Meiosis and mitosis
In order to faithfully transmit their genetic information to their progeny, eukaryotic species possess a specialized form of cell division called meiosis. During meiosis, the chromosome number is accurately halved through two successive rounds of cell division following a single round of DNA replication, and the result is haploid germ cells. The characteristic events during meiosis are the pairing/synapsis and recombination of homologous chromosomes in prophase I. These steps ensure the formation of bivalent chromosomes specific in meiosis. Misregulation of these processes results in various genetic disorders including aneuploidy, Down Syndrome, infertility, and azoospermia.
Our studies try to address the fundamental question of how pairing/synapsis and recombination of homologous chromosomes are regulated on the molecular level during mammalian meiosis using the mouse as our model system. We employ a combination of genetic, molecular, cytological, and biochemical approaches.
Telomere in meiosis
Telomeres are composed of repetitive DNA sequences, to which the shelterin complex constitutively binds to protect the terminal DNA. To achieve pairing/synapsis of homologous chromosomes, meiotic chromosomes move within the nucleus by attachment of the telomeres to the nuclear membrane. Recent studies have identified a multi-subunit DNA-binding complex, TERB1-TERB2-MAJIN, that binds to telomeric DNA in mammalian germ cells,,,. TERB1-TERB2-MAJIN is responsible for the attachment of telomeres to the nuclear membrane and drives the subsequent chromosome movement along the membrane. We are interested in the molecular regulations directing TERB1-TERB2-MAJIN complex assembly as well the identification of novel factors regulating telomere dynamics in germ cells.
Meiotic homologous recombination
The DNA double strand breaks (DSBs) are well recognized as a major driving force in cancer development and progression. Despite its potential threatening, DSBs are intentionally introduced genome-wide specifically in germ cells. Meiotic DSBs are needed to exchange DNA strand between homologous chromosomes, as an initial step of meiotic homologous recombination. The meiotic DSBs are repaired by the invasion of the single-stranded DNA into the homologous DNA catalyzed by the RAD51 and DMC1 recombinases. The strand invasion results in the formation of a joint molecule structure that is ultimately resolved into a crossover.
Recently, we have identified novel proteins termed Meiotic localizer of BRCA2 (MEILB2) and BRCA2 and MEILB2-associating protein 1 (BRME1), which bind to the meiotic DSBs and form complex with RAD51 as well as its mediator protein BRCA2. BRCA2 is a potent tumor suppressor involved in DNA break repair in cancer cells, however, its meiotic functions has been largely elusive. We found that MEILB2 and BRME1 promote the localization of BRCA2, RAD51, and DMC1 at meiotic DSBs and ensures the proper DSB repair in meiosis.
Telomere length defines the proliferative capacity of a cell, thus cancer cells activate either telomerase or the alternative lengthening of telomeres (ALT) pathway to maintain their telomere length. The inhibition of telomerase by anti-cancer drugs tends to lead to the activation of the ALT pathway as a way for the cancer cell to survive, but the detailed mechanisms behind ALT activation remain poorly understood. The nematode Caenorhabditis elegans is the only multicellular organism so far reported that can survive perpetually through the activation of ALT after the deletion of the telomerase gene. The analysis of C. elegans telomeres will provide evolutionarily conserved mechanisms underlying the regulation of telomerase activity and the ALT pathway.
Telomeric double-stranded DNA (dsDNA)-binding proteins in a wide variety of species typically have a single MYB-like DNA binding (MYB) domain at their C-terminus that directly recognizes the telomeric dsDNA in a sequence-specific manner. Deviations from this are found in budding yeast, where telomeric dsDNA is bound by the Rap1 protein with two tandem MYB domains. This deviation can be explained by the atypical telomeric dsDNA sequence (heterogeneous and not GC-rich) in this organism. Another deviation is found in C. elegans, which has a typical telomeric dsDNA (TTAGGC)n, but its genome does not have any TRF-like MYB domain proteins or RAP1-like telomeric proteins.
In a recent study, we identified the two double-strand telomeric DNA-binding proteins (DTN-1 and DTN-2) in C. elegans, which are essential for the maintenance of germline immortality. DTN-1/2 have three tandem MYB domains on its N-termini and directly bind to telomeric dsDNA. Further, we have identified the POT-1-binding regions (PBRs) on DTN-1/2’s C-termini and showed that DTN-1/2 serve the direct molecular linkage between telomeric dsDNA and ssDNA.
Preliminarily, we have found that DTN-1/2 have redundant roles in the suppression of the DNA damage response (DDR), while they play opposing roles in the regulation of telomere length. Through the analysis of these factors using a combination of biochemical and cell biology approaches as well as protein crystallography, we will seek to determine how DDR is repressed and how telomere length is maintained in order to describe the evolutionarily conserved mechanisms underlying the regulation of telomerase activity and the ALT pathway.
 Shibuya H, Hernández-Hernández A, Morimoto A, Lumi Negishi, Höög C, Watanabe Y.
MAJIN links telomeric DNA to the nuclear membrane by exchanging telomere cap.
 Shibuya H, Morimoto A, Watanabe Y.
The Dissection of Meiotic Chromosome Movement in Mice Using an In Vivo Electroporation Technique.
Plos Genetics. 2014
 Shibuya H, Ishiguro K, Watanabe Y.
The TRF1-binding protein TERB1 promotes chromosome movement and telomere rigidity in meiosis.
Nature Cell Biology. 2014
 Morimoto A*, Shibuya H*, Zhu X, Kim J, Ishiguro K, Han M, Watanabe Y (*Equal contributions).
A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis.
The Journal of Cell Biology. 2012
 Zhang J, Tu Z, Watanabe Y, Shibuya H.
Distinct TERB1 domains regulate different protein interactions for meiotic telomere movement.
Cell Reports. 2017
 Zhang J*, Fujiwara Y*, Yamamoto S, Shibuya H. (*co-first authors)
A meiosis-specific BRCA2 binding protein recruits recombinases to DNA double-strand breaks to ensure homologous recombination.
Nature Communications. 2019
 Zhang J*, Gurusaran M*, Fujiwara Y*, Zhang K, Echbarthi M, Vorontsov E, Guo R, Pendlebury D, Alam I, Livera G, Emmanuelle M, Wang P, Nandakumar J, Davies O, Shibuya H. (*co-first authors)
The BRCA2-MEILB2-BRME1 complex governs meiotic recombination and impairs the mitotic BRCA2-RAD51 function in cancer cells.
Nature Communications. 2020
 Yamamoto I, Zhang K, Zhang J, Vorontsov E, Shibuya H.
Telomeric double-strand DNA-binding proteins DTN-1 and DTN-2 ensure germline immortality in Caenorhabditis elegans.