Speedy A–Cdk2 binding mediates initial telomere– nuclear envelope attachment during meiotic prophase I independent of Cdk2 activation Zhaowei Tua,1, Mustafa Bilal Bayazita,1, Hongbin Liub, Jingjing Zhanga, Kiran Busayavalasaa, Sanjiv Risala, Jingchen Shaoa, Ande Satyanarayanac,2,3, Vincenzo Coppolac,4, Lino Tessarolloc, Meenakshi Singha, Chunwei Zhengd, Chunsheng Hand, Zijiang Chenb, Philipp Kaldise,f,5, Jan-Åke Gustafssong,5, and Kui Liua,b,5 aDepartment of Chemistry and Molecular Biology, University of Gothenburg, SE-40530 Gothenburg, Sweden; bThe Key Laboratory of Reproductive Endocrinology, Shandong University, Ministry of Education, Jinan 250001, China; cMouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201; dState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; eInstitute of Molecular and Cell Biology, Agency for Science, Technology, and Research (A*STAR), Singapore 138673, Republic of Singapore; fDepartment of Biochemistry, National University of Singapore, Singapore 117599, Republic of Singapore; and gCenter for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX 77204 Contributed by Jan-Åke Gustafsson, November 16, 2016 (sent for review September 12, 2016; reviewed by Heng-Yu Fan and Hengbin Wang) Telomere attachment to the nuclear envelope (NE) is a prerequisite are characterized by their highly conserved, ∼140-aa central Cdk- for chromosome movement during meiotic prophase I that is required binding core, called the Ringo domain (14, 17). In mice, four ho- for pairing of homologous chromosomes, synapsis, and homolo- mologs of Speedy have been identified: Speedy A, Speedy B1a, gous recombination. Here we show that Speedy A, a noncanonical Speedy B1b, and Speedy B3 (14, 16, 17). Both mouse Speedy A activator of cyclin-dependent kinases (Cdks), is specifically localized to and the human homolog Spy1 are able to induce meiotic re- telomeres in prophase I male and female germ cells in mice, and plays sumption when injected into Xenopus oocytes (14, 17). However, an essential role in the telomere–NE attachment. Deletion of Spdya in the in vivo physiological role of mammalian Speedy A is unknown. mice disrupts telomere–NE attachment, and this impairs homologous In the present study, we generated Spdya knockout mice and pairing and synapsis and leads to zygotene arrest in male and female studied the functional roles of Speedy A in mammals. We found germ cells. In addition, we have identified a telomere localization that distinct from Xenopus, Speedy A in mice is not involved in domain on Speedy A covering the distal N terminus and the Cdk2- oocyte maturation. Instead, Speedy A is only specifically ex- binding Ringo domain, and this domain is essential for the localization pressed in male and female germ cells at meiotic prophase I, and of Speedy A to telomeres. Furthermore, we found that the binding of it is localized to the telomeres. Loss of Speedy A in mice impairs Cdk2 to Speedy A is indispensable for Cdk2’s localization on telo- telomere–NE attachment in early meiosis, perturbs homologous meres, suggesting that Speedy A and Cdk2 might be the initial com- recombination, and leads to infertility in both sexes. Moreover, ponents that are recruited to the NE for forming the meiotic telomere complex. However, Speedy A-Cdk2–mediated telomere–NE attach- ment is independent of Cdk2 activation. Our results thus indicate that Significance Speedy A and Cdk2 might mediate the initial telomere–NE attachment for the efficient assembly of the telomere complex that is essential for In meiotic prophase I, telomere attachment to the nuclear enve- meiotic prophase I progression. lope is a prerequisite for subsequent prophase events, such as homologous pairing and recombination. In this study, we show meiosis | telomere | Speedy A | Cdk2 | germ cells that Speedy A, a noncanonical activator of cyclin-dependent ki- nases (Cdks), is essential for telomere attachment to the nuclear n mammals, the progression of meiotic prophase I is largely envelope in mice. We have identified a telomere localization ’ Idependent on the dynamic movement of chromosomes along domain in Speedy A, which covers the protein s distal N-terminus the nuclear envelope (NE) (1, 2). A prerequisite for faithful and Cdk2-binding Ringo domain but excludes its Cdk-activation chromosome movement is the anchoring of telomeres to the domain. Furthermore, we found that the binding of Cdk2 to Speedy A is essential for Cdk2’s localization to telomeres. Our transmembrane LINC (linker of nucleoskeleton and cytoskeleton) results suggest that Speedy A–Cdk2 binding might mediate the complex that bridges chromatin to the cytoskeleton (3). initial assembly of the meiotic telomere complex, a process that is In recent years, several meiosis-specific structural molecules that independent of Cdk2 activation. mediate telomere–NE attachment have been identified in mice, such as TERB1 (telomere repeat binding bouquet formation pro- Author contributions: P.K., J.-Å.G., and K.L. designed research; Z.T., M.B.B., H.L., J.Z., K.B., tein 1), SUN1 (Sad1 and UNC84 domain containing 1), KASH5 S.R., J.S., A.S., V.C., L.T., M.S., and C.Z. performed research; C.H., Z.C., P.K., J.-Å.G., and K.L. (Klarsicht/ANC-1/Syne/homology 5), TERB2 (telomere repeat analyzed data; and Z.T., M.B.B., P.K., and K.L. wrote the paper. binding bouquet formation protein 2), and MAJIN (membrane- Reviewers: H.-Y.F., Zhejiang University; and H.W., University of Alabama at Birmingham. anchored junction protein), and mice lacking any one of SUN1, The authors declare no conflict of interest. KASH5, TERB1, TERB2, or MAJIN display impaired telomere 1Z.T. and M.B.B. contributed equally to this work. attachment and are sterile (4–8). Moreover, cyclin-dependent ki- 2Present address: Department of Biochemistry and Molecular Biology, Molecular Oncol- nase 2 (Cdk2) is localized to telomeres in mouse spermatocytes ogy and Biomarkers Program, Georgia Regents University Cancer Center, Georgia Re- and prophase I oocytes (9), and loss of Cdk2 leads to sterility in gents University, Augusta, GA 30912. 3 both male and female mice (10, 11). Present address: Department of Pathology, Program in Pulmonary Vascular Disease, Speedy/RINGO (Rapid inducer of G2/M progression in oo- Vascular Biology Center, Georgia Regents University, Augusta, GA 30912. 4Present address: Department of Molecular Virology, Immunology & Medical Genetics, cytes) proteins are atypical noncyclin Cdk activators that were first The Ohio State University, Columbus, OH 43210. discovered in Xenopus laevis as proteins that induce G2/M transi- 5To whom correspondence may be addressed. Email: [email protected], jgustafs@ tion during oocyte maturation (12, 13). Multiple members of the central.uh.edu, or [email protected]. – Speedy family have since been identified in mammals (14 16). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Speedy proteins activate Cdks independently of cyclins, and they 1073/pnas.1618465114/-/DCSupplemental. 592–597 | PNAS | January 17, 2017 | vol. 114 | no. 3 www.pnas.org/cgi/doi/10.1073/pnas.1618465114 Downloaded by guest on September 28, 2021 we found that the proper attachment of telomeres to the NE is dependent on a telomere localization domain (TLD) on Speedy A, as defined by this study, whereas the C terminus of Speedy A, which is required for Cdk2 activation, is dispensible for the at- tachment of telomeres to the NE. Furthermore, we showed that the binding of Cdk2 to Speedy A is required for Cdk2 to localize onto telomeres. These results suggest that the function of Speedy A goes beyond that of a noncanonical activator of Cdk as pre- viously suggested (14, 15, 18, 19). Rather, the binding between Speedy A and Cdk2 might mediate the initial assembly of the telomere–NE complex that is essential for meiotic prophase I progression. Results Speedy A Is Specifically Expressed in Meiotic Germ Cells and Is Localized to Telomeres. To study the functional roles of Speedy A in devel- opment, we first investigated the expression of Speedy A protein in various tissues by Western blotting. We found that Speedy A was specifically expressed in the adult testis and embryonic ovary when meiotic prophase I occurs, but not in the adult ovary or in the adult liver, lung, spleen, kidney, or intestine (Fig. 1A). To further reveal the expression pattern of Speedy A in germ cells, we isolated different types of male germ cells by BSA gra- dient sedimentation (20). In the subsequent Western blotting, we found that Speedy A expression was undetectable in spermatogo- nia, but its expression began to beobservedinpreleptotenegerm cells (Fig. 1B). The expression of Speedy A then decreased in pachytene cells and became almost undetectable in round sper- matids and elongated spermatids (Fig. 1B). In contrast, the protein level of the 33-kDa isoform of Cdk2 (p33) remained constant be- tween spermatogonia and spermatocytes, but the level of the 39-kDa isoform of Cdk2 (p39) (21) increased from preleptotene to pachytene cells (Fig. 1B). Moreover, Speedy A was first observed in testes at postnatal day (PD) 12 (Fig. 1C), which was concurrent with up-regulation of p39 Cdk2 (Fig. 1C). At PD12, Speedy A exhibits as a doublet and it is likely that this is caused by phos- phorylation (Fig. 1C). We also examined Spdya mRNA expression in the female germ cells at various developmental time points by RT-PCR. Spdya mRNA was not expressed in mitotic female primordial germ cells at 11.5 days postcoitum (dpc), but its expression was up-regulated in female germ cells once they entered meiosis at 14.5 and 17.5 dpc (Fig. 1D). The Spdya mRNA levels then decreased in prenatal oocytes at 18.5 and 19.5 dpc, when they became arrested at dic- tyate, and the Spdya mRNA levels were almost undetectable in the adult ovary (Fig.