Saito TT et al. (2014) Worm "Crossover recombination mediated by HIM-18/SLX4-associated nucleases."

Saito TT, Colaiacovo MP

[Worm, 2014]

Meiosis is a specialized cell division program that results in the formation of haploid gametes (i.e., sperm and eggs) from diploid parental cells, and is essential for all sexually reproducing organisms. Crossover formation, the reciprocal exchange of genetic information during recombination, is critical for accurate meiotic chromosome segregation. Misregulation of crossover formation leads to genomic instability and aneuploidy (cells with the incorrect number of chromosomes), resulting in tumorigenesis, birth defects, miscarriages, and infertility in humans. Recently, a shuriken/Swiss army knife-like multi-nuclease complex has been implicated in processing various types of DNA repair intermediates. However, how these nucleases coordinate their functions during repair remained unclear. Our studies in C. elegans revealed genetic redundancies between these nucleases for meiotic crossover formation and that they promote distinct crossover control at different chromosome regions. Specifically, XPF-1 acts redundantly with both MUS-81 and SLX-1 to resolve Holliday junction recombination intermediates into crossover products at designated future crossover sites on chromosome arms. In contrast, SLX-1 is required for suppression of crossovers at the center region of chromosomes. Altogether, our studies have shed light on the interplay between structure-specific endonucleases and uncovered their ability to exert either positive or negative meiotic crossover control on a chromosome region-specific basis.

Saito RM et al. (1998) Midwest Worm Meeting "Chromosomal dynamics visualized with Histone::GFP"

Godbey A, Saito RM, Schedl TB

[Midwest Worm Meeting, 1998]

During our characterization of Oof mutations (Oocyte fertility, 97' meeting abstract), the need for a dynamic method of analyzing chromosomal morphology became apparent. Previously, the characterization of germline phenotypes has relied heavily on the interpretation of chromosomal morphologies revealed by the DAPI-staining of fixed animals. Analysis of fixed samples likely overlooks important dynamic processes. The isolation of the gene encoding the green fluorescent protein (GFP) and the development of 4-D imaging technologies have aided the visualization of a variety of proteins within living cells. However, two technical barriers limit our application of these technologies in viewing chromosomes of the C. elegans germline: the selection of an appropriate fusion partner and, more importantly, the current inability of researchers to efficiently express transgenes within germ cells. The poster will present our approaches to these problems. In order to visualize the dynamic motions of chromosomes within living cells we are producing histone::GFP fusions. The core nucleosome is composed of an octamer of four histones (H2A, H2B, H3 and H4) that is wrapped by approximately 2 turns of DNA. Linker histone (H1) and the functionally related HMG (high mobility group) proteins bind the DNA located between adjacent nucleosomes. The condensation of the nucleosomes into higher order chromatin stuctures is mediated by covalent modifications of both the core subunits and the linker proteins. Various data, including the recent report of the high-resolution crystal structure of the core nucleosome, suggest locations to position the GFP fusion that may have the least effect on histone function. As the targets of the fusion, we have chosen the C-terminus of H2A, which protrudes from the core nucleosome, and the C-terminus of HMG-1 and following the suggestion of G. Seydoux, the C-terminus of histone H1. To date, database searches have revealed at least twenty sequences related to histones H1, H2A or HMG-1. We have not been able to predict the probability of germline expression of any locus using the available data (nucleotide or amino acid sequence). Therefore, we are systematically producing fusions with these identified loci until germline expression is observed. Histone::GFP hybrids have allowed the somatic chromosomes to be viewed throughout the cell cycle. Spatial and temporal expression patterns of complex array (Kelly et al, 1997) transgenes vary with the target loci. For example, while expression of the histone H1.1::GFP (kindly provided by G. Seydoux) is detectable from the 8-cell stage of embryogenesis through L2/L3, the histone H2A::GFP is observed prior to the lima bean stage and is maintained throughout adulthood. Unfortunately, we have not yet detected expression from transgenes within any of the cells of the germ lineage. Current studies involve the insertion of the GFP coding region into the intact histone loci. If required, future studies may involve histone::GFP driven by a characterized germline promoter sequence.

Saito TL et al. (2013) Genome Res "The transcription start site landscape of C. elegans."

Morishita S, Fire A, Gu SG, Stadler M, Hashimoto SI, Saito TL, Morton JJ, Blumenthal T

[Genome Res, 2013]

More than half of Caenorhabditis elegans pre-mRNAs lose their original 5' ends in a process termed "trans-splicing" in which the RNA extending from the transcription start site (TSS) to the site of trans-splicing of the primary transcript, termed the "outron," is replaced with a 22-nt spliced leader. This complicates the mapping of TSSs, leading to a lack of available TSS mapping data for these genes. We used growth at low temperature and nuclear isolation to enrich for transcripts still containing outrons, applying a modified SAGE capture procedure and high-throughput sequencing to characterize 5' termini in this transcript population. We report from this data both a landscape of 5'-end utilization for C. elegans and a representative collection of TSSs for 7351 trans-spliced genes. TSS distributions for individual genes were often dispersed, with a greater average number of TSSs for trans-spliced genes, suggesting that trans-splicing may remove selective pressure for a single TSS. Upstream of newly defined TSSs, we observed well-known motifs (including TATAA-box and SP1) as well as novel motifs. Several of these motifs showed association with tissue-specific expression and/or conservation among six worm species. Comparing TSS features between trans-spliced and non-trans-spliced genes, we found stronger signals among outron TSSs for preferentially positioning of flanking nucleosomes and for downstream Pol II enrichment. Our data provide an enabling resource for both experimental and theoretical analysis of gene structure and function in C. elegans.

Saito RM et al. (1997) International C. elegans Meeting "a genetic screen for oocyte arrest mutations"

Schedl TB, Saito RM

[International C. elegans Meeting, 1997]

The Schedl lab has been studying the process of oocyte meiotic maturation and ovulation which can be observed in real time using video Nomarski DIC microscopy (see McCarter et al. abstract; see also Iwasaki et al., 1996 and McCarter et al., 1997). Oocytes in the proximal gonad arm are in diakinesis of meiotic prophase. In female mutants and hermaphrodites purged of sperm these oocytes can be arrested in diakinesis for hours to days. Under normal circumstances, the most proximal oocyte matures, beginning nuclear envelope breakdown (NEBD) and undergoing a transformation from a cylindrical shape to an egg shape (cortical rearrangement), in response to an unknown sperm supplied factor. The maturing oocyte is then ovulated into the spermatheca where fertilization occurs. A collection of Emo mutants (for endomitotic oocytes in the gonad arm) have been characterized which appear to be primarily defective in either ovulation or coupling maturation with ovulation (see McCarter et al. abstract; Greenstein et al., 1994; Meyer et al., 1996). To gain insight into the process of oocyte meiotic maturation and late prophase arrest, I am screening for recessive sterile mutations which fail to undergo meiotic maturation. The F2 progeny of EMS-mutagenized hermaphrodites are initially screened for the retention of oocytes in the gonad arm and few or no embryos/oocytes in the uterus (similar to fem/fog females or fer/spe mutants). Mutants are then characterized for 1) presence of sperm, 2) sterility even when mated with N2 males and 3) failure of the oocyte to undergo NEBD and cortical rearrangement. Mutant alleles displaying all these phenotypes are likely to identify genes required for normal maturation. The poster will present my progress of the screen which, to date, numbers over 5,000 genomes. This work was supported by NSF Grant 9506220

Takamune Saito et al. (2008) Development & Evolution Meeting "HIM-18 is required for genome integrity in the C. elegans germline"

Monica Colaiacovo, Takamune Saito

[Development & Evolution Meeting, 2008]

The formation of haploid gametes through the process of meiosis is crucial for sexual reproduction and the continuity of life. However, the diploid genome is subjected to DNA damage within the germline both as a result of non-programmed double-strand breaks (DSBs) (i.e. following the collapse of stalled replication forks) and programmed DSBs (i.e. SPO-11-mediated meiotic DSBs). To generate inheritable gametes, these DSBs have to be properly repaired. We recently identified him-18 (high incidence of males) through an RNAi screen of germline-enriched genes performed in the laboratory. HIM-18 is conserved throughout metazoans and is involved in maintaining genome integrity in the germline. Immunostaining reveals the presence of HIM-18 foci in nuclei at the mitotic proliferative zone. These foci are no longer apparent upon entry into meiosis, but re-emerge in nuclei at late pachytene, persisting through diakinesis. Chromosome non-disjunction is severely increased in him-18 mutants as suggested by the high embryonic lethality (82%; n=1585) and high incidence of males (13%) observed among its progeny. Although an extended transition zone is observed in him-18 mutants, both homologous chromosome pairing and the assembly of the synaptonemal complex (SC) are apparently normal. However, both SC disassembly and the formation of foci of a putative chiasma marker (ZHP-3) are delayed in him-18 mutants. Moreover, the physical connection between the six pairs of attached homologous chromosomes observed in him-18 mutants is more fragile than in wild type, judging by immunostaining with homolog boundary markers such as AIR-2 and Phospho histone H3. Several pieces of evidence suggest that this may result from defects in DSB repair. First, crossover (CO) frequencies are reduced in both Chromosome I (73% of wild type) and the X (50% of wild type) in him18 mutants. Moreover, the distribution of COs is biased to the pairing center end on the X chromosome in him-18 compared to wild type. Second, increased levels of RAD-51 foci are observed both in the mitotic zone and during meiotic prophase in him-18, along with increased levels of germ cell apoptosis. Interestingly, these RAD-51 foci were not completely suppressed in him-18; spo-11 mutants suggesting they originated from collapsed replication forks. Taken together, our data suggests a function for HIM-18 in replication fork maintenance at the mitotic proliferative zone and in crossover control during late meiotic prophase.

R Mako Saito et al. (2002) East Coast Worm Meeting "Developmental control of cell cycle in C. elegans"

R Mako Saito, Sander Van Den Heuvel

[East Coast Worm Meeting, 2002]

The decision to enter a new division cycle in response to extracellular signals occurs during the G1 phase of the cell cycle. How the basic cell-cycle machinery is regulated by these signals is poorly understood. We use the development of the C. elegans vulva as a model to study the regulation of cell cycle entry during development, since the vulva precursor cells (VPCs) display a developmentally regulated period of cell cycle arrest. We have previously described a screen to identify negative regulators of VPC divisions. Briefly, mutants are identified in the lin-12(gf) background as animals with greater than six pseudovulvae. By observing the vulval lineages during the L2 and L3 stages, the origin of these extra pseudovulvae can be verified to result from ectopic divisions of the VPCs. To date, we have identified at least four elm (enhancer of lin-12(gf) multivulvae) loci that result in extra cell divisions.

R Mako Saito et al. (2002) West Coast Worm Meeting "Developmental control of cell cycle in C. elegans"

Sander Van Den Heuvel, R Mako Saito

[West Coast Worm Meeting, 2002]

The decision to enter a new division cycle in response to extracellular signals occurs during the G1 phase of the cell cycle. How the basic cell-cycle machinery is regulated by these signals is poorly understood. We use the development of the C. elegans vulva as a model to study the regulation of cell cycle entry during development, since the vulva precursor cells (VPCs) display a developmentally regulated period of cell cycle arrest. We have previously described a screen to identify negative regulators of VPC divisions. Briefly, mutants are identified in the lin-12(gf) background as animals with greater than six pseudovulvae. By observing the vulval lineages during the L2 and L3 stages, the origin of these extra pseudovulvae can be verified to result from ectopic divisions of the VPCs. To date, we have identified at least four elm (enhancer of lin-12(gf) multivulvae) loci that result in extra cell divisions.

Takamune T. Saito et al. (2007) International Worm Meeting "HIM-18, a new regulator of meiotic progression and chromosome segregation."

Takamune T. Saito, Monica P. Colaiacovo

[International Worm Meeting, 2007]

Sexually reproducing organisms generate haploid gametes through a specialized form of cell division known as meiosis. Reduction of the genome complement by half is achieved by following a single round of DNA replication with two consecutive rounds of chromosome segregation (meiosis I and II). During meiosis I, several events ensure that homologous chromosomes segregate away from each other. Homologs pair, a tripartite proteinaceous structure known as the synaptonemal complex (SC) forms between these paired and aligned homologs, and repair of programmed DNA double-strand breaks (DSBs) occurs. Repair of DSBs via a reciprocal strand exchange event (crossover) results in a physical connection between the homologs (chiasmata) which is important for their proper alignment at the metaphase plate and subsequent segregation to opposite poles of the meiotic spindle. This reductional division is then followed by meiosis II during which sister chromatid partitioning occurs. Despite the fundamental significance of meiosis several of its molecular mechanisms remain poorly understood. Here we introduce him-18 (high incidence of males), a new meiotic candidate identified through an RNAi based screen of germline enriched genes performed in the laboratory. In C. elegans, approximately 0.2% of the progeny from a self-fertilizing XX hermaphrodite are XO males due to spontaneous X chromosome non-disjunction during meiosis I. him-18 mutants produce a slightly reduced number of embryos and 82% fail to hatch. Among the surviving adults, nearly 13% are males. Altogether these observations indicate a severe increase in chromosome non-disjunction. Cytological analysis of chromosome organization/morphogenesis during meiotic prophase indicates a ~2-fold extension of transition zone compared to wild type. Our preliminary analysis suggests that the persistence of chromosomes in a clustered organization characteristic of entrance into meiosis is not, however, a result of either impaired homologous pairing or SC formation. Instead, him-18 mutants have a slightly delayed SC disassembly and the six bivalents observed in diakinesis oocytes have an altered morphology. Specifically, the cruciform display of attached homologs characteristic of this stage is altered suggesting that either the position or number of crossover events along paired homologs may be altered in him-18 mutants. To further address this, we are currently investigating the progression of meiotic recombination as well as both the frequency and distribution of crossovers in him-18 mutants. Taken together, our data suggests a function for HIM-18 in promoting proper meiotic progression and chromosome segregation.

Saito A et al. (2006) Genome Inform "Cell fate simulation model of gustatory neurons with MicroRNAs double-negative feedback loop by hybrid functional Petri net with extension."

Ueno K, Miyano S, Doi A, Saito A, Nagasaki M

[Genome Inform, 2006]

Biological regulatory networks have been extensively researched. Recently, the microRNA regulation has been analyzed and its importance has increasingly emerged. We have applied the Hybrid Functional Petri net with extension (HFPNe) model and succeeded in creating model biological pathways, e.g. metabolic pathways, gene regulatory networks, cell signaling networks, and cell-cell interaction models with one of the HFPNe implementations Cell Illustrator. Thus, we have applied HFPNe to model regulatory networks that involve a new key regulator microRNA. As a test case, we selected the cell fate determination model of two gustatory neurons of Caenorhabditis elegans-ASE left (ASEL) and ASE right (ASER). These neurons are morphologically bilaterally symmetric but physically asymmetric in function. Johnston et al. have suggested that their cell fate is determined by the double-negative feedback loop involving the lsy-6 and mir-273 microRNAs. Our simulation model confirms their hypothesis. In addition, other well-known mutants that are related with the double-negative feedback loop are also well-modeled. The new upstream regulator of lsy-6 (lsy-2) that is mentioned in another paper is also integrated into this model for the mechanism of switching between ASEL and ASER without any contradictions. Therefore, the HFPNe-based modeling will be one of the promising modeling methods and simulation architectures that illustrate microRNA regulatory networks.

R Mako Saito et al. (2004) East Coast Worm Meeting "cdc-14 regulates cki-1 to control cell-cycle arrest"

John S Satterlee, Audrey Perreault, Bethan Peach, Sander Van Den Heuvel, R Mako Saito

[East Coast Worm Meeting, 2004]

The development of multicellular organisms requires proper temporal and spatial control of cell divisions. To reveal underlying mechanisms, we screened for cell-cycle mutants that disrupt the reproducible pattern of somatic divisions in the nematode C. elegans . The screen for mutations that allowed extra divisions of the vulva precursor cells (VPC) led to the identification of genes that are required for their developmental arrest. Surprisingly, we found a novel role for the cdc-14 phosphatase in establishing the temporary quiescence of the VPCs. While budding yeast Cdc14p is essential for mitotic exit, inactivation of C. elegans cdc-14 resulted in extra cell divisions within multiple lineages, with no apparent defects in mitosis or cell-fate determination. The localization of a functional CDC-14 reporter was dynamic and cell-cycle dependent. Several lines of genetic evidence suggest that loss of cdc-14 function disrupts a cki-1 dependent pathway to arrest cell divisions during development. Moreover, we show that cdc-14 acts upstream of cki-1 and elevates CKI-1 nuclear accumulation. These data demonstrate that cdc-14 contributes to developmental regulation of cell-cycle arrest by promoting cki-1 activity. If conserved, a role for Cdc14 in Cip/Kip stabilization may have important implications for human cancer.