A system for inducible mitochondria-specific protein degradation in vivo.

Targeted protein degradation systems developed for eukaryotes employ cytoplasmic machineries to perform proteolysis. This has prevented mitochondria-specific analysis of proteins that localize to multiple locations, for example, the mitochondria and the nucleus. Here, we present an inducible mitochondria-specific protein degradation system in Saccharomyces cerevisiae based on the Mesoplasma florum Lon (mf-Lon) protease and its corresponding ssrA tag (called PDT). We show that mitochondrially targeted mf-Lon protease efficiently and selectively degrades a PDT-tagged reporter protein localized to the mitochondrial matrix. The degradation can be induced by depleting adenine from the medium, and tuned by altering the promoter strength of the MF-LON gene. We furthermore demonstrate that mf-Lon specifically degrades endogenous, PDT-tagged mitochondrial proteins. Finally, we show that mf-Lon-dependent PDT degradation can also be achieved in human mitochondria. In summary, this system provides an efficient tool to selectively analyze the mitochondrial function of dually localized proteins.

Age-dependent aneuploidy in mammalian oocytes instigated at the second meiotic division.

Ageing severely affects the chromosome segregation process in human oocytes resulting in aneuploidy, infertility and developmental disorders. A considerable amount of segregation errors in humans are introduced at the second meiotic division. We have here compared the chromosome segregation process in young adult and aged female mice during the second meiotic division. More than half of the oocytes in aged mice displayed chromosome segregation irregularities at anaphase II, resulting in dramatically increased level of aneuploidy in haploid gametes, from 4% in young adult mice to 30% in aged mice. We find that the post-metaphase II process that efficiently corrects aberrant kinetochore-microtubule attachments in oocytes in young adult mice is approximately 10-fold less efficient in aged mice, in particular affecting chromosomes that show small inter-centromere distances at the metaphase II stage in aged mice. Our results reveal that post-metaphase II processes have critical impact on age-dependent aneuploidy in mammalian eggs.

Epigenetic Dysregulation of Mammalian Male Meiosis Caused by Interference of Recombination and Synapsis.

Meiosis involves a series of specific chromosome events, namely homologous synapsis, recombination, and segregation. Disruption of either recombination or synapsis in mammals results in the interruption of meiosis progression during the first meiotic prophase. This is usually accompanied by a defective transcriptional inactivation of the X and Y chromosomes, which triggers a meiosis breakdown in many mutant models. However, epigenetic changes and transcriptional regulation are also expected to affect autosomes. In this work, we studied the dynamics of epigenetic markers related to chromatin silencing, transcriptional regulation, and meiotic sex chromosome inactivation throughout meiosis in knockout mice for genes encoding for recombination proteins SPO11, DMC1, HOP2 and MLH1, and the synaptonemal complex proteins SYCP1 and SYCP3. These models are defective in recombination and/or synapsis and promote apoptosis at different stages of progression. Our results indicate that impairment of recombination and synapsis alter the dynamics and localization pattern of epigenetic marks, as well as the transcriptional regulation of both autosomes and sex chromosomes throughout prophase-I progression. We also observed that the morphological progression of spermatocytes throughout meiosis and the dynamics of epigenetic marks are processes that can be desynchronized upon synapsis or recombination alteration. Moreover, we detected an overlap of early and late epigenetic signatures in most mutants, indicating that the normal epigenetic transitions are disrupted. This can alter the transcriptional shift that occurs in spermatocytes in mid prophase-I and suggest that the epigenetic regulation of sex chromosomes, but also of autosomes, is an important factor in the impairment of meiosis progression in mammals.

Post-metaphase correction of aberrant kinetochore-microtubule attachments in mammalian eggs.

The accuracy of the two sequential meiotic divisions in oocytes is essential for creating a haploid gamete with a normal chromosomal content. Here, we have analysed the 3D dynamics of chromosomes during the second meiotic division in live mouse oocytes. We find that chromosomes form stable kinetochore-microtubule attachments at the end of prometaphase II stage that are retained until anaphase II onset. Remarkably, we observe that more than 20% of the kinetochore-microtubule attachments at the metaphase II stage are merotelic or lateral. However, < 1% of all chromosomes at onset of anaphase II are found to lag at the spindle equator and < 10% of the laggards missegregate and give rise to aneuploid gametes. Our results demonstrate that aberrant kinetochore-microtubule attachments are not corrected at the metaphase stage of the second meiotic division. Thus, the accuracy of the chromosome segregation process in mouse oocytes during meiosis II is ensured by an efficient correction process acting at the anaphase stage.

Sexual dimorphism in the width of the mouse synaptonemal complex.

Sexual dimorphism has been used to describe morphological differences between the sexes, but can be extended to any biologically related process that varies between males and females. The synaptonemal complex (SC) is a tripartite structure that connects homologous chromosomes in meiosis. Here, aided by super-resolution microscopy techniques, we show that the SC is subject to sexual dimorphism, in mouse germ cells. We have identified a significantly narrower SC in oocytes and have established that this difference does not arise from a different organization of the lateral elements nor from a different isoform of transverse filament protein SYCP1. Instead, we provide evidence for the existence of a narrower central element and a different integration site for the C-termini of SYCP1, in females. In addition to these female-specific features, we speculate that post-translation modifications affecting the SYCP1 coiled-coil region could render a more compact conformation, thus contributing to the narrower SC observed in females.

High density of REC8 constrains sister chromatid axes and prevents illegitimate synaptonemal complex formation.

During meiosis, cohesin complexes mediate sister chromatid cohesion (SCC), synaptonemal complex (SC) assembly and synapsis. Here, using super-resolution microscopy, we imaged sister chromatid axes in mouse meiocytes that have normal or reduced levels of cohesin complexes, assessing the relationship between localization of cohesin complexes, SCC and SC formation. We show that REC8 foci are separated from each other by a distance smaller than 15% of the total chromosome axis length in wild-type meiocytes. Reduced levels of cohesin complexes result in a local separation of sister chromatid axial elements (LSAEs), as well as illegitimate SC formation at these sites. REC8 but not RAD21 or RAD21L cohesin complexes flank sites of LSAEs, whereas RAD21 and RAD21L appear predominantly along the separated sister-chromatid axes. Based on these observations and a quantitative distribution analysis of REC8 along sister chromatid axes, we propose that the high density of randomly distributed REC8 cohesin complexes promotes SCC and prevents illegitimate SC formation.

The width of the lateral element of the synaptonemal complex is determined by a multilayered organization of its components.

The synaptonemal complex (SC) is a proteinaceous structure that holds the homologous chromosomes in close proximity while they exchange genetic material in a process known as meiotic recombination. This meiotic recombination leads to genetic variability in sexually reproducing organisms. The ultrastructure of the SC is studied by electron microscopy and it is observed as a tripartite structure. Two lateral elements (LE) separated by a central region (CR) confer its classical tripartite organization. The LEs are the anchoring platform for the replicated homologous chromosomes to properly exchange genetic material with one another. An accurate assembly of the LE is indispensable for the proper completion of meiosis. Ultrastructural studies suggested that the LE is organized as a multilayered unit. However, no validation of this model has been previously provided. In this ultrastructural study, by using mice with different genetic backgrounds that affect the LE width, we provide further evidence that support a multilayered organization of the LE. Additionally, we provide data suggesting additional roles of the different cohesin complex components in the structure of the LEs of the SC.

The central element of the synaptonemal complex in mice is organized as a bilayered junction structure.

The synaptonemal complex transiently stabilizes pairing interactions between homologous chromosomes during meiosis. Assembly of the synaptonemal complex is mediated through integration of opposing transverse filaments into a central element, a process that is poorly understood. We have, here, analyzed the localization of the transverse filament protein SYCP1 and the central element proteins SYCE1, SYCE2 and SYCE3 within the central region of the synaptonemal complex in mouse spermatocytes using immunoelectron microscopy. Distribution of immuno-gold particles in a lateral view of the synaptonemal complex, supported by protein interaction data, suggest that the N-terminal region of SYCP1 and SYCE3 form a joint bilayered central structure, and that SYCE1 and SYCE2 localize in between the two layers. We find that disruption of SYCE2 and TEX12 (a fourth central element protein) localization to the central element abolishes central alignment of the N-terminal region of SYCP1. Thus, our results show that all four central element proteins, in an interdependent manner, contribute to stabilization of opposing N-terminal regions of SYCP1, forming a bilayered transverse-filament-central-element junction structure that promotes synaptonemal complex formation and synapsis.

Bivalent separation into univalents precedes age-related meiosis I errors in oocytes.

The frequency of chromosome segregation errors during meiosis I (MI) in oocytes increases with age. The two-hit model suggests that errors are caused by the combination of a first hit that creates susceptible crossover configurations and a second hit comprising an age-related reduction in chromosome cohesion. This model predicts an age-related increase in univalents, but direct evidence of this phenomenon as a major cause of segregation errors has been lacking. Here, we provide the first live analysis of single chromosomes undergoing segregation errors during MI in the oocytes of naturally aged mice. Chromosome tracking reveals that 80% of the errors are preceded by bivalent separation into univalents. The set of the univalents is biased towards balanced and unbalanced predivision of sister chromatids during MI. Moreover, we find univalents predisposed to predivision in human oocytes. This study defines premature bivalent separation into univalents as the primary defect responsible for age-related aneuploidy.

MEI4 ­ a central player in the regulation of meiotic DNA double-strand break formation in the mouse.

The formation of programmed DNA double-strand breaks (DSBs) at the beginning of meiotic prophase marks the initiation of meiotic recombination. Meiotic DSB formation is catalyzed by SPO11 and their repair takes place on meiotic chromosome axes. The evolutionarily conserved MEI4 protein is required for meiotic DSB formation and is localized on chromosome axes. Here, we show that HORMAD1, one of the meiotic chromosome axis components, is required for MEI4 localization. Importantly, the quantitative correlation between the level of axis-associated MEI4 and DSB formation suggests that axis-associated MEI4 could be a limiting factor for DSB formation. We also show that MEI1, REC8 and RAD21L are important for proper MEI4 localization. These findings on MEI4 dynamics during meiotic prophase suggest that the association of MEI4 to chromosome axes is required for DSB formation, and that the loss of this association upon DSB repair could contribute to turning off meiotic DSB formation.

Merotelic attachments allow alignment and stabilization of chromatids in meiosis II oocytes.

The chromosome segregation process in human oocytes is highly error-prone, generating meiosis II (MII) oocytes with unbalanced chromatids that contribute to aneuploidy in offspring. This raises questions regarding the mechanism for transmission of chromatids and how chromatids evade the error correction mechanisms in MII oocytes. Here, we analyse the behaviour of chromatids in mouse MII oocytes. We find that chromatids align at the spindle equator at the metaphase stage of MII and that their presence does not obstruct entry into the anaphase stage. The alignment process is mediated by merotelic (bi-directional) microtubule-kinetochore attachments, revealing a multi-domain organization of the kinetochore of mammalian meiotic chromosomes. Our results suggest that biorientation of chromatids stabilize microtubule attachments at the kinetochores in a tension-dependent manner. Our results also suggest that merotelic attachments contribute to chromosome mis-segregation in wild-type MII oocytes. Thus, merotely is an important promoter of aneuploidy in mammalian oocytes.

STAG3-mediated stabilization of REC8 cohesin complexes promotes chromosome synapsis during meiosis.

Cohesion between sister chromatids in mitotic and meiotic cells is promoted by a ring-shaped protein structure, the cohesin complex. The cohesin core complex is composed of four subunits, including two structural maintenance of chromosome (SMC) proteins, one α-kleisin protein, and one SA protein. Meiotic cells express both mitotic and meiosis-specific cohesin core subunits, generating cohesin complexes with different subunit composition and possibly separate meiotic functions. Here, we have analyzed the in vivo function of STAG3, a vertebrate meiosis-specific SA protein. Mice with a hypomorphic allele of Stag3, which display a severely reduced level of STAG3, are viable but infertile. We show that meiocytes in homozygous mutant Stag3 mice display chromosome axis compaction, aberrant synapsis, impaired recombination and developmental arrest. We find that the three different α-kleisins present in meiotic cells show different dosage-dependent requirements for STAG3 and that STAG3-REC8 cohesin complexes have a critical role in supporting meiotic chromosome structure and functions.

Human disease locus discovery and mapping to molecular pathways through phylogenetic profiling.

Genes with common profiles of the presence and absence in disparate genomes tend to function in the same pathway. By mapping all human genes into about 1000 clusters of genes with similar patterns of conservation across eukaryotic phylogeny, we determined that sets of genes associated with particular diseases have similar phylogenetic profiles. By focusing on those human phylogenetic gene clusters that significantly overlap some of the thousands of human gene sets defined by their coexpression or annotation to pathways or other molecular attributes, we reveal the evolutionary map that connects molecular pathways and human diseases. The other genes in the phylogenetic clusters enriched for particular known disease genes or molecular pathways identify candidate genes for roles in those same disorders and pathways. Focusing on proteins coevolved with the microphthalmia-associated transcription factor (MITF), we identified the Notch pathway suppressor of hairless (RBP-Jk/SuH) transcription factor, and showed that RBP-Jk functions as an MITF cofactor.

Synaptonemal complex components persist at centromeres and are required for homologous centromere pairing in mouse spermatocytes.

Recent studies in simple model organisms have shown that centromere pairing is important for ensuring high-fidelity meiotic chromosome segregation. However, this process and the mechanisms regulating it in higher eukaryotes are unknown. Here we present the first detailed study of meiotic centromere pairing in mouse spermatogenesis and link it with key events of the G2/metaphase I transition. In mouse we observed no evidence of the persistent coupling of centromeres that has been observed in several model organisms. We do however find that telomeres associate in non-homologous pairs or small groups in B type spermatogonia and pre-leptotene spermatocytes, and this association is disrupted by deletion of the synaptonemal complex component SYCP3. Intriguingly, we found that, in mid prophase, chromosome synapsis is not initiated at centromeres, and centromeric regions are the last to pair in the zygotene-pachytene transition. In late prophase, we first identified the proteins that reside at paired centromeres. We found that components of the central and lateral element and transverse filaments of the synaptonemal complex are retained at paired centromeres after disassembly of the synaptonemal complex along diplotene chromosome arms. The absence of SYCP1 prevents centromere pairing in knockout mouse spermatocytes. The localization dynamics of SYCP1 and SYCP3 suggest that they play different roles in promoting homologous centromere pairing. SYCP1 remains only at paired centromeres coincident with the time at which some kinetochore proteins begin loading at centromeres, consistent with a role in assembly of meiosis-specific kinetochores. After removal of SYCP1 from centromeres, SYCP3 then accumulates at paired centromeres where it may promote bi-orientation of homologous centromeres. We propose that, in addition to their roles as synaptonemal complex components, SYCP1 and SYCP3 act at the centromeres to promote the establishment and/or maintenance of centromere pairing and, by doing so, improve the segregation fidelity of mammalian meiotic chromosomes.

Meiosis in mice without a synaptonemal complex.

The synaptonemal complex (SC) promotes fusion of the homologous chromosomes (synapsis) and crossover recombination events during meiosis. The SC displays an extensive structural conservation between species; however, a few organisms lack SC and execute meiotic process in a SC-independent manner. To clarify the SC function in mammals, we have generated a mutant mouse strain (Sycp1(-/-)Sycp3(-/-), here called SC-null) in which all known SC proteins have been displaced from meiotic chromosomes. While transmission electron microscopy failed to identify any remnants of the SC in SC-null spermatocytes, neither formation of the cohesion axes nor attachment of the chromosomes to the nuclear membrane was perturbed. Furthermore, the meiotic chromosomes in SC-null meiocytes achieved pre-synaptic pairing, underwent early homologous recombination events and sustained a residual crossover formation. In contrast, in SC-null meiocytes synapsis and MLH1-MLH3-dependent crossovers maturation were abolished, whereas the structural integrity of chromosomes was drastically impaired. The variable consequences that SC inactivation has on the meiotic process in different organisms, together with the absence of SC in some unrelated species, imply that the SC could have originated independently in different taxonomic groups.

Age-related meiotic segregation errors in mammalian oocytes are preceded by depletion of cohesin and Sgo2.

BACKGROUND: The growing trend for women to postpone childbearing has resulted in a dramatic increase in the incidence of trisomic pregnancies. Maternal age-related miscarriage and birth defects are predominantly a consequence of chromosome segregation errors during the first meiotic division (MI), which involves the segregation of replicated recombined homologous chromosomes. Despite the importance to human reproductive health, the events precipitating female age-related meiotic errors are poorly understood. RESULTS: Here we use a long-lived wild-type mouse strain to show that the ability to segregate chromosomes synchronously during anaphase of MI declines dramatically during female aging. This is preceded by depletion of chromosome-associated cohesin in association with destabilization of chiasmata, the physical linkages between homologous chromosomes, and loss of the tight association between sister centromeres. Loss of cohesin is not due to an age-related decline in the ability of the spindle checkpoint to delay separase-mediated cleavage of cohesin until entry into anaphase I. However, we find that reduced cohesin is accompanied by depletion of Sgo2, which protects centromeric cohesin during MI. CONCLUSIONS: The data indicate that cohesin declines gradually during the long prophase arrest that precedes MI in female mammals. In aged oocytes, cohesin levels fall below the level required to stabilize chiasmata and to hold sister centromeres tightly together, leading to chromosome missegregation during MI. Cohesin loss may be amplified by a concomitant decline in the levels of the centromeric cohesin protector Sgo2. These findings indicate that cohesin is a key molecular link between female aging and chromosome missegregation during MI.

Synaptonemal complex assembly and H3K4Me3 demethylation determine DIDO3 localization in meiosis.

Synapsis of homologous chromosomes is a key meiotic event, mediated by a large proteinaceous structure termed the synaptonemal complex. Here, we describe a role in meiosis for the murine death-inducer obliterator (Dido) gene. The Dido gene codes for three proteins that recognize trimethylated histone H3 lysine 4 through their amino-terminal plant homeodomain domain. DIDO3, the largest of the three isoforms, localizes to the central region of the synaptonemal complex in germ cells. DIDO3 follows the distribution of the central region protein SYCP1 in Sycp3-/- spermatocytes, which lack the axial elements of the synaptonemal complex. This indicates that synapsis is a requirement for DIDO3 incorporation. Interestingly, DIDO3 is missing from the synaptonemal complex in Atm mutant spermatocytes, which form synapses but show persistent trimethylation of histone H3 lysine 4. In order to further address a role of epigenetic modifications in DIDO3 localization, we made a mutant of the Dido gene that produces a truncated DIDO3 protein. This truncated protein, which lacks the histone-binding domain, is incorporated in the synaptonemal complex irrespective of histone trimethylation status. DIDO3 protein truncation in Dido mutant mice causes mild meiotic defects, visible as gaps in the synaptonemal complex, but allows for normal meiotic progression. Our results indicate that histone H3 lysine 4 demethylation modulates DIDO3 localization in meiosis and suggest epigenetic regulation of the synaptonemal complex.

BRCA1-mediated chromatin silencing is limited to oocytes with a small number of asynapsed chromosomes.

Transcriptional silencing of the sex chromosomes during male meiosis is regarded as a manifestation of a general mechanism active in both male and female germ cells, called meiotic silencing of unsynapsed chromatin (MSUC). MSUC is initiated by the recruitment of the tumor suppressor protein BRCA1 to the axes of unsynapsed chromosomes. We now show that Sycp3, a structural component of the chromosome axis, is required for localization of BRCA1 to unsynapsed pachytene chromosomes. Importantly, we find that oocytes carrying an excess of two to three pairs of asynapsed homologous chromosomes fail to recruit enough BRCA1 to the asynapsed axes to activate MSUC. Furthermore, loss of MSUC function only transiently rescues oocytes from elimination during early postnatal development. The fact that the BRCA1-dependent synapsis surveillance system cannot respond to higher degrees of asynapsis and is dispensable for removal of aberrant oocytes argues that MSUC has a limited input as a quality control mechanism in female germ cells.

Regulation of APC/C activity in oocytes by a Bub1-dependent spindle assembly checkpoint.

BACKGROUND: Missegregation of chromosomes during meiosis in human females causes aneuploidy, including trisomy 21, and is thought also to be the major cause of age-related infertility. Most errors are thought to occur at the first meiotic division. The high frequency of errors raises questions as to whether the surveillance mechanism known as the spindle assembly checkpoint (SAC) that controls the anaphase-promoting complex or cyclosome (APC/C) operates effectively in oocytes. Experimental approaches hitherto used to inactivate the SAC in oocytes suffer from a number of drawbacks. RESULTS: Bub1 protein was depleted specifically in oocytes with a Zp3-Cre transgene to delete exons 7 and 8 from a floxed BUB1(F) allele. Loss of Bub1 greatly accelerates resolution of chiasmata and extrusion of polar bodies. It also causes defective biorientation of bivalents, massive chromosome missegregation at meiosis I, and precocious loss of cohesion between sister centromeres. By using a quantitative assay for APC/C-mediated securin destruction, we show that the APC/C is activated in an exponential fashion, with activity peaking 12-13 hr after GVBD, and that this process is advanced by 5 hr in oocytes lacking Bub1. Importantly, premature chiasmata resolution does not occur in Bub1-deficient oocytes also lacking either the APC/C's Apc2 subunit or separase. Finally, we show that Bub1's kinase domain is not required to delay APC/C activation. CONCLUSIONS: We conclude that far from being absent or ineffective, the SAC largely determines the timing of APC/C and hence separase activation in oocytes, delaying it for about 5 hr.