Conditional control of fluorescent protein degradation by an auxin-dependent nanobody.

The conditional and reversible depletion of proteins by auxin-mediated degradation is a powerful tool to investigate protein functions in cells and whole organisms. However, its wider applications require fusing the auxin-inducible degron (AID) to individual target proteins. Thus, establishing the auxin system for multiple proteins can be challenging. Another approach for directed protein degradation are anti-GFP nanobodies, which can be applied to GFP stock collections that are readily available in different experimental models. Here, we combine the advantages of auxin and nanobody-based degradation technologies creating an AID-nanobody to degrade GFP-tagged proteins at different cellular structures in a conditional and reversible manner in human cells. We demonstrate efficient and reversible inactivation of the anaphase promoting complex/cyclosome (APC/C) and thus provide new means to study the functions of this essential ubiquitin E3 ligase. Further, we establish auxin degradation in a vertebrate model organism by employing AID-nanobodies in zebrafish.

Quantitative Cell Cycle Analysis Based on an Endogenous All-in-One Reporter for Cell Tracking and Classification.

Cell cycle kinetics are crucial to cell fate decisions. Although live imaging has provided extensive insights into this relationship at the single-cell level, the limited number of fluorescent markers that can be used in a single experiment has hindered efforts to link the dynamics of individual proteins responsible for decision making directly to cell cycle progression. Here, we present fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA) as an all-in-one cell cycle reporter that allows simultaneous analysis of cell cycle progression, including the transition into quiescence, and the dynamics of individual fate determinants. We also provide an image analysis pipeline for automated segmentation, tracking, and classification of all cell cycle phases. Combining the all-in-one reporter with labeled endogenous cyclin D1 and p21 as prime examples of cell-cycle-regulated fate determinants, we show how cell cycle and quantitative protein dynamics can be simultaneously extracted to gain insights into G1 phase regulation and responses to perturbations.

The enigmatic meiotic dense body and its newly discovered component, SCML1, are dispensable for fertility and gametogenesis in mice.

Meiosis is a critical phase in the life cycle of sexually reproducing organisms. Chromosome numbers are halved during meiosis, which requires meiosis-specific modification of chromosome behaviour. Furthermore, suppression of transposons is particularly important during meiosis to allow the transmission of undamaged genomic information between generations. Correspondingly, specialized genome defence mechanisms and nuclear structures characterize the germ line during meiosis. Survival of mammalian spermatocytes requires that the sex chromosomes form a distinct silenced chromatin domain, called the sex body. An enigmatic spherical DNA-negative structure, called the meiotic dense body, forms in association with the sex body. The dense body contains small non-coding RNAs including microRNAs and PIWI-associated RNAs. These observations gave rise to speculations that the dense body may be involved in sex body formation and or small non-coding RNA functions, e.g. the silencing of transposons. Nevertheless, the function of the dense body has remained mysterious because no protein essential for dense body formation has been reported yet. We discovered that the polycomb-related sex comb on midleg-like 1 (SCML1) is a meiosis-specific protein and is an essential component of the meiotic dense body. Despite abolished dense body formation, Scml1-deficient mice are fertile and proficient in sex body formation, transposon silencing and in timely progression through meiosis and gametogenesis. Thus, we conclude that dense body formation is not an essential component of the gametogenetic program in the mammalian germ line.

Implementation of meiosis prophase I programme requires a conserved retinoid-independent stabilizer of meiotic transcripts.

Sexual reproduction is crucially dependent on meiosis, a conserved, specialized cell division programme that is essential for the production of haploid gametes. Here we demonstrate that fertility and the implementation of the meiotic programme require a previously uncharacterized meiosis-specific protein, MEIOC. Meioc invalidation in mice induces early and pleiotropic meiotic defects in males and females. MEIOC prevents meiotic transcript degradation and interacts with an RNA helicase that binds numerous meiotic mRNAs. Our results indicate that proper engagement into meiosis necessitates the specific stabilization of meiotic transcripts, a previously little-appreciated feature in mammals. Remarkably, the upregulation of MEIOC at the onset of meiosis does not require retinoic acid and STRA8 signalling. Thus, we propose that the complete induction of the meiotic programme requires both retinoic acid-dependent and -independent mechanisms. The latter process involving post-transcriptional regulation likely represents an ancestral mechanism, given that MEIOC homologues are conserved throughout multicellular animals.

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.

Strong artificial selection in domestic mammals did not result in an increased recombination rate.

Recombination rates vary in intensity and location at the species, individual, sex and chromosome levels. Despite the fundamental biological importance of this process, the selective forces that operate to shape recombination rate and patterns are unclear. Domestication offers a unique opportunity to study the interplay between recombination and selection. In domesticates, intense selection for particular traits is imposed on small populations over many generations, resulting in organisms that differ, sometimes dramatically, in morphology and physiology from their wild ancestor. Although earlier studies suggested increased recombination rate in domesticates, a formal comparison of recombination rates between domestic mammals and their wild congeners was missing. In order to determine broad-scale recombination rate, we used immunolabeling detection of MLH1 foci as crossover markers in spermatocytes in three pairs of closely related wild and domestic species (dog and wolf, goat and ibex, and sheep and mouflon). In the three pairs, and contrary to previous suggestions, our data show that contemporary recombination rate is higher in the wild species. Subsequently, we inferred recombination breakpoints in sequence data for 16 genomic regions in dogs and wolves, each containing a locus associated with a dog phenotype potentially under selection during domestication. No difference in the number and distribution of recombination breakpoints was found between dogs and wolves. We conclude that our data indicate that strong directional selection did not result in changes in recombination in domestic mammals, and that both upper and lower bounds for crossover rates may be tightly regulated.

Mouse CCDC79 (TERB1) is a meiosis-specific telomere associated protein.

BACKGROUND: Telomeres have crucial meiosis-specific roles in the orderly reduction of chromosome numbers and in ensuring the integrity of the genome during meiosis. One such role is the attachment of telomeres to trans-nuclear envelope protein complexes that connect telomeres to motor proteins in the cytoplasm. These trans-nuclear envelope connections between telomeres and cytoplasmic motor proteins permit the active movement of telomeres and chromosomes during the first meiotic prophase. Movements of chromosomes/telomeres facilitate the meiotic recombination process, and allow high fidelity pairing of homologous chromosomes. Pairing of homologous chromosomes is a prerequisite for their correct segregation during the first meiotic division. Although inner-nuclear envelope proteins, such as SUN1 and potentially SUN2, are known to bind and recruit meiotic telomeres, these proteins are not meiosis-specific, therefore cannot solely account for telomere-nuclear envelope attachment and/or for other meiosis-specific characteristics of telomeres in mammals. RESULTS: We identify CCDC79, alternatively named TERB1, as a meiosis-specific protein that localizes to telomeres from leptotene to diplotene stages of the first meiotic prophase. CCDC79 and SUN1 associate with telomeres almost concurrently at the onset of prophase, indicating a possible role for CCDC79 in telomere-nuclear envelope interactions and/or telomere movements. Consistent with this scenario, CCDC79 is missing from most telomeres that fail to connect to SUN1 protein in spermatocytes lacking the meiosis-specific cohesin SMC1B. SMC1B-deficient spermatocytes display both reduced efficiency in telomere-nuclear envelope attachment and reduced stability of telomeres specifically during meiotic prophase. Importantly, CCDC79 associates with telomeres in SUN1-deficient spermatocytes, which strongly indicates that localization of CCDC79 to telomeres does not require telomere-nuclear envelope attachment. CONCLUSION: CCDC79 is a meiosis-specific telomere associated protein. Based on our findings we propose that CCDC79 plays a role in meiosis-specific telomere functions. In particular, we favour the possibility that CCDC79 is involved in telomere-nuclear envelope attachment and/or the stabilization of meiotic telomeres. These conclusions are consistent with the findings of an independently initiated study that analysed CCDC79/TERB1 functions.

Meiotic DNA double-strand breaks and chromosome asynapsis in mice are monitored by distinct HORMAD2-independent and -dependent mechanisms.

Meiotic crossover formation involves the repair of programmed DNA double-strand breaks (DSBs) and synaptonemal complex (SC) formation. Completion of these processes must precede the meiotic divisions in order to avoid chromosome abnormalities in gametes. Enduring key questions in meiosis have been how meiotic progression and crossover formation are coordinated, whether inappropriate asynapsis is monitored, and whether asynapsis elicits prophase arrest via mechanisms that are distinct from the surveillance of unrepaired DNA DSBs. We disrupted the meiosis-specific mouse HORMAD2 (Hop1, Rev7, and Mad2 domain 2) protein, which preferentially associates with unsynapsed chromosome axes. We show that HORMAD2 is required for the accumulation of the checkpoint kinase ATR along unsynapsed axes, but not at DNA DSBs or on DNA DSB-associated chromatin loops. Consistent with the hypothesis that ATR activity on chromatin plays important roles in the quality control of meiotic prophase, HORMAD2 is required for the elimination of the asynaptic Spo11(-/-), but not the asynaptic and DSB repair-defective Dmc1(-/-) oocytes. Our observations strongly suggest that HORMAD2-dependent recruitment of ATR to unsynapsed chromosome axes constitutes a mechanism for the surveillance of asynapsis. Thus, we provide convincing evidence for the existence of a distinct asynapsis surveillance mechanism that safeguards the ploidy of the mammalian germline.

Meiotic homologue alignment and its quality surveillance are controlled by mouse HORMAD1.

Meiotic crossover formation between homologous chromosomes (homologues) entails DNA double-strand break (DSB) formation, homology search using DSB ends, and synaptonemal-complex formation coupled with DSB repair. Meiotic progression must be prevented until DSB repair and homologue alignment are completed, to avoid the formation of aneuploid gametes. Here we show that mouse HORMAD1 ensures that sufficient numbers of processed DSBs are available for successful homology search. HORMAD1 is needed for normal synaptonemal-complex formation and for the efficient recruitment of ATR checkpoint kinase activity to unsynapsed chromatin. The latter phenomenon was proposed to be important in meiotic prophase checkpoints in both sexes. Consistent with this hypothesis, HORMAD1 is essential for the elimination of synaptonemal-complex-defective oocytes. Synaptonemal-complex formation results in HORMAD1 depletion from chromosome axes. Thus, we propose that the synaptonemal complex and HORMAD1 are key components of a negative feedback loop that coordinates meiotic progression with homologue alignment: HORMAD1 promotes homologue alignment and synaptonemal-complex formation, and synaptonemal complexes downregulate HORMAD1 function, thereby permitting progression past meiotic prophase checkpoints.

A novel mammalian HORMA domain-containing protein, HORMAD1, preferentially associates with unsynapsed meiotic chromosomes.

HORMA domain-containing proteins regulate interactions between homologous chromosomes (homologs) during meiosis in a wide range of eukaryotes. We have identified a mouse HORMA domain-containing protein, HORMAD1, and biochemically and cytologically shown it to be associated with the meiotic chromosome axis. HORMAD1 first accumulates on the chromosomes during the leptotene to zygotene stages of meiotic prophase I. As germ cells progress into the pachytene stage, HORMAD1 disappears from the synapsed chromosomal regions. However, once the chromosomes desynapse during the diplotene stage, HORMAD1 again accumulates on the chromosome axis of the desynapsed homologs. HORMAD1 thus preferentially localizes to unsynapsed or desynapsed chromosomal regions during the prophase I stage of meiosis. Analysis of mutant strains lacking different components of the synaptonemal complex (SC) revealed that establishment of the SC is required for the displacement of HORMAD1 from the chromosome axis. Our results therefore strongly suggest that also mammalian cells use a HORMA domain-containing protein as part of a surveillance system that monitors synapsis or other interactions between homologs.

Mouse HORMAD1 and HORMAD2, two conserved meiotic chromosomal proteins, are depleted from synapsed chromosome axes with the help of TRIP13 AAA-ATPase.

Meiotic crossovers are produced when programmed double-strand breaks (DSBs) are repaired by recombination from homologous chromosomes (homologues). In a wide variety of organisms, meiotic HORMA-domain proteins are required to direct DSB repair towards homologues. This inter-homologue bias is required for efficient homology search, homologue alignment, and crossover formation. HORMA-domain proteins are also implicated in other processes related to crossover formation, including DSB formation, inhibition of promiscuous formation of the synaptonemal complex (SC), and the meiotic prophase checkpoint that monitors both DSB processing and SCs. We examined the behavior of two previously uncharacterized meiosis-specific mouse HORMA-domain proteins--HORMAD1 and HORMAD2--in wild-type mice and in mutants defective in DSB processing or SC formation. HORMADs are preferentially associated with unsynapsed chromosome axes throughout meiotic prophase. We observe a strong negative correlation between SC formation and presence of HORMADs on axes, and a positive correlation between the presumptive sites of high checkpoint-kinase ATR activity and hyper-accumulation of HORMADs on axes. HORMADs are not depleted from chromosomes in mutants that lack SCs. In contrast, DSB formation and DSB repair are not absolutely required for depletion of HORMADs from synapsed axes. A simple interpretation of these findings is that SC formation directly or indirectly promotes depletion of HORMADs from chromosome axes. We also find that TRIP13 protein is required for reciprocal distribution of HORMADs and the SYCP1/SC-component along chromosome axes. Similarities in mouse and budding yeast meiosis suggest that TRIP13/Pch2 proteins have a conserved role in establishing mutually exclusive HORMAD-rich and synapsed chromatin domains in both mouse and yeast. Taken together, our observations raise the possibility that involvement of meiotic HORMA-domain proteins in the regulation of homologue interactions is conserved in mammals.

Fluorescence activated cell sorting of live female germ cells and somatic cells of the mouse fetal gonad based on forward and side scattering.

Analysis of female mammalian germ cells has been hindered by difficulties in isolating high purity germ cell populations from embryonic and fetal gonads. Meiotic prophase stage oocytes are particularly difficult to isolate due to the lack of suitable surface markers. Oct4 promoter driven GFP expression has been used to distinguish germ cells/oocytes (GFP positive) from somatic cells (GFP negative), however, the requirement for transgenic animals has limited the use of this technique. We analyzed the side- and forward scattering properties of living cell populations obtained from fetal ovaries of Oct4-GFP transgenic and wild-type mice. On the basis of these measurements, we defined criteria that allow the discrimination and identification of germ cells and somatic cells within cell suspensions of nontransgenic female fetal gonads. The described method is suitable for the isolation of populations of germ cells and somatic cells of higher than 90% purity. We also demonstrated that the sorted cells can be used in downstream immunofluorescence and RT-PCR applications. Hence, we conclude that side and forward scattering based sorting of female germ cells is a valuable tool that will benefit the understanding of female gametogenesis.