The THO complex is required for nucleolar integrity in Drosophila spermatocytes

The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. Here, we isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, our study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes.     

A novel eIF4G homolog, Off-schedule, couples translational control to meiosis and differentiation in Drosophila spermatocytes

During spermatogenesis, cells coordinate differentiation with the meiotic cell cycle to generate functional gametes. We identified a novel gene, which we named off-schedule (ofs), as being essential for this coordinated control. During the meiotic G(2) phase, Drosophila ofs mutant germ cells do not reach their proper size and fail to execute meiosis or significant differentiation. The accumulation of four cell cycle regulators--Cyclin A, Boule, Twine and Roughex--is altered in these mutants, indicating that ofs reveals a novel branch of the pathway controlling meiosis and differentiation. Ofs is homologous to eukaryotic translation initiation factor eIF4G. The level of ofs expression in spermatocytes is much higher than for the known eIF4G ortholog (known as eIF-4G or eIF4G), suggesting that Ofs substitutes for this protein. Consistent with this, assays for association with mRNA cap complexes, as well as RNA-interference and phenotypic-rescue experiments, demonstrate that Ofs has eIF4G activity. Based on these studies, we speculate that spermatocytes monitor G(2) growth as one means to coordinate the initiation of meiotic division and differentiation.     

Novel response to microtubule perturbation in meiosis

During the mitotic cell cycle, microtubule depolymerization leads to a cell cycle arrest in metaphase, due to activation of the spindle checkpoint. Here, we show that under microtubule-destabilizing conditions, such as low temperature or the presence of the spindle-depolymerizing drug benomyl, meiotic budding yeast cells arrest in G(1) or G(2), instead of metaphase. Cells arrest in G(1) if microtubule perturbation occurs as they enter the meiotic cell cycle and in G(2) if cells are already undergoing premeiotic S phase. Concomitantly, cells down-regulate genes required for cell cycle progression, meiotic differentiation, and spore formation in a highly coordinated manner. Decreased expression of these genes is likely to be responsible for halting both cell cycle progression and meiotic development. Our results point towards the existence of a novel surveillance mechanism of microtubule integrity that may be particularly important during specialized cell cycles when coordination of cell cycle progression with a developmental program is necessary.     

Male-specific IDGF, a novel gene encoding a membrane-bound extracellular signaling molecule expressed exclusively in testis of Drosophila melanogaster

We identified a novel gene of Drosophila melanogaster, Male-specific IDGF (MSI), encoding a transmembrane signaling molecule with exclusive expression in the testis. This molecule (MSI) contains a single transmembrane domain and has 35% amino acid identity with insect-derived growth factor (IDGF), a soluble growth factor for embryonic cells of the flesh fly, Sarcophaga peregrina. When MSI was exogenously expressed in Schneiders's line 2 cells, it was shown to be localized on the cell surface and exhibits growth factor activity, suggesting that MSI is a membrane-bound extracellular signaling molecule. Gene expression studies revealed that MSI mRNA was restricted to mature primary spermatocytes, whereas MSI was detected in the cells at the later developmental stages. Analysis using four meiotic arrest mutants, aly, can, mia, and sa suggested that MSI is involved in spermiogenesis, the final differentiation step of spermatogenesis. These results suggest that MSI is an extracellular signaling molecule participating in spermatogenesis and is a new member of the IDGF family.     

asunder Is a Critical Regulator of Dynein–Dynactin Localization during Drosophila Spermatogenesis

Spermatogenesis uses mitotic and meiotic cell cycles coordinated with growth and differentiation programs to generate functional sperm. Our analysis of a Drosophila mutant has revealed that asunder (asun), which encodes a conserved protein, is an essential regulator of spermatogenesis. asun spermatocytes arrest during prophase of meiosis I. Strikingly, arrested spermatocytes contain free centrosomes that fail to stably associate with the nucleus. Spermatocytes that overcome arrest exhibit severe defects in meiotic spindle assembly, chromosome segregation, and cytokinesis. Furthermore, the centriole-derived basal body is detached from the nucleus in asun postmeiotic spermatids, resulting in abnormalities later in spermatogenesis. We find that asun spermatocytes and spermatids exhibit drastic reduction of perinuclear dynein–dynactin, a microtubule motor complex. We propose a model in which asun coordinates spermatogenesis by promoting dynein–dynactin recruitment to the nuclear surface, a poorly understood process required for nucleus–centrosome coupling at M phase entry and fidelity of meiotic divisions.     

Mutation of Zebrafish caf-1b Results in S Phase Arrest,Defective Differentiation,and p53-Mediated Apoptosis During Organogenesis

The cell cycle of multicellular organisms must be tightly coordinated with organogenesis and differentiation. Experiments done in vitro have identified chromatin assembly factor 1 (CAF-1) as a protein complex promoting chromatin assembly during DNA replication, but the in vivo role of CAF-1 in multicellular animals is still poorly understood. Here we describe the characterization of a zebrafish mutant disrupting CAF-1b activity, and show that it leads to defective cell cycle progression and differentiation in several organs, including the retina, optic tectum, pectoral fins, and head skeleton. Retinal precursor cells mutant for caf-1b arrest in S phase and undergo p53-mediated apoptosis. While p53 deficiency is able to rescue apoptosis in caf-1b mutants, it fails to rescue differentiation, indicating that CAF-1 activity is essential for differentiation in these organs. In addition, we also show that regulation of caf-1b expression in the retina depends on a group of genes that regulate the switch from proliferation to differentiation.     

A proposed role for the Polycomb group protein dRING in meiotic sister-chromatid cohesion

ORD protein is required for accurate chromosome segregation during male and female meiosis in Drosophila melanogaster. Null ord mutations result in random segregation of sister chromatids during both meiotic divisions because cohesion is completely abolished prior to kinetochore capture of microtubules during meiosis I. Previous analyses of mutant ord alleles have led us to propose that the C-terminal half of the ORD protein mediates protein-protein interactions that are essential for sister-chromatid cohesion. To identify proteins that interact with ORD, we conducted a yeast two-hybrid screen using an ORD bait and isolated dRING, a core subunit of the Drosophila Polycomb repressive complex 1. We show that a missense mutation in ORD completely ablates the two-hybrid interaction with dRING and prevents nuclear retention of the mutant ORD protein in male meiotic cells. Using affinity-purified antibodies generated against full-length recombinant dRING, we demonstrate that dRING protein is expressed in the male and female gonads and colocalizes extensively with ORD on the chromatin of primary spermatocytes during G2 of meiosis. Our results suggest a novel role for the Polycomb group protein dRING and are consistent with the model that interaction of dRING and ORD is required to promote the proper segregation of meiotic chromosomes.Communicated by R. Paro     

The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility

During meiosis, the homologous chromosomes pair and recombine. An evolutionarily conserved protein structure, the synaptonemal complex (SC), is located along the paired meiotic chromosomes. We have studied the function of a structural component in the axial/lateral element of the SC, the synaptonemal complex protein 3 (SCP3). A null mutation in the SCP3 gene was generated, and we noted that homozygous mutant males were sterile due to massive apoptotic cell death during meiotic prophase. The SCP3-deficient male mice failed to form axial/lateral elements and SCs, and the chromosomes in the mutant spermatocytes did not synapse. While the absence of SCP3 affected the nuclear distribution of DNA repair and recombination proteins (Rad51 and RPA), as well as synaptonemal complex protein 1 (SCP1), a residual chromatin organization remained in the mutant meiotic cells.     

Mutational Analyses of Fs(1)ya, an Essential, Developmentally Regulated, Nuclear Envelope Protein in Drosophila

The fs(1)Ya protein (YA) is an essential, maternally encoded, nuclear lamina protein that is under both developmental and cell cycle control. A strong Ya mutation results in early arrest of embryos. To define the function of YA in the nuclear envelope during early embryonic development, we characterized the phenotypes of four Ya mutant alleles and determined their molecular lesions. Ya mutant embryos arrest with abnormal nuclear envelopes prior to the first mitotic division; a proportion of embryos from two leaky Ya mutants proceed beyond this but arrest after several abnormal divisions. Ya unfertilized eggs contain nuclei of different sizes and condensation states, apparently due to abnormal fusion of the meiotic products immediately after meiosis. Lamin is localized at the periphery of the uncondensed nuclei in these eggs. These results suggest that YA function is required during and after egg maturation to facilitate proper chromatin condensation, rather than to allow a lamin-containing nuclear envelope to form. Two leaky Ya alleles that partially complement have lesions at opposite ends of the YA protein, suggesting that the N- and C-termini are important for YA function and that YA might interact with itself either directly or indirectly.     

The DUET gene is necessary for chromosome organization and progression during male meiosis in Arabidopsis and encodes a PHD finger protein

Progression through the meiotic cell cycle is an essential part of the developmental program of sporogenesis in plants. The duet mutant of Arabidopsis was identified as a male sterile mutant that lacked pollen and underwent an aberrant male meiosis. Male meiocyte division resulted in the formation of two cells instead of a normal tetrad. In wild type, male meiosis extends across two successive bud positions in an inflorescence whereas in duet, meiotic stages covered three to five bud positions indicating defective progression. Normal microspores were absent in the mutant and the products of the aberrant meiosis were uni- to tri-nucleate cells that later degenerated, resulting in anthers containing largely empty locules. Defects in male meiotic chromosome organization were observed starting from diplotene and extending to subsequent stages of meiosis. There was an accumulation of meiotic structures at metaphase 1, suggesting an arrest in cell cycle progression. Double mutant analysis revealed interaction with dyad, a mutation causing chromosome cohesion during female meiosis. Cloning and molecular analysis of DUET indicated that it potentially encodes a PHD-finger protein and shows specific expression in male meiocytes. Taken together these data suggest that DUET is required for male meiotic chromosome organization and progression.     

Retinoblastoma tumor suppressor protein-dependent methylation of histone H3 lysine 27 is associated with irreversible cell cycle exit

The retinoblastoma tumor suppressor protein (pRb) is involved in mitotic exit, promoting the arrest of myoblasts, and myogenic differentiation. However, it is unclear how permanent cell cycle exit is maintained in differentiated muscle. Using RNA interference, expression profiling, and chromatin immunoprecipitations, we show that pRb is essential for cell cycle exit and the differentiation of myoblasts and is also uniquely required to maintain this arrest in myotubes. Remarkably, we also uncover a function for the pRb-related proteins p107 and p130 as enforcers of a G2/M phase checkpoint that prevents progression into mitosis in cells that have lost pRb. We further demonstrate that pRb effects permanent cell cycle exit in part by maintaining trimethylation of histone H3 lysine 27 (H3K27) on cell cycle genes. H3K27 trimethylation silences other genes, including Cyclin D1, in a pRb-independent but polycomb-dependent manner. Thus, our data distinguish two distinct chromatin-based regulatory mechanisms that lead to terminal differentiation.