-Trf1 chromosome spreads of spermatocytes were immunostained with antibodies against SYCP3 (green) and γH2AX (red). γH2AX marked DSBs in leptotene and zygotene spermatocytes. No difference Flox/ was observed between Trf1 Flox −/− and Stra8-Trf1 at these Flox/Flox stages. In the Trf1 spermatocytes at the pachytene stage, DSBs were mostly repaired and γH2AX was confined to the XY body, whereas at the pachytene-like −/− stage in Stra8-Trf1 spermatocytes, γH2AX remained on some of the Flox/Flox autosomal regions. b Trf1 −/− and Stra8-Trf1 chromosome spreads of spermatocytes were immunostained with antibodies against SYCP3 (green) and ATR Flox/Flox (red). In the Trf1 spermatocytes at the pachytene stage, DSBs were mostly repaired and ATR was confined to the XY body, while the ATR signal persisted on the −/− autosomes in Stra8-Trf1 Flox/Flox spermatocytes. c Trf1 and −/− Stra8-Trf1 chromosome spreads of spermatocytes were immunostained with antibodies against SYCP3 (green) and DMC1 (red). The DMC1 signals were dramatically increased in −/− the Stra8-Trf1 spermatocytes at the zygotene stage compared Flox/Flox with those in the Trf1 Flox/Flox spermatocytes. d Trf1 and −/− Stra8-Trf1 chromosome spreads of spermatocytes were immunostained with antibodies against SYCP3 (green) and MLH1 (red). MLH1 is a recombination marker in WT spermatocytes and decreased MLH1 staining was observed in −/− Stra8-Trf1 spermatocytes. e MLH1 signals were counted in spreads. Quantification of the MLH1 foci numbers per cell in Flox/Flox the Trf1 and Stra8- −/− Trf1 spermatocytes. Flox/Flox Trf1 , 25.0 ± 1.7; −/− Stra8-Trf1 , 10.6 ± 2.8. Data are presented as mean ± SEM. ***P < 0.001. Dual roles of TRF1 in tethering telomeres 1181 Fig. 5 TRF1 is required for preventing telomere fusion at the bouquet Quantitative PCR was performed to measure telomere length and the stage. a Telomere fusions occurred after Trf1 depletion. Meiotic relative telomere to reference single copy gene (T/R) ratio was cal- Flox/Flox −/− Flox/Flox chromosome spreads from the Trf1 and Stra8-Trf1 testis culated to represent the average telomere length in the Trf1 and −/− were immunostained with antibody against SYCP3 (red) and Tel-FISH Stra8-Trf1 spermatocytes at pachytene or pachytene-like stage. −/− Flox/Flox −/− (green). In the pachytene-like Stra8-Trf1 spermatocytes, FISH Trf1 (gray bar, 0.78 ± 0.12), Stra8-Trf1 (white bar, 1.23 ± signal can be found in the middle of the SYCP3 threads, instead of at 0.09). Data are presented as mean ± SEM. ***P < 0.001. e and f the ends. Schematic illustrations represent the structures. b and c Speedy A and Cdk2 are not recruited to the telomeres in the Trf1- Flox/Flox −/− Telomeric FISH signal intensities and areas per telomere were deficient spermatocytes. Trf1 and Stra8-Trf1 chromosome −/− increased in Stra8-Trf1 spermatocytes. Telomeric FISH signal spreads of spermatocytes were immunolabelled with antibodies against intensities and areas were measured, and the mean Tel-FISH signal SYCP3 (red) and Speedy A (green) or Cdk2 (green). g Selected Flox/Flox intensities and areas were shown. Intensity: Trf1 (gray bar, shelterin and LINC complexes protein levels were decreased after Trf1 −/− Flox/ 116.22 ± 5.42) Stra8-Trf1 (white bar, 142.77 ± 5.35). Area: depletion. Immunoblotting for shelterin and LINC proteins in Trf1 Flox/Flox 2 −/− Flox −/− Trf1 (gray bar, 121.98 ± 7.58 px ), Stra8-Trf1 (white bar, and Stra8-Trf1 spermatocytes. TIN2, TRF2, POT1, Speedy A, 2 −/− 158.28 ± 8.87 px ). Data are presented as mean ± SEM. ***P < 0.001. and Cdk2 were all decreased to some extent in the Stra8-Trf1 d Telomere length was increased in the Trf1-deficient spermatocytes. spermatocytes. GAPDH was used as loading control. 1182 L. Wang et al. Fig. 6 Direct physical interaction between TRF1 and Speedy A. proteins were detected by immunoblotting analysis with anti-FLAG a Coomassie blue-stained gels showing the purification of relevant and anti-GST antibodies. e Speedy A-mediated interaction between proteins. b Direct physical interaction between TRF1 and Speedy A TRF1 and Cdk2. GST-TRF1 or GST conjugated sepharose beads were were determined by GST pull-down assays. GST-TRF1 or GST con- used to pull down His-FLAG-Speedy A and (or) His-MYC-Cdk2. jugated sepharose beads were used to pull down His-FLAG-Speedy A. Bound proteins were detected by immunoblotting analysis with anti- Bound proteins were detected by immunoblotting analysis with anti- FLAG or anti-MYC antibody. Asterisks indicate the bands of Speedy FLAG and anti-GST antibodies. c No direct physical interaction A. Input for His-FLAG-Speedy A and His-MYC-Cdk2 were 10% and between TRF1 and Cdk2, which was determined by GST pull-down 1%, respectively. f TRF1 and Cdk2 do not compete for interaction assays. GST-TRF1 or GST conjugated sepharose beads were used to with Speedy A. GST-TRF1 or GST conjugated sepharose beads were pull down His-MYC-Cdk2. Bound proteins were detected by immu- used to pull down His-FLAG-Speedy A in the presence of increasing noblotting analysis with anti-MYC and anti-GST antibodies. d Direct amounts of His-MYC-Cdk2. Bound proteins were detected by physical interaction between CDK2 and Speedy A was determined by immunoblotting analysis with anti-FLAG and anti-MYC antibodies. GST pull-down assays. GST-CDK2 or GST conjugated sepharose Asterisks indicate signals of Speedy A. beads were used to pull down purified His-FLAG-Speedy A. Bound Direct physical interaction between TRF1 and suggested that in the presence of Speedy A, Cdk2 could be Speedy A pulled down by TRF1 (Fig. 6e). To further demonstrate that Speedy A was a scaffold protein, we did a competition To further investigate the relationship between TRF1, experiment by adding excess Speedy A or Cdk2. When we Speedy A and Cdk2, we purified TRF1, Speedy A and increased the levels of Cdk2 in the reaction, the binding of Cdk2 proteins (Fig. 6a) and performed the GST pull-down Speedy A to TRF1 was unchanged, suggesting that TRF1 and Cdk2 might bind Speedy A at different non-overlapping experiments. Our results indicated that TRF1 could directly interact with Speedy A, but not Cdk2 (Fig. 6b-c). We fur- sites (Fig. 6f). Thus, TRF1 interacts directly with Speedy A, ther confirmed the known interaction between Cdk2 and and Speedy A binds to Cdk2, thereby recruiting Cdk2 to the Speedy A, and found that GST-Cdk2 could directly bind telomeres. to Speedy A (Fig. 6d). Since previous reports indicated that Speedy A was necessary for Cdk2’s localization on telo- Speedy A and Cdk2 are required for protecting meres during meiosis, [36] we speculated Speedy A might telomere fusion at the bouquet stage work as a scaffold protein to facilitate the indirect interac- tion between TRF1 and Cdk2. To test this possibility, GST- To investigate whether Speedy A and Cdk2 are also TRF1 was used to pull-down Cdk2 in the presence or required for preventing telomere fusion during meiosis, we absence of Speedy A. The GST pull-down experiments performed telomere-FISH and SYCP3 staining of Speedy A, Dual roles of TRF1 in tethering telomeres 1183 Fig. 7 TRF1-mediated Speedy A and Cdk2 recruitment are required for MEFs. Telomere numbers were counted and no significant changes +/+ preventing telomere fusion at the bouquet stage. a Telomere fusions were detected after Speedy A knockout. Speedy A , 90.25 ± 3.84; −/− −/− were detected in Speedy A chromosome spreads of spermatocytes. Speedy A , 94.13 ± 3.23. Data are presented as mean ± SEM. +/+ −/− −/− Speedy A and Speedy A spreads were immunolabelled with d TRF1 is still localized on telomeres in Speedy A spermatocytes. +/+ −/− antibodies against SYCP3 (red) and telomere FISH (green). Chro- Speedy A and Speedy A metaphase spreads were immunostained mosome fusions are indicated by arrows and a schematic illustrations with Telomere FISH (red) and DAPI (blue). e Immunoblotting for −/− of the structures is shown. b The telomere numbers in WT, Speedy telomere related proteins in Speedy A spermatocytes. SUN1 and −/− −/− −/− −/− A , Cdk2 , and Sun1 knockout mice. The telomere numbers TRF1 in Speedy A spermatocytes remained unchanged; β-Actin was −/− −/− were decreased significantly in Speedy A and Cdk2 spermato- used as the loading control. f A model of the meiosis specific telomere −/− cytes, but increased in Sun1 spermatocytes. The numbers of telo- fusion protection complex, with TRF1, Speedy A, and Cdk2 as the key mere (FISH) foci are presented as mean ± SEM. ***P < 0.001. players to protect telomeres from fusion. +/+ −/− c Quantification of Tel-FISH signals in Speedy A and Speedy A Cdk2, and Sun1-deficient spermatocytes and found some Speedy A and Cdk2 are required for preventing telomere −/− −/− fused telomeres in Speedy A and Cdk2 spermatocytes fusion during meiosis, and telomere fusion in Trf1-deficient −/− but not in Sun1 spermatocytes (Fig. 7a, Supplementary spermatocytes might be caused by the absence of Speedy A Figures 5a and 5c). Consistent with the telomere fusion and Cdk2 on telomeres. results, we found that the total number of telomeres in the Since the disruption of Trf1, Speedy A, and Cdk2 could −/− −/− Speedy A and Cdk2 spermatocytes was significantly cause telomere fusion during meiosis and Trf1 depletion decreased compared with their control groups (Fig. 7b), but resulted in the exclusion of Speedy A and Cdk2 from tel- we did not observe this phenomenon in somatic cells since omeres, TRF1 might be an upstream telomeric recruiter for −/− the total number of telomeres in Speedy A mouse Speedy A and Cdk2. To test this hypothesis, we stained for −/− −/− embryonic fibroblasts (MEFs) was similar to that of the TRF1 in Speedy A and Cdk2 spermatocytes and −/− −/− control (Fig. 7c). In contrast to Speedy A and Cdk2 , found that TRF1 was still localized on telomeres albeit the −/− the total number of telomeres in Sun1 spermatocytes was TRF1 signal was decreased compared to the control (Fig. 7d normal at pachytene-like stage, but was increased at zygo- and Supplementary Figure 5b). The lower TRF1 signaling tene stage, presumably due to homologous chromosome might be due to structural changes of the telomeres because synapsis failure (Fig. 7b). These results are similar to those the deletion of Speedy A or Cdk2 leads to telomere fusions findings in the Trf1-deficient spermatocytes, indicating that in spermatocytes. Immunoblotting analysis of telomeric 1184 L. Wang et al. complex components indicated that SUN1 and TRF1 levels telomeres. Thus, we propose that Speedy A may act as a −/− were not changed in Speedy A spermatocytes (Fig. 7e), scaffold protein for TRF1 and Cdk2, thus recruiting Cdk2 to thus suggesting that TRF1 and Speedy A may be central for telomeres. On the other hand, the RINGO domain of the mechanism of protecting telomeres from fusion at the Speedy A interacts with Cdk2 and promotes Cdk2 activa- bouquet stage. tion to protect telomere from fusion during meiosis. Telomere-NE attachment is essential for homologous chromosome synapsis and recombination and many key Discussion proteins have been identified for this attachment such as TERB1, TERB2, MAJIN, Speedy A, Cdk2, SUN1, SUN2, The cell interior is densely crowded with thousands of and KASH5. [24, 26, 28, 29, 36, 37] TRF1 can interact with macromolecules such as proteins, DNA, RNA, and other TERB1 to form the chimeric complex, TRF1-TERB1/2- molecules, where biological macromolecules have to func- MAJIN, which is indispensable for telomere-NE attach- tion in crowded cellular environment. [45, 46] Chromatin, a ment. [28, 29] During mid prophase, telomere cap exchange huge macromolecule, usually distributes all over the is achieved by removing the shelterin complex, including nucleus in most somatic cells. [47] In contrast in germ cells, TRF1, from the membrane-anchored telomeres, which is chromatin packs into a relative small space, and chromo- dependent on CDK activity. [29] However, TRF1 is still some ends cluster in close proximity at the bouquet stage localized near the membrane-anchored telomere region, during meiosis, [17, 20, 21] thus creating a crowded indicating additional roles of TRF1 during meiosis. Our microenvironment for telomeres. It is not known how to results demonstrate that in addition to the telomere-NE protect the telomere from fusion in this special micro- attachment, TRF1, Speedy A, and Cdk2 are required for environment. Here, we demonstrate that the disruption of protecting telomeres from fusion at the bouquet stage during the shelterin component Trf1 drives telomere fusion during meiosis. However, the disruption of Sun1, Terb1, Terb2 and meiosis (Fig. 5a) and TRF1 is required for the telomeric Majin do not affect telomere stability because telomere −/− localization of Speedy A and Cdk2 (Fig. 5e-f). Further fusions were not observed in Sun1 spermatocytes analysis indicated that the absence of Speedy A and Cdk2 (Supplementary Figure 5c), and the number of telomere led to severe telomere fusions in spermatocytes (Fig. 7a and does not decrease in Terb1 and Terb2 deficient spermato- Supplementary Figure 5a). Thus, TRF1, Speedy A, and cytes. [28, 29] Considering that SUN1-dependent chromatin Cdk2 are found to be involved in protecting telomeres from mobility is important for fusion of dysfunctional telomeres fusion in this crowded microenvironment (Fig. 7f). in somatic cells, [51] it is possible that the absence of SUN1 TRF1 is important for the functional telomere structure or other LINC complex components do not cause telomere and it can directly bind double-stranded telomeres by con- fusion during meiosis. Thus, the functions of LINC com- necting with TRF2 through TIN2. [4, 15] Depletion of ponents and their adaptor proteins in preventing telomere TRF1 affects the telomeric association of TRF2, [48] which fusion and telomere-NE attachment are not the same pro- has a crucial role in chromosome end protection. [12] cess. Whereas SUN1 only participates in telomere-NE Similarly, cyclin E deletion causes TRF2 depletion from the attachments, TRF1, Speedy A, and Cdk2 are involved in telomeres and aberrant telomere structures. [49] Speedy A both of these two processes (Figs. 5 and 7, Supplementary was dissociated from telomeres in Trf1-deficient spermato- Figures 5a-b). Therefore TRF1, Speedy A, and Cdk2 are cytes and TRF1 could directly bind to Speedy A, suggesting key players of the meiosis-specific mechanism in protecting that TRF1 might recruit Speedy A to telomeres during telomeres from fusion at bouquet stage. meiosis. Speedy A then serves as a scaffold protein to further recruit Cdk2 to telomeres. Recently, a “telomere localization domain” of Speedy A was identified and it is Materials and methods sufficient for Speedy A to co-localize with TRF1 on telo- meres. [36] The C-terminal RINGO domain of Speedy A is Animals’ experiments also necessary for Cdk2’s localization to telomeres during Flox/Flox meiosis. [26, 36] In addition, it has been reported that the The Trf1 mice (C57BL/6J) were a gift from Prof. formation of cyclin E/Cdk2 complex is necessary for the Maria A. Blasco from the Spanish National Cancer Centre Flox/Flox telomeric localization of Cdk2 and protection of the telo- (CNIO). [13]The Trf1 Stra8-Cre mice were bred Flox/Flox meres from end-to-end fusions. [49, 50] We found that from intercrosses of Trf1 mice and Stra8-Cre mice −/− −/− cyclin E levels were increased in Trf1-deficient spermato- [30]. The Speedy A and Cdk2 mice have been −/− cytes and cyclin E was still recruited to chromosomes at the reported previously. [36, 52]The Sun1 mice were pachytene stage (Supplementary Figure 6), suggesting that purchased from Jackson Laboratory, numbered 012715- tm1Mhan cyclin E itself is not sufficient to recruit Cdk2 to the B6; 129S6-Sun1 /J (Bar Harbor, ME). All animal Dual roles of TRF1 in tethering telomeres 1185 studies were carried out in accordance with the protocols mouse (A21057) and Alexa Fluor® 680-conjugated rabbit approved by the Institutional Animal Care and Use secondary antibodies for goat IgGs (A21088) for immuno- Committee of the Institute of Zoology, Chinese Academy blotting were purchased from Invitrogen (Carlsbad, CA), of Sciences. IRDye® 800CW-conjugated goat secondary antibodies for rabbit (926-32211) for immunoblotting was purchased from Tissue collection and histological analysis LI-COR (Lincoln, NE). For histological examination, at least three adult mice for Immunoblotting each genotype were analyzed. The testes were dissected and fixed with Bouin’s fixative for up to 24 h. Next, the testes To prepare protein extracts, the testis albuginea was peeled were dehydrated using graded ethanol and embedded in and the testis or the isolated spermatocytes was suspended paraffin. 5 μm sections were collected and covered on glass in cold RIPA buffer (R0010 Solarbio) supplemented slides. After deparaffinization, sections were stained with with a protein inhibitor cocktail (Roche Diagnostics, H&E for histological analysis, or stained with Periodic Acid 04693116001, Rotkreuz, Switzerland) and 1 mM phe- Schiff (PAS)-hematoxylin for determining the seminiferous nylmethylsulfonyl fluoride (PMSF, 0754, Amresco). After epithelia cycle stages. homogenization and transient sonication, cell extracts were incubated on ice for 30 min. The samples were then cen- Epididymal sperm count trifuged at 12,000×g for 20 min at 4 °C. The supernatant was transferred to a new tube for immunoblotting. Protein The cauda epididymis was isolated from 10-week-old mice. samples were separated via SDS-PAGE and electro- Sperm were released from the cauda epididymis and incu- transferred to a nitrocellulose membrane. After incubation bated at 37 °C for 30 min under 5% CO2. The sperm with primary and secondary antibodies, the membrane was solution was diluted and sperm number was counted with a scanned using an ODYSSEY Sa Infrared Imaging System hemocytometer. (LI-COR Biosciences, Lincoln, NE). Antibodies Immunofluorescence and TUNEL assay The FLAG (1:2000, M20008L), GST (1:2000, M20007L), MYC (1:1000, M20002M) antibodies were purchased from The chromosome spreads of spermatocytes were washed with PBS for 3 times and blocked with 5% bovine serum Abmart (Shanghai, China). Mouse anti-TRF1 (ab10579), rabbit antibodies against SYCP3 (150292) and rabbit anti- albumin (AP0027, Amresco, Solon, OH). Primary anti- bodies were incubated at 4 °C overnight, followed by SUN1 (ab103021) were purchased from Abcam (Cam- bridge, MA). Mouse antibodies against γH2AX (05-636) incubation with the secondary antibodies. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). The were purchased from Merck Millipore (Darmstadt, Germany). Mouse antibodies against MLH1 (51-1327GR) images were taken immediately using an LSM 780 micro- scope (Zeiss, Oberkochen, Germany) or a TCS SP8 were purchased from BD Pharmingen (SanDiego, CA). Rabbit antibodies against SYCP1 (NB300-228c) and rabbit microscope (Leica, Wetzlar, Germany). 5-μm sections mounted on glass slides were first deparaffinized and then anti-TRF2 (NB110-57130) were purchased from Novus boiled for 15 min in sodium citrate buffer for antigen Biologicals (Littleton, CO). Mouse antibodies against SYCP3 (SC-74569), goat antibodies against ATR (SC- retrieval. After washing with PBS, sections were blocked and followed by antibody incubation as described above. To 1187), rabbit antibodies against DMC1 (SC-22768) were purchased from Santa Cruz Biotechnology (Dallas, TX). detect apoptotic cells in testis, we used the terminal deox- ynucleotidyl transferase dUTP nick end-labeling (TUNEL) Mouse anti-RAP1 (A300-306A) was purchased from Bethyl (Montgomery, TX). Mouse antibodies against assay kit (In Situ Cell Death Detection Kit; Roche, 11684795910) according to the manufacturer’s instructions. Cyclin E (MA5-14336) were purchased from Thermo fisher (Rockford, USA). Rabbit anti-Speedy A was generated as [53] Briefly, sections of the testes were deparaffinized and boiled for 15 min in sodium citrate buffer for antigen previously reported. [36] Goat FITC-conjugated secondary antibodies for rabbit, donkey FITC-conjugated secondary retrieval. After treated with H O for 10 min at room tem- 2 2 perature and sodium citrate for 2 min on ice, the slides were antibodies for mouse, rabbit TRITC-conjugated rinsed twice with PBS, the TUNEL reaction mixture was secondary antibodies for goat, and goat TRITC- conjugated secondary antibodies for mouse IgGs were added and incubated in a humidified atmosphere for 60 min at 37 °C in the dark, following by immunofluorescence purchased from Zhong Shan Jin Qiao (Beijing, China). Alexa Fluor®680-conjugated goat secondary antibodies for staining as detailed above. 1186 L. Wang et al. Immuno-FISH assay pH 7.4, 500 mM NaCl, 10 mM imidazole, 10% glycerol for hexahistidine-tagged fusion protein; 50 mM Tris, pH 7.4, The immuno-FISH assay was carried out based on a 500 mM NaCl, 2 mM MgCl , 5% glycerol for GST-tagged FISH protocol with minor modifications. [54] In brief, fusion proteins) supplemented with 1 mM PMSF. Sonica- spermatocyte spreads were treated with DNase free tion was used to lyse the bacteria. Then we collected the RNase A (V900498, SIGMA, 100 g/ml) at 37 °C for 30 min supernatant by high-speed centrifugation, and incubated and then dehydrated with 70, 85, and 100% alcohol. them with Ni Sepharose 6 Fast Flow (GE Healthcare, After denaturation at 85 °C for 10 min, they were hybridized Marlborough, MA) or Glutathione Sepharose 4B (GE for 2 h at 37 °C with fluorescein isothiocyanate (FITC)- Healthcare, Marlborough, MA) for 2 h at 4 °C. The beads labeled Tel (CCCTAA)3 PNA probe (F1009 PNA BIO). were washed, and the protein was eluted using the lysis The slices were washed sequentially in 2X saline sodium buffer supplemented with 250 mM imidazole or 10 mM citrate (SSC) with 0.1% Tween (twice) at 65 °C and glutathione. The pull-down assays were performed as with 2X SSC (twice) at room temperature for 5 min described previously [58]. Briefly, GST-TRF1 and GST- each time. The preparations were then co-labeled with Cdk2 were diluted in a buffer containing 20 mM Tris, pH antibodies. 7.4, 150 mM NaCl, 0.1% Triton X-100, 2 mM MgCl , 0.1% BSA and incubated with Glutathione Sepharose 4B agarose beads (GE Healthcare, Marlborough, MA) at 4 °C for 2 h, Genomic DNA extraction and qPCR assay to followed by further incubation for 2 h at 4 °C with FLAG- measure telomere length Speedy A or MYC-Cdk2 or both of them. The beads were washed three times with high-salt buffer, and then subjected The average telomere length was measured from total to immunoblotting analysis with anti-GST, anti-MYC and genomic DNA of pachytene and pachytene-like spermato- anti-FLAG antibodies. Flox/Flox −/− cytes in Trf1 and Stra8-Trf1 mice. Genomic DNA used for average telomere length measurement was extrac- Transmission electron microscopy ted following a previous report. [55]Briefly, cells were harvested and lysed in fresh lysis buffer (10 mM Tris-HCL Adult mouse testis was dissected and fixed with 2.5% (vol/ pH 8.0, 0.1 M EDTA, 0.5% SDS, 20 ug/ml RNase) for 3 h vol) glutaraldehyde in 0.2 M cacodylate buffer (50 mM and gently inverted every 30 min to release the RNA. cacodylate pH 7.2, 50 mM KCl, 2.5 mM MgCl ) overnight. Then 200 ug/ml Proteinase K (P6556-100MG, SIGMA) After washing in cacodylate buffer, the tissues were cut into was added and incubated the mixture at 55 °C overnight. small pieces of approximately 1 mm and immersed in 1% After that, phenol:chloroform:isoamyl alcohol = 25:24:1 OsO in 0.2 M cacodylate buffer for 2 h at 4 °C. Next, the was used to extract the total genomic DNA, followed samples were washed and submerged in 0.5% uranyl acetate by ethanol precipitation. The air-dried DNA was overnight. Dehydrated through a graded ethanol series and dissolved in 30 µl TE buffer for 2 h in a 37 °C water bath. embedded in resin (Low Viscosity Embedding Media Purity and integrity of the genomic DNA was tested Spurr’s Kit, EMS, 14300). Ultrathin sections were cut on an with NanoDrop and agarose gel electrophoresis. The ultramicrotome and mounted on copper grids. Then the OD260/OD280 ratio was between 1.60 to 1.90. Telomere sections were stained with uranyl acetate and lead citrate for length was measured by the qPCR method as previously 10 min and observed using a JEM-1400 transmission elec- described. [56, 57] tron microscope (JEOL, Tokyo, Japan). Protein purification and GST pull-down assay Chromosome spreads of spermatocyte For purification of GST-TRF1, GST-Cdk2, His-FLAG- Spermatocyte surface spreading was conducted according to Speedy A, and His-MYC-Cdk2, cDNA encoding mouse the drying-down technique as previously described. [59] TRF1, Cdk2 (variant 1) and Speedy A were cloned into Briefly, testes were dissected and the tubules were washed pGEX-4t-1 and modified pET28a (one MYC or FLAG tag in phosphate-buffered saline (PBS) pH 7.4 at room tem- was first cloned in to the vector) respectively. Briefly, the perature. Next, the tubules were submerged in a hypotonic plasmid was transformed into BL21 (DE3) cells and grown extraction buffer (30 mM Tris pH 8.2, 50 mM sucrose, in Terrific Broth at 37 °C. When the optical density reached 17 mM trisodium citrate dihydrate, 5 mM EDTA, 0.5 mM 1.0, they were transferred to the low temperature (16 °C) DTT and 0.5 mM PMSF) for 30–45 min. Subsequently, the shaker, and induced with 0.25 mM isopropyl-D- tubules were torn into pieces in 100 mM sucrose pH 8.2 on thiogalactoside (IPTG) for 16 h. After that, cells were har- a clean glass slide and then pipetted gently to make a sus- vested and then resuspended in lysis buffer (20 mM Tris, pension. The cell suspensions were loaded on slides Dual roles of TRF1 in tethering telomeres 1187 containing 1% paraformaldehyde (PFA) pH 9.2 and 0.15% Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, Triton X-100. The slides were dried for at least 2 h in a which permits any non-commercial use, sharing, adaptation, closed box with high humidity. Finally, the slides distribution and reproduction in any medium or format, as long as were washed with 0.4% Photoflo (Kodak, 1464510, you give appropriate credit to the original author(s) and the source, Rochester, NY) for 10 min and immunostained with anti- provide a link to the Creative Commons license, and indicate if changes were made. If you remix, transform, or build upon this article bodies according to the standard protocols mentioned or a part thereof, you must distribute your contributions under the same above. license as the original. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not −/− Speedy A MEF isolation and metaphase spread included in the article’s Creative Commons license and your intended karyotyping use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. −/− Speedy A mice was generated from intercrossing of org/licenses/by-nc-sa/4.0/. +/- Speedy A mice. We obtained mouse embryonic fibro- blasts (MEF) from 13.5-day-old embryos as previously References described. [60] The cells were incubated at 37 °C with 5% CO . For karyotype determination of metaphase spreads, 1. Lundblad V, Szostak JW. A mutant with a defect in telomere 0.1 µg/mL Colchicine was used to synchronize MEF cells elongation leads to senescence in yeast. Cell 1989;57:633–43. 2. Sfeir A, de Lange T. Removal of shelterin reveals the telomere at metaphase, following treatment with hypotonic end-protection problem. Science 2012;336:593–7. buffer (0.075M KCl) and incubation at room temperature 3. Hayashi MT, Cesare AJ, Rivera T, Karlseder J. Cell death during for 20 min. The suspension was centrifuged and the crisis is mediated by mitotic telomere deprotection. Nature supernatant was discarded. Cell pellets were fixed twice: in 2015;522:492–6. 4. O’Sullivan RJ, Karlseder J. Telomeres: protecting chromosomes methanol/glacial acetic acid (3:1) and methanol/glacial against genome instability. Nat Rev Mol Cell Bio 2010;11:171–81. acetic acid (1:1). Fixed cells were dropped onto ice-cold 5. de Lange T. Shelterin: the protein complex that shapes and safe- slides and air dried. The slides were stained with telomere- guards human telomeres. Gene Dev 2005;19:2100–10. FISH and DAPI to calculate the proportion of fusion. 6. Palm W, de Lange T. How shelterin protects mammalian telo- meres. Annu Rev Genet 2008;42:301–34. 7. Bianchi A, Smith S, Chong L, Elias P, deLange T. TRF1 is a dimer and bends telomeric DNA. Embo J 1997;16:1785–94. Statistical analysis 8. Bilaud T, Brun C, Ancelin K, Koering CE, Laroche T, Gilson E. Telomeric localization of TRF2, a novel human telobox protein. Nat Genet 1997;17:236–9. All data are presented as the mean ± SEM. The statistical 9. Li BB, de Lange T. Rap1 affects the length and heterogeneity of significance of the differences between the mean values for human telomeres. Mol Biol Cell 2003;14:5060–8. the various genotypes was measured by Student’s t-tests 10. O’Connor MS, Safari A, Xin HW, Liu D, Songyang Z. A critical with a paired, 2-tailed distribution. The data were con- role for TPP1 and TIN2 interaction in high-order telomeric complex assembly. Proc Natl Acad Sci USA 2006;103: sidered significant when the P-value was less than 0.05 (*), 11874–9. 0.01 (**) or 0.001(***). 11. Wang F, Podell ER, Zaug AJ, Yang YT, Baciu P, Cech TR, et al The POT1-TPP1 telomere complex is a telomerase processivity Acknowledgements We thank Prof. Maria A. Blasco for providing the factor. Nature 2007;445:506–10. Trf1 floxed mice. We thank Qingyuan Sun, Kui Liu, and Nathan 12. Okamoto K, Bartocci C, Ouzounov I, Diedrich JK, Yates JR, Palmer for critical reading of the manuscript. Denchi EL. A two-step mechanism for TRF2-mediated chromo- some-end protection. Nature 2013;494:502–5. Author contributions LW, ZT, and CL performed most of the 13. Martinez P, Thanasoula M, Munoz P, Liao CY, Tejera A, McNees experiments and wrote the manuscript. HL performed part of the C, et al Increased telomere fragility and fusions resulting from −/− −/− experiment. PK constructed the Cdk2 and SpeedyA knockout TRF1 deficiency lead to degenerative pathologies and increased mouse and revised the manuscript. ZC and WL supervised the whole cancer in mice. Gene Dev 2009;23:2060–75. project and wrote the manuscript. 14. Allegra A, Innao V, Penna G, Gerace D, Allegra AG, Musolino C. Telomerase and telomere biology in hematological diseases: A Funding This work was supported by the National key R&D program new therapeutic target. Leuk Res 2017;56:60–74. of China (Grant No. 2016YFA0500901), National Nature Science of 15. Maciejowski J, de Lange T. Telomeres in cancer: tumour sup- China (Grant No. 31471277 and 91649202) to W. Li and the Bio- pression and genome instability. Nat Rev Mol Cell Bio medical Research Council of A*STAR (Agency for Science, Tech- 2017;18:175–86. nology and Research, Singapore) to P. Kaldis. 16. Martinez P, Blasco MA. Telomere-driven diseases and telomere- targeting therapies. J Cell Biol 2017;216:875–87. 17. Scherthan H. Telomere attachment and clustering during meiosis. Compliance with ethical standards Cell Mol life Sci: CMLS 2007;64:117–24. 18. de Lange T. Human telomeres are attached to the nuclear matrix. Conflict of interest The authors declare that they have no conflict of Embo J 1992;11:717–24. interest. 1188 L. Wang et al. 19. Luderus ME, van Steensel B, Chong L, Sibon OC, Cremers FF, 40. Hunter N, Borner GV, Lichten M, Kleckner N. Gamma-H2AX de Lange T. Structure, subnuclear distribution, and nuclear matrix illuminates meiosis. Nat Genet 2001;27:236–8. association of the mammalian telomeric complex. J Cell Biol 41. Wang HY, Wang ML, Wang HC, Bocker W, Iliakis G. 1996;135:867–81. Complex H2AX phosphorylation patterns by multiple kinases 20. Reig-Viader R, Garcia-Caldes M, Ruiz-Herrera A. Telomere including ATM and DNA-PK in human cells exposed to ionizing homeostasis in mammalian germ cells: a review. Chromosoma radiation and treated with kinase inhibitors. J Cell Physiol 2016;125:337–51. 2005;202:492–502. 21. Link J, Jahn D, Alsheimer M. Structural and functional adapta- 42. Garcia-Muse T, Boulton SJ. Distinct modes of ATR activation tions of the mammalian nuclear envelope to meet the meiotic after replication stress and DNA double-strand breaks in Cae- requirements. Nucl-Phila 2015;6:93–101. norhabditis elegans. Embo J 2005;24:4345–55. 22. Harper L, Golubovskaya I, Cande WZ. A bouquet of chromo- 43. Baker SM, Plug AW, Prolla TA, Bronner CE, Harris AC, Yao X, somes. J Cell Sci 2004;117:4025–32. et al Involvement of mouse Mlh1 in DNA mismatch repair and 23. Scherthan H. A bouquet makes ends meet. Nat Rev Mol Cell Bio meiotic crossing over. Nat Genet 1996;13:336–42. 2001;2:621–7. 44. Bandaria JN, Qin PW, Berk V, Chu S, Yildiz A. Shelterin protects 24. Ding X, Xu R, Yu JH, Xu T, Zhuang Y, Han M. SUN1 is required chromosome ends by compacting telomeric chromatin. Cell for telomere attachment to nuclear envelope and gametogenesis in 2016;164:735–46. mice. Dev Cell 2007;12:863–72. 45. Nakano S, Miyoshi D, Sugimoto N. Effects of molecular 25. Haque F, Mazzeo D, Patel JT, Smallwood DT, Ellis JA, Shanahan crowding on the structures, interactions, and functions of nucleic CM, et al Mammalian SUN protein interaction networks at the acids. Chem Rev 2014;114:2733–58. inner nuclear membrane and their role in laminopathy disease 46. Zimmerman SB. Macromolecular crowding effects on macro- processes. J Biol Chem 2010;285:3487–98. molecular interactions—some implications for genome structure 26. Mikolcevic P, Isoda M, Shibuya H, Barrantes ID, Igea A, Suja JA, and function. Biochim Biophys Acta 1993;1216:175–85. et al Essential role of the Cdk2 activator RingoA in meiotic tel- 47. Miyoshi D, Sugimoto N. Molecular crowding effects on structure omere tethering to the nuclear envelope. Nat Commun and stability of DNA. Biochimie 2008;90:1040–51. 2016;7:11084. 48. Iwano T, Tachibana M, Reth M, Shinkai Y. Importance of 27. Viera A, Alsheimer M, Gomez R, Berenguer I, Ortega S, TRF1 for functional telomere structure. J Biol Chem Symonds CE, et al CDK2 regulates nuclear envelope protein 2004;279:1442–8. dynamics and telomere attachment in mouse meiotic prophase. J 49. Manterola M, Sicinski P, Wolgemuth DJ. E-type cyclins modulate Cell Sci 2015;128:88–99. telomere integrity in mammalian male meiosis. Chromosoma 28. Shibuya H, Ishiguro K, Watanabe Y. The TRF1-binding protein 2016;125:253–64. TERB1 promotes chromosome movement and telomere rigidity in 50. Martinerie L. Mammalian E-type cyclins control chromosome meiosis. Nat Cell Biol 2014;16:145–56. pairing, telomere stability and CDK2 localization in male meiosis. 29. Shibuya H, Hernandez-Hernandez A, Morimoto A, Negishi L, Plos Genet 2014;10:e1004165. Hoog C, Watanabe Y. MAJIN links telomeric DNA to the nuclear 51. Lottersberger F, Karssemeijer RA, Dimitrova N, de Lange T. membrane by exchanging telomere cap. Cell 2015;163:1252–66. 53BP1 and the LINC complex promote microtubule-dependent 30. Sadate-Ngatchou PI, Payne CJ, Dearth AT, Braun RE. Cre DSB mobility and DNA repair. Cell 2015;163:880–93. recombinase activity specific to postnatal, premeiotic male germ 52. Berthet C, Aleem E, Coppola V, Tessarollo L, Kaldis P. Cdk2 cells in transgenic mice. Genesis 2008;46:738–42. knockout mice are viable. Curr Biol 2003;13:1775–85. 31. Roosen-Runge EC. The process of spermatogenesis in mammals. 53. Song ZH, Yu HY, Wang P, Mao GK, Liu WX, Li MN, et al Germ Biol Rev Camb Philos Soc 1962;37:343–77. cell-specific Atg7 knockout results in primary ovarian insuffi- 32. Hess RA, Renato de Franca L. Spermatogenesis and cycle of the ciency in female mice. Cell Death Dis 2015;6:e1589. seminiferous epithelium. Adv Exp Med Biol 2008;636:1–15. 54. Bolzan AD, Bianchi MS. Detection of incomplete chromosome 33. Ahmed EA, de Rooij DG. Staging of mouse seminiferous tubule elements and interstitial fragments induced by bleomycin in cross-sections. Methods Mol Biol 2009;558:263–77. hamster cells using a telomeric PNA probe. Mutat Res-Fund Mol 34. Zickler D, Kleckner N. Meiotic chromosomes: integrating struc- M 2004;554:1–8. ture and function. Annu Rev Genet 1999;33:603–754. 55. Wang F, Pan XH, Kalmbach K, Seth-Smith ML, Ye XY, Antumes 35. Roeder GS, Bailis JM. The pachytene checkpoint. Trends Genet DMF, et al Robust measurement of telomere length in single cells. 2000;16:395–403. Proc Natl Acad Sci USA 2013;110:E1906–E1912. 36. Tu ZW, Bayazit MB, Liu HB, Zhang JJ, Busayavalasa K, Risal S, 56. Callicott RJ, Womack JE. Real-time PCR assay for measurement et al Speedy A-Cdk2 binding mediates initial telomere-nuclear of mouse telomeres. Comp Med 2006;56:17–22. envelope attachment during meiotic prophase I independent of 57. Cawthon RM. Telomere measurement by quantitative PCR. Cdk2 activation. Proc Natl Acad Sci USA 2017;114:592–7. Nucleic Acids Res 2002;30:e47. 37. Horn HF, Kim DI, Wright GD, Wong ESM, Stewart CL, Burke B, 58. Liu C, Liu WX, Ye YH, Li W. Ufd2p synthesizes branched et al A mammalian KASH domain protein coupling meiotic ubiquitin chains to promote the degradation of substrates modified chromosomes to the cytoskeleton. J Cell Biol 2013;202: with atypical chains. Nat Commun 2017;8:14274. 1023–39. 59. Peters AH, Plug AW, van Vugt MJ, de Boer P. A drying- 38. Meuwissen RL, Offenberg HH, Dietrich AJ, Riesewijk A, van down technique for the spreading of mammalian meiocytes Iersel M, Heyting C. A coiled-coil related protein specific for from the male and female germline. Chromosome Res synapsed regions of meiotic prophase chromosomes. Embo J 1997;5:66–68. 1992;11:5091–5100. 60. Conner DA. Mouse embryo fibroblast (MEF) feeder cell pre- 39. Neale MJ, Keeney S. Clarifying the mechanics of DNA strand paration. Curr Protoc Mol Biol/Ed Frederick M Ausubel [Et al] exchange in meiotic recombination. Nature 2006;442:153–8. 2001;Chapter 23:Unit23 22. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death & Differentiation Springer Journals

Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis

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Copyright © 2018 by The Author(s)
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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1350-9047
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10.1038/s41418-017-0037-8
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Abstract

Telomeres integrity is indispensable for chromosomal stability by preventing chromosome erosion and end-to-end fusions. During meiosis, telomeres attach to the inner nuclear envelope and cluster into a highly crowded microenvironment at the bouquet stage, which requires specific mechanisms to protect the telomeres from fusion. Here, we demonstrate that germ cell-specific knockout of a shelterin complex subunit, Trf1, results in arrest of spermatocytes at two different stages. The obliterated telomere-nuclear envelope attachment in Trf1-deficient spermatocytes impairs homologue synapsis and recombination, resulting in a pachytene-like arrest, while the meiotic division arrest might stem from chromosome end-to- end fusion due to the failure of recruiting meiosis specific telomere associated proteins. Further investigations uncovered that TRF1 could directly interact with Speedy A, and Speedy A might work as a scaffold protein to further recruit Cdk2, thus protecting telomeres from fusion at this stage. Together, our results reveal a novel mechanism of TRF1, Speedy A, and Cdk2 in protecting telomere from fusion in a highly crowded microenvironment during meiosis. Introduction progressively shorter during mitotic cycles or due to environmental stresses. When telomeres are shortened and Telomeres are nucleoprotein structures located at the end become dysfunctional, cellular senescence is triggered and of chromosomes and telomere integrity is essential for tissue/organ aging might be accelerated. [2] Dysfunctional genome stability and cell survival. [1] Telomeres become telomeres also tend to form chromosomal end-to-end fusion, eventually causing genomic instability, cell cycle arrest, and finally cell death. [3] Protection of telomere fusion is mainly regulated by the telomerase complex and Edited by R.A. Knight the shelterin complex, which includes six subunits (TRF1, Lina Wang, Zhaowei Tu and Chao Liu contributed equally to this TRF2, RAP1, TIN2, TPP1, and POT1). [4–11] TRF1 and work. TRF2 directly bind to double-stranded telomeric DNA and Electronic supplementary material The online version of this article prevent the activation of both ATM- and ATR-dependent (https://doi.org/10.1038/s41418-017-0037-8) contains supplementary DNA damage signaling pathways. [12, 13] Mutations in material, which is available to authorized users. * Zijiang Chen Ministry of Education, Jinan, Shandong 250001, China chenzijiang@hotmail.com Institute of Molecular and Cell Biology (IMCB), Agency for * Wei Li Science, Technology, and Research (A*STAR), leways@ioz.ac.cn Singapore 138673, Republic of Singapore Department of Biochemistry, National University of Singapore, State Key Laboratory of Stem Cell and Reproductive Biology, Singapore 117599, Republic of Singapore Institute of Zoology, Chinese Academy of Sciences, University of the Chinese Academy of Sciences, Beijing 100049, Beijing 100101, China China Department of Chemistry and Molecular Biology, University of Key Laboratory for Major Obstetric Diseases of Guangdong Gothenburg, Gothenburg SE-405 30, Sweden Province, Key Laboratory of Reproduction and Genetics of Center for Reproductive Medicine, Shandong Provincial Hospital Guangdong Higher Education Institutes, The Third Affiliated Affiliated to Shandong University, Jinan, Shandong 250001, China Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, China The Key Laboratory for Reproductive Endocrinology of the 1234567890 Dual roles of TRF1 in tethering telomeres 1175 telomere related genes are associated with many diseases, crossing mice with a floxed Trf1 allele to Stra8-Cre mice, including Hoyeraal-Hreidarsson syndrome, dyskeratosis which express Cre recombinase during early-stage sperma- congenita, pulmonary fibrosis, and cancer. [14–16] togenesis beginning on day 3 after birth. [30] Germ cell- Telomeres are attached to the nuclear matrix and scattered specific Trf1 knockout mice will be referred to as Stra8- −/− −/− throughout the nucleus in somatic cells. [17–19]In contrast Trf1 . We found that the Stra8-Trf1 male mice were in germ cells, telomeres display an unprecedented behavior completely infertile. The size and weight of their testes were Flox/Flox during meiotic prophase I where they attach to the inner significantly reduced compared with that of Trf1 and +/− nuclear envelope (NE) and cluster to form a structure which Stra8-Trf1 mice (Fig. 1a-b) due to efficient Trf1 deletion resembles a bouquet of flowers, termed the bouquet stage. (Fig. 1c). Further histological examination revealed that Trf1- [17, 20, 21] This phenomenon is conserved in diverse deficient testes lacked post-meiotic cells (Fig. 1d) and the eukaryote species and required for homologous chromosome diameter of Trf1-deficient seminiferous tubules was decreased pairing, synapsis, and proper resolution of recombination significantly (Fig. 1e). Few spermatozoa could be detected in events. [22, 23] During the bouquet stage, telomeres are in the cauda epididymis and the total number of spermatozoa −/− close proximity to each other, thus creating a crowded was dramatically reduced in the Stra8-Trf1 mice compared microenvironment. Since the prerequisite for telomere fusion with that of the control group, indicating that the disruption of is that two telomeres are in close proximity to each other to Trf1 severely impairs spermatogenesis (Fig. 1f-g). The allow the reaction to occur, telomeres are vulnerable to amount of degenerated cells with highly condensed nuclei −/− unwanted end-to-end fusions at the bouquet stage. In this found in the seminiferous epithelium of Stra8-Trf1 mice crowded microenvironment, protection mechanisms must (Fig. 1d), which are resembling the characteristic of apoptotic have been evolved to protect them fusing with each other. cells, let us speculate that the reduction of post-meiotic cells in The attachment of telomeres to the NE is dependent on the Trf1-deficient testes is due to apoptosis. To verify this pos- transmembrane linker of nucleoskeleton and cytoskeleton sibility, we performed TUNEL assays and found that the (LINC)-complex, which contains SUN1, SUN2, KASH5, number of TUNEL positive cells per seminiferous tubule in −/− and adaptor proteins like TERB1, TERB2, MAJIN, Speedy the Stra8-Trf1 mice was significantly increased compared A, and TRF1. [24–29] Since TRF1 is a core subunit of the with that of the control group (Supplementary Figure 1), shelterin complex and also involved in anchoring telomeres suggesting that spermatocytes in Trf1-deficient testes are to the NE by interacting with TERB1, [28]weselected TRF1 eliminated through apoptosis. Altogether, these results indi- as the starting point to investigate the telomere fusion pro- cate that germ cell-specific disruption of Trf1 results in tection mechanism at the bouquet stage. We knocked out spermatogenetic failure and that TRF1 is essential for Flox/Flox Trf1 in germ cells by crossing Trf1 mice [13]with spermatogenesis. Stra8-Cre transgenic mice. [30] We found that Trf1 is essential for spermatogenesis since the knockout of Trf1 in male germ cells led to arrest at two stages: the pachytene-like The Trf1-deficient spermatocytes arrest at two different stages. and the meiotic division stages. It is the defective attachment of telomeres to NE that results in the pachytene-like arrest, Spermatogenesis is a process in the seminiferous epithelium whereas the meiotic division arrest stems from chromosome end-to-end fusions in Trf1 knockout spermatocytes. Further where various generations of germ cells undergo a series of developmental steps, including spermatogonial mitosis, investigations uncovered that Speedy A and cyclin- spermatocytic meiosis, and spermiogenesis. [31, 32] Sper- dependent kinase 2 (Cdk2) but not SUN1 were involved in protection from telomere fusion at the bouquet stage. Thus, in matogenesis can be subdivided into 12 stages in mouse testes using Periodic Acid Schiff (PAS) and hematoxylin addition to their roles in tethering telomeres to NE, our work defines novel functions for TRF1, Speedy A, and Cdk2 in staining of sections. [33] To identify which stages of sper- matogenesis were affected after Trf1 deletion, sections were protecting telomeres from fusion in a crowded micro- environment during meiosis. stained with PAS and hematoxylin and we determined that spermatogenesis of Trf1-deficient mice was blocked at stages IV and XII (Fig. 2a). Since early spermatocytes gradually develop into mid-pachytene spermatocytes at Results stage III–V and spermatocytes in either the first or the second meiotic division are present in stage XII, [33] our TRF1 is essential for spermatogenesis −/− results indicate that the Stra8-Trf1 spermatocytes might To study the molecular mechanism of protection from telo- be arrested at the pachytene and meiotic division stages. To confirm this hypothesis, we identified the various mere fusion at the bouquet stage, the core subunit of the shelterin complex, Trf1, was knocked out in germ cells by stages of meiotic prophase I by staining for a component of 1176 L. Wang et al. −/− Fig. 1 TRF1 is required for spermatogenesis. a The Stra8-Trf1 mice Arrows indicate apoptotic cells. e The diameter of the Flox/Flox +/− −/− testes were smaller than those of the Trf1 and Stra8-Trf1 seminiferous tubules in Stra8-Trf1 mice was smaller than that of Flox/Flox Flox/Flox mice (10-week-old, and the same to the below). b Quantification of the Trf1 mice. Trf1 (gray bar, 195.50 ± 4.49 μm), Flox/Flox +/− −/− testis weight of the Trf1 and Stra8-Trf1 mice. Testis weight: Stra8-Trf1 (white bar, 102.40 ± 3.62 μm). Data are presented as Flox/Flox +/− Trf1 (grey bar, 151.20 ± 1.42 mg), Stra8-Trf1 (black bar, mean ± SEM. ***P < 0.001. f Histological analysis of the caudal −/− Flox/Flox −/− 148.40 ± 1.96 mg), Stra8-Trf1 (white bar, 27.40 ± 1.69 mg). Data epididymides from 10-week-old Trf1 and Stra8-Trf1 mice. are presented as mean ± SEM. ***P < 0.001. c The TRF1 protein g The total number of sperm in the cauda epididymis was −/− −/− Flox/Flox levels were reduced in the testes of the Stra8-Trf1 mice. Histone significantly decreased in the Stra8-Trf1 mice. Trf1 (grey 6 −/− 6 H2A was used as the loading control. d PAS-hematoxylin analysis of bar, 19.03 ± 1.22 × 10 ), Stra8-Trf1 (white bar, 0.06 ± 0.11 × 10 ). Flox/Flox −/− the seminiferous tubules of Trf1 and Stra8-Trf1 mice. Data are presented as mean ± SEM. **P < 0.01. the synaptonemal complex, SYCP3. [34] All meiotic pro- TUNEL positive tubules and spermatocytes in stage IV phase I stages could be identified in the spermatocyte nuclei significantly higher than that of the control (Supplementary Flox/Flox of Trf1 testes, whereas only spermatocytes from the Figure 1d-e), suggesting that the deletion of Trf1 leads to a leptotene to the pachytene-like stages were observed in the pachytene-like arrest and results in spermatocyte death. −/− Stra8-Trf1 mice testes (Fig. 2b). Further quantification of Since germ cells of Trf1-deficient mice also displayed the meiotic prophase I stages in testes indicated that the stage XII arrest (Fig. 2a) and some diplotene and diakinesis −/− proportion of the zygotene cells was significantly increased spermatocytes could be identified in the Stra8-Trf1 and that of diplotene cells was decreased in the Trf1-defi- mouse testes, we speculated that some Trf1-deficient sper- cient testes (Fig. 2c). The percentage of spermatocytes in matocytes could bypass the pachytene checkpoint due to the the pachytene stage was not accumulated in Stra8- variation of TRF1 expression, TRF1 protein half-life, or −/− Trf1 mouse testes (Fig. 2c), most likely because the other reasons. We found that some of the germ cells were pachytene checkpoint-arrested spermatocytes undergoing then blocked at the meiotic division stage. To determine the apoptosis. [35] To further confirm this possibility, TUNEL fate of the meiotic division stage-arrested germ cells, we staining was performed, and TUNEL-positive signal was performed TUNEL staining and found that some sperma- detected in the pachytene-like spermatocytes in testes from tocytes undergoing meiotic division were TUNEL positive −/− Stra8-Trf1 mice (Fig. 2d), with the percentages of (Fig. 2d), with both the percentages of TUNEL positive Dual roles of TRF1 in tethering telomeres 1177 Fig. 2 Trf1-deficient spermatocytes arrest at two stages. a Seminiferous tubules Flox/Flox paraffinsections from Trf1 −/− and Stra8-Trf1 testis were stained with PAS-hematoxylin. A type A spermatogonia, In intermediate spermatogonia, B type B spermatogonia, PL preleptotene spermatocytes, L leptotene spermatocytes, Z zygotene spermatocytes, P pachytene spermatocytes, aP apoptotic pachytene spermatocytes, M meiotic divisions, aM abnormal meiotic divisions, sS secondary spermatocytes, rSt round spermatids, S spermatids. b Spermatocyte stages in pre- Flox/Flox metaphase in Trf1 and −/− Stra8-Trf1 spermatocytes. Flox/Flox −/− Trf1 and Stra8-Trf1 chromosome spreads of spermatocytes were immunostained with antibodies against SYCP3 (green) and DAPI (blue). c Meiotic stage Flox/Flox frequencies in Trf1 and −/− Stra8-Trf1 testes. Lep Flox/Flox (Leptotene): Trf1 (gray bar, 12.07 ± 0.94%), Stra8- −/− Trf1 (white bar, 20.85 ± 1.30%). Zyg (Zygotene): Flox/Flox Trf1 (gray bar, 6.71 ± −/− 1.34%), Stra8-Trf1 (white bar, 23.63 ± 6.42%). Pac (Pachytene): Flox/Flox Trf1 (gray bar, 55.31 ± −/− 2.50%), Stra8-Trf1 (white bar, 47.60 ± 2.75%). Dip (Diplotene): Flox/Flox Trf1 (gray bar, 23.00 ± −/− 2.71%), Stra8-Trf1 (white bar, 7.00 ± 0.89%). Dia (Diakinesis): Flox/Flox Trf1 (gray bar, 2.88 ± −/− 1.49%), Stra8-Trf1 (white bar, 0.89 ± 0.52%). Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001. d Representative Flox/Flox TUNEL results in Trf1 and −/− Stra8-Trf1 testes. Paraffin Flox/Flox sections from Trf1 and −/− Stra8-Trf1 testes were stained with TUNEL (green) and DAPI (blue) to determine apoptotic cells in stage IV and stage XII tubules together with pachytene and metaphase spermatocytes. tubules and spermatocytes in stage XII significantly higher pachytene-like stage but some of them bypass the pachytene than that of the control (Supplementary Figure 1f-g). Thus, checkpoint and are later blocked at the meiotic division Trf1 disruption leads to most spermatocytes arresting at the stage. 1178 L. Wang et al. Fig. 3 TRF1 is required for the attachment of telomeres to the nuclear control group. Zygotene to pachytene spermatocytes were counted for envelope in spermatocytes. a Increased detachment of telomeres from wild-type, whereas pachytene-like spermatocytes were counted for the −/− Flox/Flox −/− NE after Trf1 deletion. An increased number of telomeres were dis- Stra8-Trf1 spermatocytes. Trf1 , 1.17 ± 1.38; Stra8-Trf1 , −/− tributed at the inner site of nucleus in Stra8-Trf1 spermatocytes 9.56 ± 1.62. The numbers of internal telomere (FISH) foci are pre- Flox/Flox compared with the control group. Paraffin sections from Trf1 sented as mean ± SEM. **P < 0.01. c An increased number of telo- −/− and Stra8-Trf1 testes were immunostained with antibodies against meres were detached from NE in the Trf1-deficient spermatocytes. SYCP3 (red), Tel-FISH (green), and Lamin B (gray). An increased Electron micrographs showing telomeres (red arrowheads) and inner Flox/Flox −/− number of telomere (FISH) signals was observed at the inner site of the NE (white arrows) in the Trf1 pachytene and Stra8-Trf1 nucleus and did not co-localize with the NE (Lamin B) signals. White pachytene-like nuclei; asterisks indicate membrane vesicles. Schematic arrowheads indicate the inner telomeres. Zyg zygotene spermatocytes, illustrations represent the structures. AP attachment plate, CH chro- Pac pachytene spermatocytes. b The number of internal telomeres in matin, NE nuclear envelope, SC synaptonemal complex. −/− Stra8-Trf1 spermatocytes was increased compared with that of the Germ cell-specific Trf1 knockout impairs the pachytene stage, whereas several telomeres were still −/− attachment of telomeres to the nuclear membrane detached from the NE in the Stra8-Trf1 spermatocytes (Fig. 3a-b). Electron microscopy analysis indicated that the Since it has been reported that TERB1 is involved in telomeres either did not reach the NE, did not form an anchoring telomeres to the NE by interacting with TRF1, attachment plate (AP), or were bound to a membrane [28] we speculated that knocking out Trf1 might impair NE vesicle just before reaching the NE (Fig. 3c), which is attachment. To verify this hypothesis, we performed similar to the reported phenomenon in other telomere-NE immunofluorescence staining of the NE marker Lamin B attachment-deficient mice. [24–29, 36, 37] Thus, the dis- Flox/Flox and telomere FISH. In Trf1 spermatocytes, most of ruption of Trf1 seems to affect the telomere-NE attachment the telomeres were attached to the NE at the zygotene and during meiosis. Dual roles of TRF1 in tethering telomeres 1179 TRF1 is essential for homologue synapsis and Impaired telomere fusion protection in recombination Trf1-deficient spermatocytes leads to meiotic division arrest During meiotic prophase I, telomeres associate with the LINC complex to attach to the nuclear envelope and the It has been reported that the depletion of shelterin complex disruption of this attachment might affect homologous subunits, such as TRF1, leads to telomere de-protection and chromosome synapsis and recombination, thereby leading initiation of DNA damage response at the end of chromo- to pachytene-like arrest. [24, 26–29, 36, 37] To investigate somes with telomere fusions and cell cycle checkpoint homologous chromosomes synapsis, we stained chromo- activation in somatic cells. [5, 12, 15, 16, 44] Thus, it is some spreads with antibodies against SYCP3 and SYCP1, a possible that TRF1 depletion in germ cells could lead to key component of the synaptonemal complex [38] and chromosomal end-to-end fusions and result in meiotic found that the SYCP1 signals were not continuous with division arrest. We noticed fused chromosomes in Stra8- −/− SYCP3 and many regions of homologous chromosomes Trf1 pachytene-like spermatocytes (Figs. 2b, 4a-b). failed to synapse in the Trf1-deficient spermatocytes (Sup- Therefore, we performed telomeric FISH experiment toge- plementary Figure 2), suggesting the knockout of Trf1 ther with SYCP3 staining in chromosome spreads of sper- impairs homologue synapsis. matocyte nuclei. Indeed we found end-to-end fusions in the Chromosome synapsis and recombination are facilitated Trf1-deficient spermatocytes (Fig. 5a), which might origi- −/− by the introduction of DNA double-strand breaks (DSBs), nate from the bouquet stage in Stra8-Trf1 spermatocytes [39] which can be monitored by detecting γH2AX-positive (Supplementary Figure 3). In support of the above results, foci at the leptotene and zygotene stages and these are we uncovered that both the Tel-FISH intensity and area in decreased on autosomes after DSB repaired at the pachytene the Trf1-deficient spermatocytes were increased compared −/− stage (Fig. 4a). [40]Inthe Stra8-Trf1 spermatocytes, the to those in the control group (Fig. 5b-c), most likely due to γH2AX signal was detected at leptotene and zygotene stages, their elongation by telomere fusions (Fig. 5d). These results Flox/Flox which was similar to Trf1 spermatocytes, indicating suggest that germ cell-specific knockout of Trf1 may impair the normal production of DSBs (Fig. 4a). However, in the telomere integrity, leading to chromosomal instability with Trf1-deficient spermatocytes, the γH2AX signal was still telomere end-to-end fusions, finally resulting in a meiotic diffused on the autosomes at the pachytene-like stage, rather division arrest. than accumulated only on the XY bodies (Fig. 4a). Con- To investigate the protection mechanism of TRF1 in sistent with the γH2AX staining, ATR, which is integral to preventing telomere fusion during meiosis, we stained for the regulation of programmed DSB formation, [41, 42]was other shelterin complex subunits. Surprisingly, the protein −/− also detected on the autosomes of Stra8-Trf1 spermato- levels of the shelterin complex proteins TRF2, TIN2, and −/− cytes (Fig. 4b). These results indicate that the programmed POT1 were dramatically reduced in the Stra8-Trf1 DSBs are properly introduced, but DSB repair is impaired in mouse testes but TRF2 was still localized on telomeres, the Trf1-deficient spermatocytes. Since the DSB repair in even when fusion occurred (Fig. 5g and Supplementary meiosis is mainly dependent on homologous recombination, Figure 4a), which suggests that telomere might be protected we stained for the meiosis specific recombination related during meiosis through a meiosis-specific mechanism. −/− protein, DMC1 [39]in Stra8-Trf1 spermatocytes. The Recently, several LINC complex proteins and their adaptor number of DMC1 foci in the Trf1-deficient spermatocytes proteins have been identified, such as Speedy A, Cdk2, was dramatically increased compared with that of the TERB1, MAJIN, and SUN1. [24, 26, 28, 29, 36, 37] control group (Fig. 4c), indicating impaired homologous We screened potential effectors of telomere fusion at the recombination. The late-recombination marker MLH1 bouquet stage, and found that TRF2, RAP1, and SUN1 normally appeared at the designated crossing-over sites, were still localized on telomeres, but Speedy A and [43] and MLH1 immunofluorescence in Trf1-deficient Cdk2 were not recruited to the telomeres in Trf1-deficient spermatocytes indicated that the number of MLH1 foci spermatocytes (Fig. 5e-f and Supplementary Figures 4a-c). was significantly reduced compared with that of the control In addition, the protein levels of Speedy A and Cdk2 −/− group (Fig. 4d-e), suggesting the crossover formation were substantially decreased in the Stra8-Trf1 mouse was also impaired. Altogether, TRF1 is required for testes compared to those of the control groups (Fig. 5g). homologue synapsis and recombination, and the disruption These results indicate that the disruption of Trf1 impairs of Trf1 might affect the telomere-nuclear envelope attach- the localization of the meiotic telomere-related proteins, ment, lead to impaired homologue synapsis, recombination Speedy A and Cdk2, on telomeres, which might be and crossover formation, finally resulting in a pachytene-like indispensable for preventing telomere fusion during arrest. meiosis. 1180 L. Wang et al. Fig. 4 Impaired meiotic −/− recombination in Stra8-Trf1 Flox/Flox spermatocytes. a Trf1 −/− and Stra8-Trf1 chromosome spreads of spermatocytes were immunostained with antibodies against SYCP3 (green) and γH2AX (red). γH2AX marked DSBs in leptotene and zygotene spermatocytes. No difference Flox/ was observed between Trf1 Flox −/− and Stra8-Trf1 at these Flox/Flox stages. In the Trf1 spermatocytes at the pachytene stage, DSBs were mostly repaired and γH2AX was confined to the XY body, whereas at the pachytene-like −/− stage in Stra8-Trf1 spermatocytes, γH2AX remained on some of the Flox/Flox autosomal regions. b Trf1 −/− and Stra8-Trf1 chromosome spreads of spermatocytes were immunostained with antibodies against SYCP3 (green) and ATR Flox/Flox (red). In the Trf1 spermatocytes at the pachytene stage, DSBs were mostly repaired and ATR was confined to the XY body, while the ATR signal persisted on the −/− autosomes in Stra8-Trf1 Flox/Flox spermatocytes. c Trf1 and −/− Stra8-Trf1 chromosome spreads of spermatocytes were immunostained with antibodies against SYCP3 (green) and DMC1 (red). The DMC1 signals were dramatically increased in −/− the Stra8-Trf1 spermatocytes at the zygotene stage compared Flox/Flox with those in the Trf1 Flox/Flox spermatocytes. d Trf1 and −/− Stra8-Trf1 chromosome spreads of spermatocytes were immunostained with antibodies against SYCP3 (green) and MLH1 (red). MLH1 is a recombination marker in WT spermatocytes and decreased MLH1 staining was observed in −/− Stra8-Trf1 spermatocytes. e MLH1 signals were counted in spreads. Quantification of the MLH1 foci numbers per cell in Flox/Flox the Trf1 and Stra8- −/− Trf1 spermatocytes. Flox/Flox Trf1 , 25.0 ± 1.7; −/− Stra8-Trf1 , 10.6 ± 2.8. Data are presented as mean ± SEM. ***P < 0.001. Dual roles of TRF1 in tethering telomeres 1181 Fig. 5 TRF1 is required for preventing telomere fusion at the bouquet Quantitative PCR was performed to measure telomere length and the stage. a Telomere fusions occurred after Trf1 depletion. Meiotic relative telomere to reference single copy gene (T/R) ratio was cal- Flox/Flox −/− Flox/Flox chromosome spreads from the Trf1 and Stra8-Trf1 testis culated to represent the average telomere length in the Trf1 and −/− were immunostained with antibody against SYCP3 (red) and Tel-FISH Stra8-Trf1 spermatocytes at pachytene or pachytene-like stage. −/− Flox/Flox −/− (green). In the pachytene-like Stra8-Trf1 spermatocytes, FISH Trf1 (gray bar, 0.78 ± 0.12), Stra8-Trf1 (white bar, 1.23 ± signal can be found in the middle of the SYCP3 threads, instead of at 0.09). Data are presented as mean ± SEM. ***P < 0.001. e and f the ends. Schematic illustrations represent the structures. b and c Speedy A and Cdk2 are not recruited to the telomeres in the Trf1- Flox/Flox −/− Telomeric FISH signal intensities and areas per telomere were deficient spermatocytes. Trf1 and Stra8-Trf1 chromosome −/− increased in Stra8-Trf1 spermatocytes. Telomeric FISH signal spreads of spermatocytes were immunolabelled with antibodies against intensities and areas were measured, and the mean Tel-FISH signal SYCP3 (red) and Speedy A (green) or Cdk2 (green). g Selected Flox/Flox intensities and areas were shown. Intensity: Trf1 (gray bar, shelterin and LINC complexes protein levels were decreased after Trf1 −/− Flox/ 116.22 ± 5.42) Stra8-Trf1 (white bar, 142.77 ± 5.35). Area: depletion. Immunoblotting for shelterin and LINC proteins in Trf1 Flox/Flox 2 −/− Flox −/− Trf1 (gray bar, 121.98 ± 7.58 px ), Stra8-Trf1 (white bar, and Stra8-Trf1 spermatocytes. TIN2, TRF2, POT1, Speedy A, 2 −/− 158.28 ± 8.87 px ). Data are presented as mean ± SEM. ***P < 0.001. and Cdk2 were all decreased to some extent in the Stra8-Trf1 d Telomere length was increased in the Trf1-deficient spermatocytes. spermatocytes. GAPDH was used as loading control. 1182 L. Wang et al. Fig. 6 Direct physical interaction between TRF1 and Speedy A. proteins were detected by immunoblotting analysis with anti-FLAG a Coomassie blue-stained gels showing the purification of relevant and anti-GST antibodies. e Speedy A-mediated interaction between proteins. b Direct physical interaction between TRF1 and Speedy A TRF1 and Cdk2. GST-TRF1 or GST conjugated sepharose beads were were determined by GST pull-down assays. GST-TRF1 or GST con- used to pull down His-FLAG-Speedy A and (or) His-MYC-Cdk2. jugated sepharose beads were used to pull down His-FLAG-Speedy A. Bound proteins were detected by immunoblotting analysis with anti- Bound proteins were detected by immunoblotting analysis with anti- FLAG or anti-MYC antibody. Asterisks indicate the bands of Speedy FLAG and anti-GST antibodies. c No direct physical interaction A. Input for His-FLAG-Speedy A and His-MYC-Cdk2 were 10% and between TRF1 and Cdk2, which was determined by GST pull-down 1%, respectively. f TRF1 and Cdk2 do not compete for interaction assays. GST-TRF1 or GST conjugated sepharose beads were used to with Speedy A. GST-TRF1 or GST conjugated sepharose beads were pull down His-MYC-Cdk2. Bound proteins were detected by immu- used to pull down His-FLAG-Speedy A in the presence of increasing noblotting analysis with anti-MYC and anti-GST antibodies. d Direct amounts of His-MYC-Cdk2. Bound proteins were detected by physical interaction between CDK2 and Speedy A was determined by immunoblotting analysis with anti-FLAG and anti-MYC antibodies. GST pull-down assays. GST-CDK2 or GST conjugated sepharose Asterisks indicate signals of Speedy A. beads were used to pull down purified His-FLAG-Speedy A. Bound Direct physical interaction between TRF1 and suggested that in the presence of Speedy A, Cdk2 could be Speedy A pulled down by TRF1 (Fig. 6e). To further demonstrate that Speedy A was a scaffold protein, we did a competition To further investigate the relationship between TRF1, experiment by adding excess Speedy A or Cdk2. When we Speedy A and Cdk2, we purified TRF1, Speedy A and increased the levels of Cdk2 in the reaction, the binding of Cdk2 proteins (Fig. 6a) and performed the GST pull-down Speedy A to TRF1 was unchanged, suggesting that TRF1 and Cdk2 might bind Speedy A at different non-overlapping experiments. Our results indicated that TRF1 could directly interact with Speedy A, but not Cdk2 (Fig. 6b-c). We fur- sites (Fig. 6f). Thus, TRF1 interacts directly with Speedy A, ther confirmed the known interaction between Cdk2 and and Speedy A binds to Cdk2, thereby recruiting Cdk2 to the Speedy A, and found that GST-Cdk2 could directly bind telomeres. to Speedy A (Fig. 6d). Since previous reports indicated that Speedy A was necessary for Cdk2’s localization on telo- Speedy A and Cdk2 are required for protecting meres during meiosis, [36] we speculated Speedy A might telomere fusion at the bouquet stage work as a scaffold protein to facilitate the indirect interac- tion between TRF1 and Cdk2. To test this possibility, GST- To investigate whether Speedy A and Cdk2 are also TRF1 was used to pull-down Cdk2 in the presence or required for preventing telomere fusion during meiosis, we absence of Speedy A. The GST pull-down experiments performed telomere-FISH and SYCP3 staining of Speedy A, Dual roles of TRF1 in tethering telomeres 1183 Fig. 7 TRF1-mediated Speedy A and Cdk2 recruitment are required for MEFs. Telomere numbers were counted and no significant changes +/+ preventing telomere fusion at the bouquet stage. a Telomere fusions were detected after Speedy A knockout. Speedy A , 90.25 ± 3.84; −/− −/− were detected in Speedy A chromosome spreads of spermatocytes. Speedy A , 94.13 ± 3.23. Data are presented as mean ± SEM. +/+ −/− −/− Speedy A and Speedy A spreads were immunolabelled with d TRF1 is still localized on telomeres in Speedy A spermatocytes. +/+ −/− antibodies against SYCP3 (red) and telomere FISH (green). Chro- Speedy A and Speedy A metaphase spreads were immunostained mosome fusions are indicated by arrows and a schematic illustrations with Telomere FISH (red) and DAPI (blue). e Immunoblotting for −/− of the structures is shown. b The telomere numbers in WT, Speedy telomere related proteins in Speedy A spermatocytes. SUN1 and −/− −/− −/− −/− A , Cdk2 , and Sun1 knockout mice. The telomere numbers TRF1 in Speedy A spermatocytes remained unchanged; β-Actin was −/− −/− were decreased significantly in Speedy A and Cdk2 spermato- used as the loading control. f A model of the meiosis specific telomere −/− cytes, but increased in Sun1 spermatocytes. The numbers of telo- fusion protection complex, with TRF1, Speedy A, and Cdk2 as the key mere (FISH) foci are presented as mean ± SEM. ***P < 0.001. players to protect telomeres from fusion. +/+ −/− c Quantification of Tel-FISH signals in Speedy A and Speedy A Cdk2, and Sun1-deficient spermatocytes and found some Speedy A and Cdk2 are required for preventing telomere −/− −/− fused telomeres in Speedy A and Cdk2 spermatocytes fusion during meiosis, and telomere fusion in Trf1-deficient −/− but not in Sun1 spermatocytes (Fig. 7a, Supplementary spermatocytes might be caused by the absence of Speedy A Figures 5a and 5c). Consistent with the telomere fusion and Cdk2 on telomeres. results, we found that the total number of telomeres in the Since the disruption of Trf1, Speedy A, and Cdk2 could −/− −/− Speedy A and Cdk2 spermatocytes was significantly cause telomere fusion during meiosis and Trf1 depletion decreased compared with their control groups (Fig. 7b), but resulted in the exclusion of Speedy A and Cdk2 from tel- we did not observe this phenomenon in somatic cells since omeres, TRF1 might be an upstream telomeric recruiter for −/− the total number of telomeres in Speedy A mouse Speedy A and Cdk2. To test this hypothesis, we stained for −/− −/− embryonic fibroblasts (MEFs) was similar to that of the TRF1 in Speedy A and Cdk2 spermatocytes and −/− −/− control (Fig. 7c). In contrast to Speedy A and Cdk2 , found that TRF1 was still localized on telomeres albeit the −/− the total number of telomeres in Sun1 spermatocytes was TRF1 signal was decreased compared to the control (Fig. 7d normal at pachytene-like stage, but was increased at zygo- and Supplementary Figure 5b). The lower TRF1 signaling tene stage, presumably due to homologous chromosome might be due to structural changes of the telomeres because synapsis failure (Fig. 7b). These results are similar to those the deletion of Speedy A or Cdk2 leads to telomere fusions findings in the Trf1-deficient spermatocytes, indicating that in spermatocytes. Immunoblotting analysis of telomeric 1184 L. Wang et al. complex components indicated that SUN1 and TRF1 levels telomeres. Thus, we propose that Speedy A may act as a −/− were not changed in Speedy A spermatocytes (Fig. 7e), scaffold protein for TRF1 and Cdk2, thus recruiting Cdk2 to thus suggesting that TRF1 and Speedy A may be central for telomeres. On the other hand, the RINGO domain of the mechanism of protecting telomeres from fusion at the Speedy A interacts with Cdk2 and promotes Cdk2 activa- bouquet stage. tion to protect telomere from fusion during meiosis. Telomere-NE attachment is essential for homologous chromosome synapsis and recombination and many key Discussion proteins have been identified for this attachment such as TERB1, TERB2, MAJIN, Speedy A, Cdk2, SUN1, SUN2, The cell interior is densely crowded with thousands of and KASH5. [24, 26, 28, 29, 36, 37] TRF1 can interact with macromolecules such as proteins, DNA, RNA, and other TERB1 to form the chimeric complex, TRF1-TERB1/2- molecules, where biological macromolecules have to func- MAJIN, which is indispensable for telomere-NE attach- tion in crowded cellular environment. [45, 46] Chromatin, a ment. [28, 29] During mid prophase, telomere cap exchange huge macromolecule, usually distributes all over the is achieved by removing the shelterin complex, including nucleus in most somatic cells. [47] In contrast in germ cells, TRF1, from the membrane-anchored telomeres, which is chromatin packs into a relative small space, and chromo- dependent on CDK activity. [29] However, TRF1 is still some ends cluster in close proximity at the bouquet stage localized near the membrane-anchored telomere region, during meiosis, [17, 20, 21] thus creating a crowded indicating additional roles of TRF1 during meiosis. Our microenvironment for telomeres. It is not known how to results demonstrate that in addition to the telomere-NE protect the telomere from fusion in this special micro- attachment, TRF1, Speedy A, and Cdk2 are required for environment. Here, we demonstrate that the disruption of protecting telomeres from fusion at the bouquet stage during the shelterin component Trf1 drives telomere fusion during meiosis. However, the disruption of Sun1, Terb1, Terb2 and meiosis (Fig. 5a) and TRF1 is required for the telomeric Majin do not affect telomere stability because telomere −/− localization of Speedy A and Cdk2 (Fig. 5e-f). Further fusions were not observed in Sun1 spermatocytes analysis indicated that the absence of Speedy A and Cdk2 (Supplementary Figure 5c), and the number of telomere led to severe telomere fusions in spermatocytes (Fig. 7a and does not decrease in Terb1 and Terb2 deficient spermato- Supplementary Figure 5a). Thus, TRF1, Speedy A, and cytes. [28, 29] Considering that SUN1-dependent chromatin Cdk2 are found to be involved in protecting telomeres from mobility is important for fusion of dysfunctional telomeres fusion in this crowded microenvironment (Fig. 7f). in somatic cells, [51] it is possible that the absence of SUN1 TRF1 is important for the functional telomere structure or other LINC complex components do not cause telomere and it can directly bind double-stranded telomeres by con- fusion during meiosis. Thus, the functions of LINC com- necting with TRF2 through TIN2. [4, 15] Depletion of ponents and their adaptor proteins in preventing telomere TRF1 affects the telomeric association of TRF2, [48] which fusion and telomere-NE attachment are not the same pro- has a crucial role in chromosome end protection. [12] cess. Whereas SUN1 only participates in telomere-NE Similarly, cyclin E deletion causes TRF2 depletion from the attachments, TRF1, Speedy A, and Cdk2 are involved in telomeres and aberrant telomere structures. [49] Speedy A both of these two processes (Figs. 5 and 7, Supplementary was dissociated from telomeres in Trf1-deficient spermato- Figures 5a-b). Therefore TRF1, Speedy A, and Cdk2 are cytes and TRF1 could directly bind to Speedy A, suggesting key players of the meiosis-specific mechanism in protecting that TRF1 might recruit Speedy A to telomeres during telomeres from fusion at bouquet stage. meiosis. Speedy A then serves as a scaffold protein to further recruit Cdk2 to telomeres. Recently, a “telomere localization domain” of Speedy A was identified and it is Materials and methods sufficient for Speedy A to co-localize with TRF1 on telo- meres. [36] The C-terminal RINGO domain of Speedy A is Animals’ experiments also necessary for Cdk2’s localization to telomeres during Flox/Flox meiosis. [26, 36] In addition, it has been reported that the The Trf1 mice (C57BL/6J) were a gift from Prof. formation of cyclin E/Cdk2 complex is necessary for the Maria A. Blasco from the Spanish National Cancer Centre Flox/Flox telomeric localization of Cdk2 and protection of the telo- (CNIO). [13]The Trf1 Stra8-Cre mice were bred Flox/Flox meres from end-to-end fusions. [49, 50] We found that from intercrosses of Trf1 mice and Stra8-Cre mice −/− −/− cyclin E levels were increased in Trf1-deficient spermato- [30]. The Speedy A and Cdk2 mice have been −/− cytes and cyclin E was still recruited to chromosomes at the reported previously. [36, 52]The Sun1 mice were pachytene stage (Supplementary Figure 6), suggesting that purchased from Jackson Laboratory, numbered 012715- tm1Mhan cyclin E itself is not sufficient to recruit Cdk2 to the B6; 129S6-Sun1 /J (Bar Harbor, ME). All animal Dual roles of TRF1 in tethering telomeres 1185 studies were carried out in accordance with the protocols mouse (A21057) and Alexa Fluor® 680-conjugated rabbit approved by the Institutional Animal Care and Use secondary antibodies for goat IgGs (A21088) for immuno- Committee of the Institute of Zoology, Chinese Academy blotting were purchased from Invitrogen (Carlsbad, CA), of Sciences. IRDye® 800CW-conjugated goat secondary antibodies for rabbit (926-32211) for immunoblotting was purchased from Tissue collection and histological analysis LI-COR (Lincoln, NE). For histological examination, at least three adult mice for Immunoblotting each genotype were analyzed. The testes were dissected and fixed with Bouin’s fixative for up to 24 h. Next, the testes To prepare protein extracts, the testis albuginea was peeled were dehydrated using graded ethanol and embedded in and the testis or the isolated spermatocytes was suspended paraffin. 5 μm sections were collected and covered on glass in cold RIPA buffer (R0010 Solarbio) supplemented slides. After deparaffinization, sections were stained with with a protein inhibitor cocktail (Roche Diagnostics, H&E for histological analysis, or stained with Periodic Acid 04693116001, Rotkreuz, Switzerland) and 1 mM phe- Schiff (PAS)-hematoxylin for determining the seminiferous nylmethylsulfonyl fluoride (PMSF, 0754, Amresco). After epithelia cycle stages. homogenization and transient sonication, cell extracts were incubated on ice for 30 min. The samples were then cen- Epididymal sperm count trifuged at 12,000×g for 20 min at 4 °C. The supernatant was transferred to a new tube for immunoblotting. Protein The cauda epididymis was isolated from 10-week-old mice. samples were separated via SDS-PAGE and electro- Sperm were released from the cauda epididymis and incu- transferred to a nitrocellulose membrane. After incubation bated at 37 °C for 30 min under 5% CO2. The sperm with primary and secondary antibodies, the membrane was solution was diluted and sperm number was counted with a scanned using an ODYSSEY Sa Infrared Imaging System hemocytometer. (LI-COR Biosciences, Lincoln, NE). Antibodies Immunofluorescence and TUNEL assay The FLAG (1:2000, M20008L), GST (1:2000, M20007L), MYC (1:1000, M20002M) antibodies were purchased from The chromosome spreads of spermatocytes were washed with PBS for 3 times and blocked with 5% bovine serum Abmart (Shanghai, China). Mouse anti-TRF1 (ab10579), rabbit antibodies against SYCP3 (150292) and rabbit anti- albumin (AP0027, Amresco, Solon, OH). Primary anti- bodies were incubated at 4 °C overnight, followed by SUN1 (ab103021) were purchased from Abcam (Cam- bridge, MA). Mouse antibodies against γH2AX (05-636) incubation with the secondary antibodies. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). The were purchased from Merck Millipore (Darmstadt, Germany). Mouse antibodies against MLH1 (51-1327GR) images were taken immediately using an LSM 780 micro- scope (Zeiss, Oberkochen, Germany) or a TCS SP8 were purchased from BD Pharmingen (SanDiego, CA). Rabbit antibodies against SYCP1 (NB300-228c) and rabbit microscope (Leica, Wetzlar, Germany). 5-μm sections mounted on glass slides were first deparaffinized and then anti-TRF2 (NB110-57130) were purchased from Novus boiled for 15 min in sodium citrate buffer for antigen Biologicals (Littleton, CO). Mouse antibodies against SYCP3 (SC-74569), goat antibodies against ATR (SC- retrieval. After washing with PBS, sections were blocked and followed by antibody incubation as described above. To 1187), rabbit antibodies against DMC1 (SC-22768) were purchased from Santa Cruz Biotechnology (Dallas, TX). detect apoptotic cells in testis, we used the terminal deox- ynucleotidyl transferase dUTP nick end-labeling (TUNEL) Mouse anti-RAP1 (A300-306A) was purchased from Bethyl (Montgomery, TX). Mouse antibodies against assay kit (In Situ Cell Death Detection Kit; Roche, 11684795910) according to the manufacturer’s instructions. Cyclin E (MA5-14336) were purchased from Thermo fisher (Rockford, USA). Rabbit anti-Speedy A was generated as [53] Briefly, sections of the testes were deparaffinized and boiled for 15 min in sodium citrate buffer for antigen previously reported. [36] Goat FITC-conjugated secondary antibodies for rabbit, donkey FITC-conjugated secondary retrieval. After treated with H O for 10 min at room tem- 2 2 perature and sodium citrate for 2 min on ice, the slides were antibodies for mouse, rabbit TRITC-conjugated rinsed twice with PBS, the TUNEL reaction mixture was secondary antibodies for goat, and goat TRITC- conjugated secondary antibodies for mouse IgGs were added and incubated in a humidified atmosphere for 60 min at 37 °C in the dark, following by immunofluorescence purchased from Zhong Shan Jin Qiao (Beijing, China). Alexa Fluor®680-conjugated goat secondary antibodies for staining as detailed above. 1186 L. Wang et al. Immuno-FISH assay pH 7.4, 500 mM NaCl, 10 mM imidazole, 10% glycerol for hexahistidine-tagged fusion protein; 50 mM Tris, pH 7.4, The immuno-FISH assay was carried out based on a 500 mM NaCl, 2 mM MgCl , 5% glycerol for GST-tagged FISH protocol with minor modifications. [54] In brief, fusion proteins) supplemented with 1 mM PMSF. Sonica- spermatocyte spreads were treated with DNase free tion was used to lyse the bacteria. Then we collected the RNase A (V900498, SIGMA, 100 g/ml) at 37 °C for 30 min supernatant by high-speed centrifugation, and incubated and then dehydrated with 70, 85, and 100% alcohol. them with Ni Sepharose 6 Fast Flow (GE Healthcare, After denaturation at 85 °C for 10 min, they were hybridized Marlborough, MA) or Glutathione Sepharose 4B (GE for 2 h at 37 °C with fluorescein isothiocyanate (FITC)- Healthcare, Marlborough, MA) for 2 h at 4 °C. The beads labeled Tel (CCCTAA)3 PNA probe (F1009 PNA BIO). were washed, and the protein was eluted using the lysis The slices were washed sequentially in 2X saline sodium buffer supplemented with 250 mM imidazole or 10 mM citrate (SSC) with 0.1% Tween (twice) at 65 °C and glutathione. The pull-down assays were performed as with 2X SSC (twice) at room temperature for 5 min described previously [58]. Briefly, GST-TRF1 and GST- each time. The preparations were then co-labeled with Cdk2 were diluted in a buffer containing 20 mM Tris, pH antibodies. 7.4, 150 mM NaCl, 0.1% Triton X-100, 2 mM MgCl , 0.1% BSA and incubated with Glutathione Sepharose 4B agarose beads (GE Healthcare, Marlborough, MA) at 4 °C for 2 h, Genomic DNA extraction and qPCR assay to followed by further incubation for 2 h at 4 °C with FLAG- measure telomere length Speedy A or MYC-Cdk2 or both of them. The beads were washed three times with high-salt buffer, and then subjected The average telomere length was measured from total to immunoblotting analysis with anti-GST, anti-MYC and genomic DNA of pachytene and pachytene-like spermato- anti-FLAG antibodies. Flox/Flox −/− cytes in Trf1 and Stra8-Trf1 mice. Genomic DNA used for average telomere length measurement was extrac- Transmission electron microscopy ted following a previous report. [55]Briefly, cells were harvested and lysed in fresh lysis buffer (10 mM Tris-HCL Adult mouse testis was dissected and fixed with 2.5% (vol/ pH 8.0, 0.1 M EDTA, 0.5% SDS, 20 ug/ml RNase) for 3 h vol) glutaraldehyde in 0.2 M cacodylate buffer (50 mM and gently inverted every 30 min to release the RNA. cacodylate pH 7.2, 50 mM KCl, 2.5 mM MgCl ) overnight. Then 200 ug/ml Proteinase K (P6556-100MG, SIGMA) After washing in cacodylate buffer, the tissues were cut into was added and incubated the mixture at 55 °C overnight. small pieces of approximately 1 mm and immersed in 1% After that, phenol:chloroform:isoamyl alcohol = 25:24:1 OsO in 0.2 M cacodylate buffer for 2 h at 4 °C. Next, the was used to extract the total genomic DNA, followed samples were washed and submerged in 0.5% uranyl acetate by ethanol precipitation. The air-dried DNA was overnight. Dehydrated through a graded ethanol series and dissolved in 30 µl TE buffer for 2 h in a 37 °C water bath. embedded in resin (Low Viscosity Embedding Media Purity and integrity of the genomic DNA was tested Spurr’s Kit, EMS, 14300). Ultrathin sections were cut on an with NanoDrop and agarose gel electrophoresis. The ultramicrotome and mounted on copper grids. Then the OD260/OD280 ratio was between 1.60 to 1.90. Telomere sections were stained with uranyl acetate and lead citrate for length was measured by the qPCR method as previously 10 min and observed using a JEM-1400 transmission elec- described. [56, 57] tron microscope (JEOL, Tokyo, Japan). Protein purification and GST pull-down assay Chromosome spreads of spermatocyte For purification of GST-TRF1, GST-Cdk2, His-FLAG- Spermatocyte surface spreading was conducted according to Speedy A, and His-MYC-Cdk2, cDNA encoding mouse the drying-down technique as previously described. [59] TRF1, Cdk2 (variant 1) and Speedy A were cloned into Briefly, testes were dissected and the tubules were washed pGEX-4t-1 and modified pET28a (one MYC or FLAG tag in phosphate-buffered saline (PBS) pH 7.4 at room tem- was first cloned in to the vector) respectively. Briefly, the perature. Next, the tubules were submerged in a hypotonic plasmid was transformed into BL21 (DE3) cells and grown extraction buffer (30 mM Tris pH 8.2, 50 mM sucrose, in Terrific Broth at 37 °C. When the optical density reached 17 mM trisodium citrate dihydrate, 5 mM EDTA, 0.5 mM 1.0, they were transferred to the low temperature (16 °C) DTT and 0.5 mM PMSF) for 30–45 min. Subsequently, the shaker, and induced with 0.25 mM isopropyl-D- tubules were torn into pieces in 100 mM sucrose pH 8.2 on thiogalactoside (IPTG) for 16 h. After that, cells were har- a clean glass slide and then pipetted gently to make a sus- vested and then resuspended in lysis buffer (20 mM Tris, pension. The cell suspensions were loaded on slides Dual roles of TRF1 in tethering telomeres 1187 containing 1% paraformaldehyde (PFA) pH 9.2 and 0.15% Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, Triton X-100. The slides were dried for at least 2 h in a which permits any non-commercial use, sharing, adaptation, closed box with high humidity. Finally, the slides distribution and reproduction in any medium or format, as long as were washed with 0.4% Photoflo (Kodak, 1464510, you give appropriate credit to the original author(s) and the source, Rochester, NY) for 10 min and immunostained with anti- provide a link to the Creative Commons license, and indicate if changes were made. If you remix, transform, or build upon this article bodies according to the standard protocols mentioned or a part thereof, you must distribute your contributions under the same above. license as the original. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not −/− Speedy A MEF isolation and metaphase spread included in the article’s Creative Commons license and your intended karyotyping use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. −/− Speedy A mice was generated from intercrossing of org/licenses/by-nc-sa/4.0/. +/- Speedy A mice. We obtained mouse embryonic fibro- blasts (MEF) from 13.5-day-old embryos as previously References described. [60] The cells were incubated at 37 °C with 5% CO . For karyotype determination of metaphase spreads, 1. Lundblad V, Szostak JW. A mutant with a defect in telomere 0.1 µg/mL Colchicine was used to synchronize MEF cells elongation leads to senescence in yeast. Cell 1989;57:633–43. 2. Sfeir A, de Lange T. Removal of shelterin reveals the telomere at metaphase, following treatment with hypotonic end-protection problem. Science 2012;336:593–7. buffer (0.075M KCl) and incubation at room temperature 3. Hayashi MT, Cesare AJ, Rivera T, Karlseder J. Cell death during for 20 min. The suspension was centrifuged and the crisis is mediated by mitotic telomere deprotection. Nature supernatant was discarded. Cell pellets were fixed twice: in 2015;522:492–6. 4. O’Sullivan RJ, Karlseder J. Telomeres: protecting chromosomes methanol/glacial acetic acid (3:1) and methanol/glacial against genome instability. Nat Rev Mol Cell Bio 2010;11:171–81. acetic acid (1:1). Fixed cells were dropped onto ice-cold 5. de Lange T. Shelterin: the protein complex that shapes and safe- slides and air dried. The slides were stained with telomere- guards human telomeres. Gene Dev 2005;19:2100–10. FISH and DAPI to calculate the proportion of fusion. 6. Palm W, de Lange T. How shelterin protects mammalian telo- meres. Annu Rev Genet 2008;42:301–34. 7. Bianchi A, Smith S, Chong L, Elias P, deLange T. TRF1 is a dimer and bends telomeric DNA. Embo J 1997;16:1785–94. Statistical analysis 8. Bilaud T, Brun C, Ancelin K, Koering CE, Laroche T, Gilson E. Telomeric localization of TRF2, a novel human telobox protein. Nat Genet 1997;17:236–9. All data are presented as the mean ± SEM. The statistical 9. Li BB, de Lange T. Rap1 affects the length and heterogeneity of significance of the differences between the mean values for human telomeres. Mol Biol Cell 2003;14:5060–8. the various genotypes was measured by Student’s t-tests 10. O’Connor MS, Safari A, Xin HW, Liu D, Songyang Z. A critical with a paired, 2-tailed distribution. The data were con- role for TPP1 and TIN2 interaction in high-order telomeric complex assembly. Proc Natl Acad Sci USA 2006;103: sidered significant when the P-value was less than 0.05 (*), 11874–9. 0.01 (**) or 0.001(***). 11. Wang F, Podell ER, Zaug AJ, Yang YT, Baciu P, Cech TR, et al The POT1-TPP1 telomere complex is a telomerase processivity Acknowledgements We thank Prof. Maria A. Blasco for providing the factor. Nature 2007;445:506–10. Trf1 floxed mice. We thank Qingyuan Sun, Kui Liu, and Nathan 12. Okamoto K, Bartocci C, Ouzounov I, Diedrich JK, Yates JR, Palmer for critical reading of the manuscript. Denchi EL. A two-step mechanism for TRF2-mediated chromo- some-end protection. Nature 2013;494:502–5. Author contributions LW, ZT, and CL performed most of the 13. Martinez P, Thanasoula M, Munoz P, Liao CY, Tejera A, McNees experiments and wrote the manuscript. HL performed part of the C, et al Increased telomere fragility and fusions resulting from −/− −/− experiment. PK constructed the Cdk2 and SpeedyA knockout TRF1 deficiency lead to degenerative pathologies and increased mouse and revised the manuscript. ZC and WL supervised the whole cancer in mice. Gene Dev 2009;23:2060–75. project and wrote the manuscript. 14. Allegra A, Innao V, Penna G, Gerace D, Allegra AG, Musolino C. Telomerase and telomere biology in hematological diseases: A Funding This work was supported by the National key R&D program new therapeutic target. Leuk Res 2017;56:60–74. of China (Grant No. 2016YFA0500901), National Nature Science of 15. Maciejowski J, de Lange T. Telomeres in cancer: tumour sup- China (Grant No. 31471277 and 91649202) to W. Li and the Bio- pression and genome instability. Nat Rev Mol Cell Bio medical Research Council of A*STAR (Agency for Science, Tech- 2017;18:175–86. nology and Research, Singapore) to P. Kaldis. 16. Martinez P, Blasco MA. Telomere-driven diseases and telomere- targeting therapies. J Cell Biol 2017;216:875–87. 17. Scherthan H. Telomere attachment and clustering during meiosis. Compliance with ethical standards Cell Mol life Sci: CMLS 2007;64:117–24. 18. de Lange T. Human telomeres are attached to the nuclear matrix. Conflict of interest The authors declare that they have no conflict of Embo J 1992;11:717–24. interest. 1188 L. Wang et al. 19. Luderus ME, van Steensel B, Chong L, Sibon OC, Cremers FF, 40. Hunter N, Borner GV, Lichten M, Kleckner N. Gamma-H2AX de Lange T. Structure, subnuclear distribution, and nuclear matrix illuminates meiosis. Nat Genet 2001;27:236–8. association of the mammalian telomeric complex. J Cell Biol 41. Wang HY, Wang ML, Wang HC, Bocker W, Iliakis G. 1996;135:867–81. Complex H2AX phosphorylation patterns by multiple kinases 20. Reig-Viader R, Garcia-Caldes M, Ruiz-Herrera A. Telomere including ATM and DNA-PK in human cells exposed to ionizing homeostasis in mammalian germ cells: a review. Chromosoma radiation and treated with kinase inhibitors. J Cell Physiol 2016;125:337–51. 2005;202:492–502. 21. Link J, Jahn D, Alsheimer M. Structural and functional adapta- 42. Garcia-Muse T, Boulton SJ. Distinct modes of ATR activation tions of the mammalian nuclear envelope to meet the meiotic after replication stress and DNA double-strand breaks in Cae- requirements. Nucl-Phila 2015;6:93–101. norhabditis elegans. Embo J 2005;24:4345–55. 22. Harper L, Golubovskaya I, Cande WZ. A bouquet of chromo- 43. Baker SM, Plug AW, Prolla TA, Bronner CE, Harris AC, Yao X, somes. J Cell Sci 2004;117:4025–32. et al Involvement of mouse Mlh1 in DNA mismatch repair and 23. Scherthan H. A bouquet makes ends meet. Nat Rev Mol Cell Bio meiotic crossing over. Nat Genet 1996;13:336–42. 2001;2:621–7. 44. Bandaria JN, Qin PW, Berk V, Chu S, Yildiz A. Shelterin protects 24. Ding X, Xu R, Yu JH, Xu T, Zhuang Y, Han M. SUN1 is required chromosome ends by compacting telomeric chromatin. Cell for telomere attachment to nuclear envelope and gametogenesis in 2016;164:735–46. mice. Dev Cell 2007;12:863–72. 45. Nakano S, Miyoshi D, Sugimoto N. Effects of molecular 25. Haque F, Mazzeo D, Patel JT, Smallwood DT, Ellis JA, Shanahan crowding on the structures, interactions, and functions of nucleic CM, et al Mammalian SUN protein interaction networks at the acids. Chem Rev 2014;114:2733–58. inner nuclear membrane and their role in laminopathy disease 46. Zimmerman SB. Macromolecular crowding effects on macro- processes. J Biol Chem 2010;285:3487–98. molecular interactions—some implications for genome structure 26. Mikolcevic P, Isoda M, Shibuya H, Barrantes ID, Igea A, Suja JA, and function. Biochim Biophys Acta 1993;1216:175–85. et al Essential role of the Cdk2 activator RingoA in meiotic tel- 47. Miyoshi D, Sugimoto N. Molecular crowding effects on structure omere tethering to the nuclear envelope. Nat Commun and stability of DNA. Biochimie 2008;90:1040–51. 2016;7:11084. 48. Iwano T, Tachibana M, Reth M, Shinkai Y. Importance of 27. Viera A, Alsheimer M, Gomez R, Berenguer I, Ortega S, TRF1 for functional telomere structure. J Biol Chem Symonds CE, et al CDK2 regulates nuclear envelope protein 2004;279:1442–8. dynamics and telomere attachment in mouse meiotic prophase. J 49. Manterola M, Sicinski P, Wolgemuth DJ. E-type cyclins modulate Cell Sci 2015;128:88–99. telomere integrity in mammalian male meiosis. Chromosoma 28. Shibuya H, Ishiguro K, Watanabe Y. The TRF1-binding protein 2016;125:253–64. TERB1 promotes chromosome movement and telomere rigidity in 50. Martinerie L. Mammalian E-type cyclins control chromosome meiosis. Nat Cell Biol 2014;16:145–56. pairing, telomere stability and CDK2 localization in male meiosis. 29. Shibuya H, Hernandez-Hernandez A, Morimoto A, Negishi L, Plos Genet 2014;10:e1004165. Hoog C, Watanabe Y. MAJIN links telomeric DNA to the nuclear 51. Lottersberger F, Karssemeijer RA, Dimitrova N, de Lange T. membrane by exchanging telomere cap. Cell 2015;163:1252–66. 53BP1 and the LINC complex promote microtubule-dependent 30. Sadate-Ngatchou PI, Payne CJ, Dearth AT, Braun RE. Cre DSB mobility and DNA repair. Cell 2015;163:880–93. recombinase activity specific to postnatal, premeiotic male germ 52. Berthet C, Aleem E, Coppola V, Tessarollo L, Kaldis P. Cdk2 cells in transgenic mice. Genesis 2008;46:738–42. knockout mice are viable. Curr Biol 2003;13:1775–85. 31. Roosen-Runge EC. The process of spermatogenesis in mammals. 53. Song ZH, Yu HY, Wang P, Mao GK, Liu WX, Li MN, et al Germ Biol Rev Camb Philos Soc 1962;37:343–77. cell-specific Atg7 knockout results in primary ovarian insuffi- 32. Hess RA, Renato de Franca L. Spermatogenesis and cycle of the ciency in female mice. Cell Death Dis 2015;6:e1589. seminiferous epithelium. Adv Exp Med Biol 2008;636:1–15. 54. Bolzan AD, Bianchi MS. Detection of incomplete chromosome 33. Ahmed EA, de Rooij DG. Staging of mouse seminiferous tubule elements and interstitial fragments induced by bleomycin in cross-sections. Methods Mol Biol 2009;558:263–77. hamster cells using a telomeric PNA probe. Mutat Res-Fund Mol 34. Zickler D, Kleckner N. Meiotic chromosomes: integrating struc- M 2004;554:1–8. ture and function. Annu Rev Genet 1999;33:603–754. 55. Wang F, Pan XH, Kalmbach K, Seth-Smith ML, Ye XY, Antumes 35. Roeder GS, Bailis JM. The pachytene checkpoint. Trends Genet DMF, et al Robust measurement of telomere length in single cells. 2000;16:395–403. Proc Natl Acad Sci USA 2013;110:E1906–E1912. 36. Tu ZW, Bayazit MB, Liu HB, Zhang JJ, Busayavalasa K, Risal S, 56. Callicott RJ, Womack JE. Real-time PCR assay for measurement et al Speedy A-Cdk2 binding mediates initial telomere-nuclear of mouse telomeres. Comp Med 2006;56:17–22. envelope attachment during meiotic prophase I independent of 57. Cawthon RM. Telomere measurement by quantitative PCR. Cdk2 activation. Proc Natl Acad Sci USA 2017;114:592–7. Nucleic Acids Res 2002;30:e47. 37. Horn HF, Kim DI, Wright GD, Wong ESM, Stewart CL, Burke B, 58. Liu C, Liu WX, Ye YH, Li W. Ufd2p synthesizes branched et al A mammalian KASH domain protein coupling meiotic ubiquitin chains to promote the degradation of substrates modified chromosomes to the cytoskeleton. J Cell Biol 2013;202: with atypical chains. Nat Commun 2017;8:14274. 1023–39. 59. Peters AH, Plug AW, van Vugt MJ, de Boer P. A drying- 38. Meuwissen RL, Offenberg HH, Dietrich AJ, Riesewijk A, van down technique for the spreading of mammalian meiocytes Iersel M, Heyting C. A coiled-coil related protein specific for from the male and female germline. Chromosome Res synapsed regions of meiotic prophase chromosomes. Embo J 1997;5:66–68. 1992;11:5091–5100. 60. Conner DA. Mouse embryo fibroblast (MEF) feeder cell pre- 39. Neale MJ, Keeney S. Clarifying the mechanics of DNA strand paration. Curr Protoc Mol Biol/Ed Frederick M Ausubel [Et al] exchange in meiotic recombination. Nature 2006;442:153–8. 2001;Chapter 23:Unit23 22.

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Cell Death & DifferentiationSpringer Journals

Published: Jan 8, 2018

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