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1.
Proc Natl Acad Sci U S A ; 121(16): e2316651121, 2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38588418

ABSTRACT

Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.


Subject(s)
Caenorhabditis elegans Proteins , Telomere-Binding Proteins , Animals , Telomere-Binding Proteins/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Dimerization , Telomeric Repeat Binding Protein 1/genetics , Telomeric Repeat Binding Protein 1/chemistry , Telomeric Repeat Binding Protein 1/metabolism , Protein Binding , Telomere/genetics , Telomere/metabolism , Shelterin Complex , DNA/metabolism , Telomeric Repeat Binding Protein 2 , Mammals/genetics
2.
Nat Commun ; 15(1): 2315, 2024 Mar 14.
Article in English | MEDLINE | ID: mdl-38485951

ABSTRACT

The cellular membrane in male meiotic germ cells contains a unique class of phospholipids and sphingolipids that is required for male reproduction. Here, we show that a conserved membrane fluidity sensor, AdipoR2, regulates the meiosis-specific lipidome in mouse testes by promoting the synthesis of sphingolipids containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs). AdipoR2 upregulates the expression of a fatty acid elongase, ELOVL2, both transcriptionally and post-transcriptionally, to synthesize VLC-PUFA. The depletion of VLC-PUFAs and subsequent accumulation of palmitic acid in AdipoR2 knockout testes stiffens the cellular membrane and causes the invagination of the nuclear envelope. This condition impairs the nuclear peripheral distribution of meiotic telomeres, leading to errors in homologous synapsis and recombination. Further, the stiffened membrane impairs the formation of intercellular bridges and the germ cell syncytium, which disrupts the orderly arrangement of cell types within the seminiferous tubules. According to our findings we propose a framework in which the highly-fluid membrane microenvironment shaped by AdipoR2-ELOVL2 underpins meiosis-specific chromosome dynamics in testes.


Subject(s)
Membrane Fluidity , Telomere , Animals , Male , Mice , Carrier Proteins/metabolism , Meiosis , Nuclear Envelope/metabolism , Sphingolipids/metabolism , Telomere/genetics , Telomere/metabolism
3.
Mol Cell ; 83(18): 3268-3282.e7, 2023 09 21.
Article in English | MEDLINE | ID: mdl-37689068

ABSTRACT

Heritable non-genetic information can regulate a variety of complex phenotypes. However, what specific non-genetic cues are transmitted from parents to their descendants are poorly understood. Here, we perform metabolic methyl-labeling experiments to track the heritable transmission of methylation from ancestors to their descendants in the nematode Caenorhabditis elegans (C. elegans). We find heritable methylation in DNA, RNA, proteins, and lipids. We find that parental starvation elicits reduced fertility, increased heat stress resistance, and extended longevity in fed, naïve progeny. This intergenerational hormesis is accompanied by a heritable increase in N6'-dimethyl adenosine (m6,2A) on the 18S ribosomal RNA at adenosines 1735 and 1736. We identified DIMT-1/DIMT1 as the m6,2A and BUD-23/BUD23 as the m7G methyltransferases in C. elegans that are both required for intergenerational hormesis, while other rRNA methyltransferases are dispensable. This study labels and tracks heritable non-genetic material across generations and demonstrates the importance of rRNA methylation for regulating epigenetic inheritance.


Subject(s)
Caenorhabditis elegans , Hormesis , Animals , RNA, Ribosomal, 18S , Caenorhabditis elegans/genetics , Methyltransferases/genetics , Adenosine
4.
Nat Commun ; 14(1): 1715, 2023 03 27.
Article in English | MEDLINE | ID: mdl-36973253

ABSTRACT

Spindle formation in male meiosis relies on the canonical centrosome system, which is distinct from acentrosomal oocyte meiosis, but its specific regulatory mechanisms remain unknown. Herein, we report that DYNLRB2 (Dynein light chain roadblock-type-2) is a male meiosis-upregulated dynein light chain that is indispensable for spindle formation in meiosis I. In Dynlrb2 KO mouse testes, meiosis progression is arrested in metaphase I due to the formation of multipolar spindles with fragmented pericentriolar material (PCM). DYNLRB2 inhibits PCM fragmentation through two distinct pathways; suppressing premature centriole disengagement and targeting NuMA (nuclear mitotic apparatus) to spindle poles. The ubiquitously expressed mitotic counterpart, DYNLRB1, has similar roles in mitotic cells and maintains spindle bipolarity by targeting NuMA and suppressing centriole overduplication. Our work demonstrates that two distinct dynein complexes containing DYNLRB1 or DYNLRB2 are separately used in mitotic and meiotic spindle formations, respectively, and that both have NuMA as a common target.


Subject(s)
Dyneins , Spindle Apparatus , Mice , Animals , Male , Dyneins/genetics , Dyneins/metabolism , Spindle Apparatus/metabolism , Centrosome/metabolism , Meiosis , Metaphase
5.
Proc Natl Acad Sci U S A ; 120(8): e2207263120, 2023 02 21.
Article in English | MEDLINE | ID: mdl-36787362

ABSTRACT

Sperm acrosomal membrane proteins, such as Izumo sperm-egg fusion 1 (IZUMO1) and sperm acrosome-associated 6 (SPACA6), play essential roles in mammalian gamete binding or fusion. How their biosynthesis is regulated during spermiogenesis has largely remained elusive. Here, we show that 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik is a type-II transmembrane protein expressed in early round spermatids but not in mature spermatozoa. It interacts with proteins involved in N-linked glycosylation, disulfide isomerization, and endoplasmic reticulum (ER)-Golgi trafficking, suggesting a potential role in nascent protein processing. The ablation of 1700029I15Rik destabilizes non-catalytic subunits of the oligosaccharyltransferase (OST) complex that are pivotal for N-glycosylation. The knockout testes exhibit normal expression of sperm plasma membrane proteins, but decreased abundance of multiple acrosomal membrane proteins involved in fertilization. The knockout sperm show upregulated chaperones related to ER-associated degradation (ERAD) and elevated protein ubiquitination; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway underpinning the biosynthesis of acrosomal membrane proteins during spermiogenesis.


Subject(s)
Acrosome , Membrane Proteins , Animals , Male , Mice , Acrosome/metabolism , Mammals/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Knockout , Semen/metabolism , Seminal Plasma Proteins/metabolism , Sperm-Ovum Interactions , Spermatozoa/metabolism , Ovum/metabolism
6.
Elife ; 112022 06 15.
Article in English | MEDLINE | ID: mdl-35703493

ABSTRACT

Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.


Subject(s)
Dyneins , Microtubule-Associated Proteins , Animals , Chromosome Segregation , Dynactin Complex/metabolism , Dyneins/genetics , Dyneins/metabolism , Male , Meiosis , Mice , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism
7.
Cell Rep ; 38(4): 110289, 2022 01 25.
Article in English | MEDLINE | ID: mdl-35081355

ABSTRACT

The meiosis-specific telomere-binding protein TERB1 anchors telomeres to the nuclear envelope and drives chromosome movements for the pairing of homologous chromosomes. TERB1 has an MYB-like DNA-binding (MYB) domain, which is a hallmark of telomeric DNA-binding proteins. Here, we demonstrate that the TERB1 MYB domain has lost its canonical DNA-binding activity. The analysis of Terb1 point mutant mice expressing TERB1 lacking its MYB domain showed that the MYB domain is dispensable for telomere localization of TERB1 and the downstream TERB2-MAJIN complex, the promotion of homologous pairing, and even fertility. Instead, the TERB1 MYB domain regulates the enrichment of cohesin and promotes the remodeling of axial elements in the early-to-late pachytene transition, which suppresses telomere erosion. Considering its conservation across metazoan phyla, the TERB1 MYB domain is likely to be important for the maintenance of telomeric DNA and thus for genomic integrity by suppressing meiotic telomere erosion over long evolutionary timescales.


Subject(s)
Meiotic Prophase I/physiology , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Animals , Female , Humans , Male , Mice , Mice, Inbred C57BL , Protein Domains
8.
Trends Cell Biol ; 32(4): 281-284, 2022 04.
Article in English | MEDLINE | ID: mdl-34625364

ABSTRACT

Breast cancer type 2 susceptibility protein (BRCA2) is a central regulator of homologous recombination in somatic cells and safeguards genomic integrity against DNA double-strand breaks (DSBs). Recent evidence suggests that association with unique meiosis-specific cofactors allows BRCA2 to facilitate homologous recombination in germ cells.


Subject(s)
Meiosis , Rad51 Recombinase , Animals , BRCA2 Protein/genetics , DNA Breaks, Double-Stranded , DNA Repair , Homologous Recombination , Humans , Mammals/metabolism , Meiosis/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism
9.
Nat Struct Mol Biol ; 28(8): 671-680, 2021 08.
Article in English | MEDLINE | ID: mdl-34373645

ABSTRACT

Meiotic cells invoke breast cancer susceptibility gene 2 (BRCA2) to repair programmed double-stranded DNA breaks and accomplish homologous recombination. The meiosis-specific protein MEILB2 facilitates BRCA2 recruitment to meiotic recombination sites. Here, we combine crystallography, biochemical analysis and a mouse meiosis model to reveal a robust architecture that ensures meiotic BRCA2 recruitment. The crystal structure of the MEILB2-BRCA2 complex reveals how two MEILB2 homodimers sandwich two chains of BRCA2 to afford a 4:2 architecture. The sandwich lacks close contact between the two MEILB2 dimers or the two BRCA2 chains. Instead, the two halves of each BRCA2 chain bridge two MEILB2 subunits from different homodimers to form the MEILB2-BRCA2-MEILB2 sandwich. Several identical residues from the two MEILB2 subunits are employed to engage the BRCA2 halves, justifying their strict conservation. Mutational analysis of the interface reveals a synergistic mechanism for MEILB2-BRCA2 recruitment during meiosis. Overall, these studies demonstrate how BRCA2 efficiently localizes in the cell to facilitate meiosis.


Subject(s)
BRCA2 Protein/metabolism , Cell Cycle Proteins/metabolism , Meiosis/genetics , Recombinational DNA Repair/genetics , Animals , BRCA2 Protein/genetics , Cell Cycle Proteins/genetics , Cell Line , Crystallography, X-Ray , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Multiprotein Complexes/metabolism , Protein Binding/genetics , Protein Structure, Tertiary
10.
Elife ; 102021 01 21.
Article in English | MEDLINE | ID: mdl-33476260

ABSTRACT

Telomeres are nucleoprotein complexes at the ends of chromosomes and are indispensable for the protection and lengthening of terminal DNA. Despite the evolutionarily conserved roles of telomeres, the telomeric double-strand DNA (dsDNA)-binding proteins have evolved rapidly. Here, we identified double-strand telomeric DNA-binding proteins (DTN-1 and DTN-2) in Caenorhabditis elegans as non-canonical telomeric dsDNA-binding proteins. DTN-1 and DTN-2 are paralogous proteins that have three putative MYB-like DNA-binding domains and bind to telomeric dsDNA in a sequence-specific manner. DTN-1 and DTN-2 form complexes with the single-strand telomeric DNA-binding proteins POT-1 and POT-2 and constitutively localize to telomeres. The dtn-1 and dtn-2 genes function redundantly, and their simultaneous deletion results in progressive germline mortality, which accompanies telomere hyper-elongation and chromosomal bridges. Our study suggests that DTN-1 and DTN-2 are core shelterin components in C. elegans telomeres that act as negative regulators of telomere length and are essential for germline immortality.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Germ Cells/metabolism , Telomere-Binding Proteins/genetics , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Telomere/metabolism , Telomere-Binding Proteins/metabolism
11.
PLoS Genet ; 16(9): e1009048, 2020 09.
Article in English | MEDLINE | ID: mdl-32931493

ABSTRACT

During meiotic prophase, sister chromatids are organized into axial element (AE), which underlies the structural framework for the meiotic events such as meiotic recombination and homolog synapsis. HORMA domain-containing proteins (HORMADs) localize along AE and play critical roles in the regulation of those meiotic events. Organization of AE is attributed to two groups of proteins: meiotic cohesins REC8 and RAD21L; and AE components SYCP2 and SYCP3. It has been elusive how these chromosome structural proteins contribute to the chromatin loading of HORMADs prior to AE formation. Here we newly generated Sycp2 null mice and showed that initial chromatin loading of HORMAD1 was mediated by meiotic cohesins prior to AE formation. HORMAD1 interacted not only with the AE components SYCP2 and SYCP3 but also with meiotic cohesins. Notably, HORMAD1 interacted with meiotic cohesins even in Sycp2-KO, and localized along cohesin axial cores independently of the AE components SYCP2 and SYCP3. Hormad1/Rad21L-double knockout (dKO) showed more severe defects in the formation of synaptonemal complex (SC) compared to Hormad1-KO or Rad21L-KO. Intriguingly, Hormad1/Rec8-dKO but not Hormad1/Rad21L-dKO showed precocious separation of sister chromatid axis. These findings suggest that meiotic cohesins REC8 and RAD21L mediate chromatin loading and the mode of action of HORMAD1 for synapsis during early meiotic prophase.


Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Animals , Chromatids/genetics , Chromatids/metabolism , Chromatin/metabolism , Chromosomes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Male , Meiosis/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/genetics , Phosphoproteins/genetics , Prophase/genetics , Spermatocytes/metabolism , Synaptonemal Complex/metabolism , Cohesins
12.
Nat Commun ; 11(1): 2055, 2020 04 28.
Article in English | MEDLINE | ID: mdl-32345962

ABSTRACT

Breast cancer susceptibility gene II (BRCA2) is central in homologous recombination (HR). In meiosis, BRCA2 binds to MEILB2 to localize to DNA double-strand breaks (DSBs). Here, we identify BRCA2 and MEILB2-associating protein 1 (BRME1), which functions as a stabilizer of MEILB2 by binding to an α-helical N-terminus of MEILB2 and preventing MEILB2 self-association. BRCA2 binds to the C-terminus of MEILB2, resulting in the formation of the BRCA2-MEILB2-BRME1 ternary complex. In Brme1 knockout (Brme1-/-) mice, the BRCA2-MEILB2 complex is destabilized, leading to defects in DSB repair, homolog synapsis, and crossover formation. Persistent DSBs in Brme1-/- reactivate the somatic-like DNA-damage response, which repairs DSBs but cannot complement the crossover formation defects. Further, MEILB2-BRME1 is activated in many human cancers, and somatically expressed MEILB2-BRME1 impairs mitotic HR. Thus, the meiotic BRCA2 complex is central in meiotic HR, and its misregulation is implicated in cancer development.


Subject(s)
BRCA2 Protein/metabolism , Homologous Recombination/genetics , Meiosis/genetics , Mitosis/genetics , Multiprotein Complexes/metabolism , Neoplasms/genetics , Rad51 Recombinase/metabolism , Alleles , Animals , Cell Line, Tumor , Chromosome Pairing , DNA Breaks, Double-Stranded , Male , Mice, Inbred C57BL , Protein Binding , Protein Stability , Spermatozoa/metabolism
13.
PLoS Genet ; 15(7): e1008252, 2019 07.
Article in English | MEDLINE | ID: mdl-31283754

ABSTRACT

The biological roles of nucleic acid methylation, other than at the C5-position of cytosines in CpG dinucleotides, are still not well understood. Here, we report genetic evidence for a critical role for the putative DNA demethylase NMAD-1 in regulating meiosis in C. elegans. nmad-1 mutants have reduced fertility. They show defects in prophase I of meiosis, which leads to reduced embryo production and an increased incidence of males due to defective chromosomal segregation. In nmad-1 mutant worms, nuclear staging beginning at the leptotene and zygotene stages is disorganized, the cohesin complex is mislocalized at the diplotene and diakinesis stages, and chromosomes are improperly condensed, fused, or lost by the end of diakinesis. RNA sequencing of the nmad-1 germline revealed reduced induction of DNA replication and DNA damage response genes during meiosis, which was coupled with delayed DNA replication, impaired DNA repair and increased apoptosis of maturing oocytes. To begin to understand how NMAD-1 regulates DNA replication and repair, we used immunoprecipitation and mass spectrometry to identify NMAD-1 binding proteins. NMAD-1 binds to multiple proteins that regulate DNA repair and replication, including topoisomerase TOP-2 and co-localizes with TOP-2 on chromatin. Moreover, the majority of TOP-2 binding to chromatin depends on NMAD-1. These results suggest that NMAD-1 functions at DNA replication sites to regulate DNA replication and repair during meiosis.


Subject(s)
Caenorhabditis elegans Proteins/genetics , DNA Repair , DNA Replication , Dioxygenases/genetics , Oxidoreductases, N-Demethylating/genetics , Animals , Caenorhabditis elegans , Caenorhabditis elegans Proteins/metabolism , Chromosome Segregation , Dioxygenases/metabolism , Male , Meiosis , Mutation , Oxidoreductases, N-Demethylating/metabolism , Sequence Analysis, RNA
14.
BMC Genomics ; 20(1): 445, 2019 Jun 03.
Article in English | MEDLINE | ID: mdl-31159718

ABSTRACT

BACKGROUND: Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. RESULTS: Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9-3.7 ppm) 6mA modifications above background. CONCLUSIONS: Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.


Subject(s)
Adenine/analogs & derivatives , Artifacts , Cytosine/analogs & derivatives , DNA Methylation , DNA/genetics , Eukaryota/genetics , Genome , Adenine/analysis , Adenine/metabolism , Animals , Cells, Cultured , Cytosine/analysis , Cytosine/metabolism , Genomics , Humans , Mice , Myoblasts/cytology , Myoblasts/metabolism
15.
Nat Commun ; 10(1): 986, 2019 02 25.
Article in English | MEDLINE | ID: mdl-30804343

ABSTRACT

The original version of this Article contained errors in Figure 5. In panel g, the male and female symbols preceding each genotype were inadvertently converted to 'B' and '≅', respectively. These errors have been corrected in both the PDF and HTML versions of the Article.

16.
Nat Commun ; 10(1): 722, 2019 02 13.
Article in English | MEDLINE | ID: mdl-30760716

ABSTRACT

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) to maintain genomic integrity. Recombinase recruited to the DSBs by the mediator protein BRCA2 catalyzes the homology-directed repair. During meiotic HR, programmed DSBs are introduced genome-wide but their repair mechanisms, including the regulation of BRCA2, have remained largely elusive. Here we identify a meiotic localizer of BRCA2, MEILB2/HSF2BP, that localizes to the site of meiotic DSBs in mice. Disruption of Meilb2 abolishes the localization of RAD51 and DMC1 recombinases in spermatocytes, leading to errors in DSB repair and male sterility. MEILB2 directly binds to BRCA2 and regulates its association to meiotic DSBs. We map the MEILB2-binding domain within BRCA2 that is distinct from the canonical DNA-binding domain but is sufficient to localize to meiotic DSBs in a MEILB2-dependent manner. We conclude that localization of BRCA2 to meiotic DSBs is mediated by MEILB2, which is an integral mechanism to repair abundant meiotic DSBs.


Subject(s)
BRCA2 Protein/metabolism , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , Homologous Recombination , Meiosis/physiology , Recombinases/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Chromosomal Proteins, Non-Histone/metabolism , DNA Repair , DNA-Binding Proteins/genetics , Female , Gene Knockout Techniques , Male , Mice , Nuclear Proteins/metabolism , Phosphate-Binding Proteins , Rad51 Recombinase/metabolism
17.
Methods Cell Biol ; 145: 269-277, 2018.
Article in English | MEDLINE | ID: mdl-29957208

ABSTRACT

For the analysis of the molecular mechanisms underlying mammalian meiosis, the establishment of a transient gene expression system for meiocytes has been long awaited. We have established an efficient in vivo electroporation method for live mouse testis and demonstrate short-term transgene expression in spermatocytes. By expressing specific marker proteins fused with GFP, this technique is applicable not only to fixed cell observations after transgene expression but also to live imaging to dissect dynamic cellular events in live spermatocytes. The protocol is also adapted to the dissection of mutant phenotypes with defective chromosome movement during meiotic prophase I, as well as a phenotype rescue assay by expressing functional cDNAs in mutant testes.


Subject(s)
Meiosis/physiology , Microscopy/methods , Spermatocytes/physiology , Animals , Electroporation/methods , Male , Meiotic Prophase I/physiology , Mice , Testis/physiology , Transgenes/genetics
19.
Cell Rep ; 21(7): 1715-1726, 2017 Nov 14.
Article in English | MEDLINE | ID: mdl-29141207

ABSTRACT

Meiotic telomeres attach to the nuclear envelope (NE) and drive the chromosome movement required for the pairing of homologous chromosomes. The meiosis-specific telomere proteins TERB1, TERB2, and MAJIN are required to regulate these events, but their assembly processes are largely unknown. Here, we developed a germ-cell-specific knockout mouse of the canonical telomere-binding protein TRF1 and revealed an essential role for TRF1 in directing the assembly of TERB1-TERB2-MAJIN. Further, we identified a TERB2 binding (T2B) domain in TERB1 that is dispensable for the TRF1-TERB1 interaction but is essential for the subsequent TERB1-TERB2 interaction and therefore for telomere attachment to the NE. Meanwhile, cohesin recruitment at telomeres, which is required for efficient telomere movement, is mediated by the MYB-like domain of TERB1, but not by TERB2-MAJIN. Our results reveal distinct protein interactions through various domains of TERB1, which enable the sequential assembly of the meiotic telomere complex for their movements.


Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Meiosis , Telomere/genetics , Animals , Binding Sites , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Nuclear Envelope/metabolism , Protein Binding , Spermatocytes/cytology , Spermatocytes/metabolism , Telomere/metabolism , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Telomeric Repeat Binding Protein 1/genetics , Telomeric Repeat Binding Protein 1/metabolism , Cohesins
20.
Nat Struct Mol Biol ; 24(12): 1064-1072, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29083414

ABSTRACT

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere-INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1-TERB1 interface to reveal the structural basis for telomere-INM linkage. Disruption of this interface abrogates binding and compromises telomere-INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1-TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere-INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere-INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.


Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Meiosis/physiology , Nuclear Envelope/metabolism , Telomere/metabolism , Telomeric Repeat Binding Protein 1/metabolism , Animals , Binding Sites/genetics , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Chromosome Pairing , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Protein Binding/physiology , rap1 GTP-Binding Proteins/metabolism
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