Alignment Of Homologous Chromosomes Research Articles

Kinesin-7 CENP-E mediates chromosome alignment and spindle assembly checkpoint in meiosis I.

In eukaryotes, meiosis is the genetic basis for sexual reproduction, which is important for chromosome stability and species evolution. The defects in meiosis usually lead to chromosome aneuploidy, reduced gamete number, and genetic diseases, but the pathogenic mechanisms are not well clarified. Kinesin-7 CENP-E is a key regulator in chromosome alignment and spindle assembly checkpoint in cell division. However, the functions and mechanisms of CENP-E in male meiosis remain largely unknown. In this study, we have revealed that the CENP-E gene was highly expressed in the rat testis. CENP-E inhibition influences chromosome alignment and spindle organization in metaphase I spermatocytes. We have found that a portion of misaligned homologous chromosomes is located at the spindle poles after CENP-E inhibition, which further activates the spindle assembly checkpoint during the metaphase-to-anaphase transition in rat spermatocytes. Furthermore, CENP-E depletion leads to abnormal spermatogenesis, reduced sperm count, and abnormal sperm head structure. Our findings have elucidated that CENP-E is essential for homologous chromosome alignment and spindle assembly checkpoint in spermatocytes, which further contribute to chromosome stability and sperm cell quality during spermatogenesis.

Hop2 Interacts with ATF4 to Promote Osteoblast Differentiation

Activating transcription factor 4 (ATF4) is a member of the basic leucine zipper (bZip) transcription factor family required for the terminal differentiation of osteoblasts. Despite its critical importance as one of the three main osteoblast differentiation transcription factors, regulators of osteoblast terminal maturation remain poorly defined. Here we report the identification of homologous pairing protein 2 (Hop2) as a dimerization partner of ATF4 in osteoblasts via the yeast two-hybrid system. Deletional mapping revealed that the Zip domain of Hop2 is necessary and sufficient to bind ATF4 and to enhance ATF4-dependent transcription. Ectopic Hop2 expression in preosteoblasts increased endogenous ATF4 protein content and accelerated osteoblast differentiation. Mice lacking Hop2 (Hop2-/- ) have a normal stature but exhibit an osteopenic phenotype similar to the one observed in Atf4-/- mice, albeit milder, which is associated with decreased Osteocalcin mRNA expression and reduced type I collagen synthesis. Compound heterozygous mice (Atf4+/- :Hop2+/- ) display identical skeletal defects to those found in Hop2-/- mice. These results indicate that Hop2 plays a previous unknown role as a determinant of osteoblast maturation via its regulation of ATF4 transcriptional activity. Our work for the first time reveals a function of Hop2 beyond its role in guiding the alignment of homologous chromosomes. © 2019 American Society for Bone and Mineral Research.

HO Endonuclease-Initiated Recombination in Yeast Meiosis Fails To Promote Homologous Centromere Pairing and Is Not Constrained To Utilize the Dmc1 Recombinase.

Crossover recombination during meiosis is accompanied by a dramatic chromosome reorganization. In Saccharomyces cerevisiae, the onset of meiotic recombination by the Spo11 transesterase leads to stable pairwise associations between previously unassociated homologous centromeres followed by the intimate alignment of homologous axes via synaptonemal complex (SC) assembly. However, the molecular relationship between recombination and global meiotic chromosome reorganization remains poorly understood. In budding yeast, one question is why SC assembly initiates earliest at centromere regions while the DNA double strand breaks (DSBs) that initiate recombination occur genome-wide. We targeted the site-specific HO endonuclease to various positions on S. cerevisiae’s longest chromosome in order to ask whether a meiotic DSB’s proximity to the centromere influences its capacity to promote homologous centromere pairing and SC assembly. We show that repair of an HO-mediated DSB does not promote homologous centromere pairing nor any extent of SC assembly in spo11 meiotic nuclei, regardless of its proximity to the centromere. DSBs induced en masse by phleomycin exposure likewise do not promote homologous centromere pairing nor robust SC assembly. Interestingly, in contrast to Spo11, HO-initiated interhomolog recombination is not affected by loss of the meiotic kinase, Mek1, and is not constrained to use the meiosis-specific Dmc1 recombinase. These results strengthen the previously proposed idea that (at least some) Spo11 DSBs may be specialized in activating mechanisms that both 1) reinforce homologous chromosome alignment via homologous centromere pairing and SC assembly, and 2) establish Dmc1 as the primary strand exchange enzyme.

Transient and Partial Nuclear Lamina Disruption Promotes Chromosome Movement in Early Meiotic Prophase

SummaryMeiotic chromosome movement is important for the pairwise alignment of homologous chromosomes, which is required for correct chromosome segregation. Movement is driven by cytoplasmic forces, transmitted to chromosome ends by nuclear membrane-spanning proteins. In animal cells, lamins form a prominent scaffold at the nuclear periphery, yet the role lamins play in meiotic chromosome movement is unclear. We show that chromosome movement correlates with reduced lamin association with the nuclear rim, which requires lamin phosphorylation at sites analogous to those that open lamina network crosslinks in mitosis. Failure to remodel the lamina results in delayed meiotic entry, altered chromatin organization, unpaired or interlocked chromosomes, and slowed chromosome movement. The remodeling kinases are delivered to lamins via chromosome ends coupled to the nuclear envelope, potentially enabling crosstalk between the lamina and chromosomal events. Thus, opening the lamina network plays a role in modulating contacts between chromosomes and the nuclear periphery during meiosis.

Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein.

Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during meiotic recombination.

Meiotic cohesin-based chromosome structure is essential for homologous chromosome pairing in Schizosaccharomyces pombe.

Chromosome structure is dramatically altered upon entering meiosis to establish chromosomal architectures necessary for the successful progression of meiosis-specific events. An early meiotic event involves the replacement of the non-SMC mitotic cohesins with their meiotic equivalents in most part of the chromosome, forming an axis on meiotic chromosomes. We previously demonstrated that the meiotic cohesin complex is required for chromosome compaction during meiotic prophase in the fission yeast Schizosaccharomyces pombe. These studies revealed that chromosomes are elongated in the absence of the meiotic cohesin subunit Rec8 and shortened in the absence of the cohesin-associated protein Pds5. In this study, using super-resolution structured illumination microscopy, we found that Rec8 forms a linear axis on chromosomes, which is required for the organized axial structure of chromatin during meiotic prophase. In the absence of Pds5, the Rec8 axis is shortened whereas chromosomes are widened. In rec8 or pds5 mutants, the frequency of homologous chromosome pairing is reduced. Thus, Rec8 and Pds5 play an essential role in building a platform to support the chromosome architecture necessary for the spatial alignment of homologous chromosomes.

Structural insight into the central element assembly of the synaptonemal complex.

The key step in meiosis is synaptonemal complex formation, which mediates homologous chromosome alignment and synapsis. False pairing between homologous chromosomes produces infertility. Here, we present a crystal structure of the mouse meiosis-specific protein SYCE3, which is a component of the synaptonemal complex central element. Our studies show that functional SYCE3 most likely forms a dimer or higher order oligomer in cells. Furthermore, we demonstrate that the SYCE3 N-helix interacts with the SYCE1 C-helix, which is another central element component. Our results suggest that helical packing may mediate intra- or inter-association of each central element protein component, thereby playing an essential role in forming the synaptonemal complex central elements.

SUMO Localizes to the Central Element of Synaptonemal Complex and Is Required for the Full Synapsis of Meiotic Chromosomes in Budding Yeast

The synaptonemal complex (SC) is a widely conserved structure that mediates the intimate alignment of homologous chromosomes during meiotic prophase and is required for proper homolog segregation at meiosis I. However, fundamental details of SC architecture and assembly remain poorly understood. The coiled-coil protein, Zip1, is the only component whose arrangement within the mature SC of budding yeast has been extensively characterized. It has been proposed that the Small Ubiquitin-like MOdifier, SUMO, plays a role in SC assembly by linking chromosome axes with Zip1's C termini. The role of SUMO in SC structure has not been directly tested, however, because cells lacking SUMO are inviable. Here, we provide direct evidence for SUMO's function in SC assembly. A meiotic smt3 reduction-of-function strain displays reduced sporulation, abnormal levels of crossover recombination, and diminished SC assembly. SC structures are nearly absent when induced at later meiotic time points in the smt3 reduction-of-function background. Using Structured Illumination Microscopy we furthermore determine the position of SUMO within budding yeast SC structure. In contrast to previous models that positioned SUMO near Zip1's C termini, we demonstrate that SUMO lies at the midline of SC central region proximal to Zip1's N termini, within a subdomain called the “central element”. The recently identified SUMOylated SC component, Ecm11, also localizes to the SC central element. Finally, we show that SUMO, Ecm11, and even unSUMOylatable Ecm11 exhibit Zip1-like ongoing incorporation into previously established SCs during meiotic prophase and that the relative abundance of SUMO and Ecm11 correlates with Zip1's abundance within SCs of varying Zip1 content. We discuss a model in which central element proteins are core building blocks that stabilize the architecture of SC near Zip1's N termini, and where SUMOylation may occur subsequent to the incorporation of components like Ecm11 into an SC precursor structure.

A Mec1- and PP4-Dependent Checkpoint Couples Centromere Pairing to Meiotic Recombination

The faithful alignment of homologous chromosomes during meiotic prophase requires the coordination of DNA double-strand break (DSB) repair with large-scale chromosome reorganization. Here we identify the phosphatase PP4 (Pph3/Psy2) as a mediator of this process in Saccharomyces cerevisiae. In pp4 mutants, early stages of crossover repair and homology-independent pairing of centromeres are coordinately blocked. We traced the loss of centromere pairing to the persistent phosphorylation of the chromosomal protein Zip1 on serine 75. Zip1-S75 is a consensus site for the ATR-like checkpoint kinase Mec1, and centromere pairing is restored in mec1 mutants. Importantly, Zip1-S75 phosphorylation does not alter chromosome synapsis or DSB repair, indicating that Mec1 separates centromere pairing from the other functions of Zip1. The centromeric localization and persistent activity of PP4 during meiotic prophase suggest a model whereby Zip1-S75 phosphorylation dynamically destabilizes homology-independent centromere pairing in response to recombination initiation, thereby coupling meiotic chromosome dynamics to DSB repair.

From meiosis to postmeiotic events: Alignment and recognition of homologous chromosomes in meiosis

Recombination of homologous chromosomes is essential for correct reductional segregation of homologous chromosomes, which characterizes meiosis. To accomplish homologous recombination, chromosomes must find their homologous partners and pair with them within the spatial constraints of the nucleus. Although various mechanisms have developed in different organisms, two major steps are involved in the process of pairing: first, alignment of homologous chromosomes to bring them close to each other for recognition; and second, recognition of the homologous partner of each chromosome so that they can form an intimate pair. Here, we discuss the various mechanisms used for alignment and recognition of homologous chromosomes in meiosis.

Mechanics of meiosis.

The present paper reviews our observations on the premeiotic DNA replication in Lilium and on the morphology and behaviour of chromosomes during the meiotic prophase in Schizophyllum, Lilium, Drosophila, Bombyx and Homo as revealed by three-dimensional reconstructions of synaptonemal complexes (SC). The main results and conclusions are the following: (1) Premeiotic DNA replication in Lilium is 6 times longer than the mitotic S-phase. (2) Presynaptic alignment of homologous chromosomes was not observed. (3) Initiation of synapsis was in Bombyx and Homo restricted to telomere regions while in Lilium complex formation was generally initiated interstitially. (4) Interlocking of chromosomes was frequent at zygotene. (5) Direct evidence for resolution of interlockings by breakage and reunion of chromosomes was obtained in Homo and Bombyx. (6) Two phases of chromosome pairing and SC formation were revealed in Bombyx. (7) A karyotype based on SC length, centromere index and chromatin markers was constructed for human spermatocytes. (8) The number and distribution of recombination nodules were in agreement with the hypothesis that these structures are involved in crossing over and/or gene conversion. (9) Fragments of the SC were found in prometaphase I bivalents of human spermatocytes corresponding in number to the number of chiasmata. (10) In the absence of crossing over, compensatory mechanisms have evolved in female Bombyx substituting for chiasmata in the retention of homologues.

SUN-domain and KASH-domain proteins during development, meiosis and disease.

SUN-domain proteins interact directly with KASH-domain proteins to form protein complexes that connect the nucleus to every major cytoskeleton network. SUN-KASH protein complexes are also required for attaching centrosomes to the nuclear periphery and for alignment of homologous chromosomes, their pairing and recombination in meiosis. Other functions that require SUN-domain proteins include the regulation of apoptosis and maturation and survival of the germline. Laminopathic diseases affect the distribution of the SUN-KASH complexes, and mutations in KASH-domain proteins can cause Emery Dreifuss muscular dystrophy and recessive cerebellar ataxia. This review describes our current knowledge of the role of SUN-KASH domain protein complexes during development, meiosis and disease.

GENETICS OF MEIOTIC PROPHASE I IN PLANTS

During meiotic prophase I, traits are reassorted as a result of a highly organized process involving sister chromatid cohesion, homologous chromosome alignment, pairing, synapsis, and recombination. In the past two years, a number of components involved in this pathway, including Structure Maintenance of Chromosomes (SMC), MRE11, the RAD51 homologs, BRCA2, MSH4, MER3, and ZIP1, have been characterized in plants; in addition, several genes that encode components unique to plants, such as POOR HOMOLOGOUS SYNAPSIS 1 and AMEIOTIC 1, have been cloned. Based on these recent data, essentially from maize and Arabidopsis, we discuss the conserved and plant-specific aspects of meiosis commitment and meiotic prophase I features.

Stimulation of DNA Strand Exchange by the Human TBPIP/Hop2-Mnd1 Complex

In Saccharomyces cerevisiae, the Hop2 protein forms a complex with the Mnd1 protein and is required for the alignment of homologous chromosomes during meiosis, probably through extensive homology matching between them. The Rad51 and Dmc1 proteins, the eukaryotic RecA orthologs, promote strand exchange and may function in the extensive matching of homology within paired DNA molecules. In the present study, we purified the human TBPIP/Hop2-Mnd1 complex and found that it significantly stimulates the Dmc1- and Rad51-mediated strand exchange. The human Hop2-Mnd1 complex preferentially binds to a three-stranded DNA branch, which mimics the strand-exchange intermediate. These findings are consistent with genetic data, which showed that the Hop2 and Mnd1 proteins are required for homology matching between homologous chromosomes. Therefore, the human TBPIP/Hop2-Mnd1 complex may ensure proper pairing between homologous chromosomes through its stimulation of strand exchange during meiosis.

Use of Two-Color Fluorescence-Tagged Transgenes to Study Interphase Chromosomes in Living Plants

Sixteen distinct sites distributed on all five Arabidopsis (Arabidopsis thaliana) chromosomes have been tagged using different fluorescent proteins and one of two different bacterial operator-repressor systems: (1) a yellow fluorescent protein-Tet repressor fusion protein bound to tet operator sequences, or (2) a green or red fluorescent protein-Lac repressor fusion protein bound to lac operator sequences. Individual homozygous lines and progeny of intercrosses between lines have been used to study various aspects of interphase chromosome organization in root cells of living, untreated seedlings. Features reported here include distances between transgene alleles, distances between transgene inserts on different chromosomes, distances between transgene inserts on the same chromatin fiber, alignment of homologous chromosomes, and chromatin movement. The overall findings are consistent with a random and largely static arrangement of interphase chromosomes in nuclei of root cells. These transgenic lines provide tools for in-depth analyses of interphase chromosome organization, expression, and dynamics in living plants.

Exploring meiotic division in Cargèse

The EMBO workshop on ‘Meiotic Divisions and Checkpoints’ was organized by C. Jessus, M.H. Verlhac, T. Hunt and A. Nebreda and took place in Cargese, France, between 16 and 20 March 2005. ![][1] Meiosis is a special type of cell division through which haploid gametes are generated from diploid parent cells. This is accomplished by a first division that induces homologous chromosome segregation followed by a second mitosis‐like division that promotes sister chromatid separation. During the prophase of meiosis I, three basic events take place: pairing of homologous chromosomes, synapsis and recombination. Pairing or alignment of homologous chromosomes allows chromosome stabilization by the formation of the synaptonemal complex, whereas synapsis facilitates the subsequent recombination events. The synaptonemal complex is a proteinaceous structure formed by lateral elements and transverse filaments (Fig 1; Page & Hawley, 2004). The lateral elements comprises cohesins (Rec8/C(2)M/SYN1, STAG3/Rec11, SMC1‐b and SMC3), the structural proteins SCP2 and SCP3, and the HORMA‐domain proteins Hop1/HIM3/Asy1 and Red1; the transverse filaments are formed by the proteins Zip1, SCP1, C(3)G and SYP1. Once the synaptonemal complex is assembled, crossover recombination events take place between homologous partners. Crossovers form temporary connections or chiasmata that hold homologues together and allow their correct attachment to the meiosis I spindle. However, in most vertebrate oocytes, spindle I formation and germinal vesicle breakdown (GVBD) is preceded by an arrest at prophase I. This arrest is maintained until progesterone stimulates re‐entry into the meiotic cycle by inducing the polyadenylation and translation of several dormant mRNAs that encode proteins such as Mos or cyclin B. Translation of Mos and cyclin B activates the maturation‐promoting factor (MPF) and thereby induces spindle formation and GVBD. Homologous chromosomes then attach to the spindle and when aligned correctly along the metaphase plate, they segregate—a process that in some species is regulated, at … [1]: /embed/graphic-1.gif

Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.)

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST-RFLP loci in the F(1)(NA(6) x AU(6)) population. A comprehensive set of EST-SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA(6) genetic map contains 88 EST-RFLP and 71 EST-SSR loci with a total map length of 963 cM, while the AU(6) genetic map contains 67 EST-RFLP and 58 EST-SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

255.Dnmt3L: a coordinator of epigenetic modifications during spermatogenesis

Spermatogenesis is a process with unique epigenetic requirements. The differentiation from diploid spermatogonia to haploid spermatozoa requires regulation of genomic imprint establishment, stage specific gene expression, meiotic division, and the histone-protamine transition. The methyltransferase regulator, Dnmt3L, is expressed during gametogenesis and is necessary for establishment of maternal methylation imprints in the oocyte. Targeted disruption of Dnmt3L does not appear to affect oogenesis, as mature oocytes are generated, however resultant heterozygous progeny die mid gestation due to biallelic expression of imprinted genes. Dnmt3L–/– males however show spermatogenic arrest. We found that this arrest occurs during prophase I of meiosis, with spermatocytes lost by both apoptosis and germ cell sloughing. A progressive degeneration ensues, resulting in a Sertoli cell phenotype. Electron microscopy of meiotic spermatocytes revealed that homologous chromosomes fail to align and form synaptonemal complexes. Furthermore, Dnmt3L–/– spermatocytes show abnormal methylation on paternally imprinted genes and abnormal global retention of histone acetylation, implicating Dnmt3L in histone deacetylase recruitment. Thus, during spermatogenesis, Dnmt3L is crucial for two distinct epigenetic modifications; imprint establishment and global histone deacetylation prior to homologous chromosome alignment. The latter defect is likely to affect the alignment of homologous chromosomes and trigger the pachytene checkpoint leading to spermatocyte death. Since Dnmt3L has no DNA methlytransferase or HDAC activity itself, we propose that Dnmt3L is essential for the coordination of epigenetic layers, at least during spermatogenesis.

The Importance of Genetic Recombination for Fidelity of Chromosome Pairing in Meiosis

In budding yeast, absence of the Hop2 protein leads to extensive synaptonemal complex (SC) formation between nonhomologous chromosomes, suggesting a crucial role for Hop2 in the proper alignment of homologous chromosomes during meiotic prophase. Genetic analysis indicates that Hop2 acts in the same pathway as the Rad51 and Dmc1 proteins, two homologs of E. coli RecA. Thus, the hop2 mutant phenotype demonstrates the importance of the recombination machinery in promoting accurate chromosome pairing. We propose that the Dmc1/Rad51 recombinases require Hop2 to distinguish homologous from nonhomologous sequences during the homology search process. Thus, when Hop2 is absent, interactions between nonhomologous sequences become inappropriately stabilized and can initiate SC formation. Overexpression of RAD51 largely suppresses the meiotic defects of the dmc1 and hop2 mutants. We conclude that Rad51 is capable of carrying out a homology search independently, whereas Dmc1 requires additional factors such as Hop2.

Synapsis-dependent and -independent mechanisms stabilize homolog pairing during meiotic prophase in C. elegans.

Analysis of Caenorhabditis elegans syp-1 mutants reveals that both synapsis-dependent and -independent mechanisms contribute to stable, productive alignment of homologous chromosomes during meiotic prophase. Early prophase nuclei undergo normal reorganization in syp-1 mutants, and chromosomes initially pair. However, the polarized nuclear organization characteristic of early prophase persists for a prolonged period, and homologs dissociate prematurely; furthermore, the synaptonemal complex (SC) is absent. The predicted structure of SYP-1, its localization at the interface between intimately paired, lengthwise-aligned pachytene homologs, and its kinetics of localization with chromosomes indicate that SYP-1 is an SC structural component. A severe reduction in crossing over together with evidence for accumulated recombination intermediates in syp-1 mutants indicate that initial pairing is not sufficient for completion of exchange and implicates the SC in promoting crossover recombination. Persistence of polarized nuclear organization in syp-1 mutants suggests that SC polymerization may provide a motive force or signal that drives redispersal of chromosomes. Whereas our analysis suggests that the SC is required to stabilize pairing along the entire lengths of chromosomes, striking differences in peak pairing levels for opposite ends of chromosomes in syp-1 mutants reveal the existence of an additional mechanism that can promote local stabilization of pairing, independent of synapsis.