Presynaptic association of Rad51 protein with selected sites in meiotic chromatin.

Eukaryotic homologs of Escherichia coli Rec-A protein have been shown to form nucleoprotein filaments with single-stranded DNA that recognize homologous sequences in duplex DNA. Several recent reports in four widely diverse species have demonstrated the association of RecA homologs with meiotic prophase chromatin. The current immunocytological study on mouse spermatocytes and oocytes shows that a eukaryotic homolog, Rad5l, associates with a subset of chromatin sites as early as premeiotic S phase, hours before either the appearance of precursors of synaptonemal complexes or the initiation of synapsis. When homologous chromosomes do begin to pair, the Rad5l-associated sequences are sites of initial contact between homologues and of localized DNA synthesis. Distribution of Rad5l foci on the chromatin of fully synapsed bivalents at early pachynema corresponds to an R-band pattern of mitotic chromosomes. R-bands are known to be preferred sites of both synaptic initiation and recombination. The time course of appearance of Rad51 association with chromatin, its distribution, and its interaction with other Rad5l-associated sequences suggests that it plays an important role preselection of sequences and synaptic initiation.

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

Regulation of meiotic chromatin loop size by chromosomal position.

At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging.

Phospholipase D signaling is essential for meiosis.

Phospholipid metabolism plays an important role in cellular regulation by generating second messengers for signal transduction. Many stimuli activate a phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine, producing phosphatidic acid and choline. Here we report that the yeast SP014 gene, which is essential for meiosis [Honigberg, S. M., Conicella, C. & Esposito, R. E. (1992) Genetics 130, 703-716], encodes a phospholipase D. SP014 RNA and protein activity are induced during late meiotic prophase, and the enzyme has properties similar to mammalian phosphatidylinositol 4,5-bisphosphate-regulated phospholipase D. Characterization of an unusual allele of SP014 defines regions of the protein important for enzyme catalysis and regulation. These results implicate phospholipase D signaling in regulating cellular differentiation.

UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression.

The UME6 gene of Saccharomyces cerevisiae was identified as a mitotic repressor of early meiosis-specific gene expression. It encodes a Zn2Cys6 DNA-binding protein which binds to URS1, a promoter element needed for both mitotic repression and meiotic induction of early meiotic genes. This paper demonstrates that a complete deletion of UME6 causes not only vegetative derepression of early meiotic genes during vegetative growth but also a significant reduction in induction of meiosis-specific genes, accompanied by a severe defect in meiotic progression. After initiating premeiotic DNA synthesis the vast majority of cells (approximately 85%) become arrested in prophase and fail to execute recombination; a minority of cells (approximately 15%) complete recombination and meiosis I, and half of these form asci. Quantitative analysis of the same early meiotic transcripts that are vegetatively derepressed in the ume6 mutant, SPO11, SPO13, IME2, and SPO1, indicates a low level of induction in meiosis above their vegetative derepressed levels. In addition, the expression of later meiotic transcripts, SPS2 and DIT1, is significantly delayed and reduced. The expression pattern of early meiotic genes in ume6-deleted cells is strikingly similar to that of early meiotic genes with promoter mutations in URS1. These results support the view that UME6 and URS1 are part of a developmental switch that controls both vegetative repression and meiotic induction of meiosis-specific genes.

Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.

Disengaging the Smc3/kleisin interface releases cohesin from Drosophila chromosomes during interphase and mitosis

Cohesin's Smc1, Smc3, and kleisin subunits create a tripartite ring within which sister DNAs are entrapped. Evidence suggests that DNA enters through a gate created by transient dissociation of the Smc1/3 interface. Release at the onset of anaphase is triggered by proteolytic cleavage of kleisin. Less well understood is the mechanism of release at other stages of the cell cycle, in particular during prophase when most cohesin dissociates from chromosome arms in a process dependent on the regulatory subunit Wapl. We show here that Wapl-dependent release from salivary gland polytene chromosomes during interphase and from neuroblast chromosome arms during prophase is blocked by translational fusion of Smc3's C-terminus to kleisin's N-terminus. Our findings imply that proteolysis-independent release of cohesin from chromatin is mediated by Wapl-dependent escape of DNAs through a gate created by transient dissociation of the Smc3/kleisin interface. Thus, cohesin's DNA entry and exit gates are distinct.

Dppa3 is a marker of pluripotency and has a human homologue that is expressed in germ cell tumours

We identified a transcript named 11M2 on the basis of its strong male-specific expression pattern in the developing mouse gonad. 11M2 was found to be expressed by gonad primordial germ cells (PGCs) of both sexes and down-regulated in female PGCs as they enter prophase I of the first meiotic division, similar to the expression of Oct4. Mouse EST analysis revealed expression only in early-stage embryos, embryonic stem cells and pre-meiotic germ cells. 11M2 corresponds to a recently reported gene variously known as PGC7, stella or Dppa3. We have identified the human orthologue of Dppa3 and find by human EST analysis that it is expressed in human testicular germ cell tumours but not in normal human somatic tissues. The expression patterns of mouse and human DPPA3, in undifferentiated embryonic cells, embryonic germ cells and adult germ cell tumours, together suggest a role for this gene in maintaining cell pluripotentiality.