Jason Randolph et al. (2006) Development & Evolution Meeting "Conserved Histone Variants Important for Fertility"

Diana Chu, Jason Randolph, Aiza Go, Colin Fitzpatrick

[Development & Evolution Meeting, 2006]

With half of all fertility failures due to malfunctions in sperm production, male infertility is a major health problem whose molecular causes are not yet well defined. One distinct feature of sperm is how it uniquely compacts and segregates its DNA; problems in proper sperm chromosome dynamics result in infertility in males. By conducting a functional proteomic analysis of C. elegans sperm chromatin, the Chu lab identified two C. elegans sperm nuclear basic proteins, HTAS-1 and HIL-4, two conserved histone variants. Histone variants like H2A in mammals control chromosome dynamics and gene expression. HTAS-1 is homologous to H2AX, a protein required for male fertility and transcriptional regulation in mouse. The chromosomes of the H2AX-deficient mouse fail to undergo compaction, inactivation or sex chromosome pairing [1, 2]. Furthermore, HAN-P is homologous to HIL-4, an H1 variant that is important for male fertility in mouse. Our studies focus on characterizing HTAS-1 and HIL-4, to determine if they are crucial for chromosome compaction and segregation. To determine if histone variants have an affect on fertility, progeny counts were carried out on htas-1D and hil-4D mutants and compared to progeny counts of N2 wild type worms. Preliminary results show a reduction in the brood size of the htas-1D mutant by 28.3%, indicating that progeny production is being hindered by the absence of the HTAS-1 protein. On the other hand, we have found that a heterozygous HIL-4 strain is easily lost through sequential generations, most likely due to the fact that a homozygous HIL-4 strain has been reported to be lethal and sterile. Therefore, these variants are necessary for optimal fertility. Additionally through antibody staining, HTAS-1 was localized to meiotic sperm chromosomes during and after sperm meiosis, specifically from pachytene to diplotene in the wild-type. To determine if the absence of HTAS-1 and HIL-4 result in observable sperm chromosome morphology we are in the process of assessing whether larger chromosomes or chromosome segregation defects are observed during spermatogenesis in the htas-1D and hil-4D mutants through DAPI staining and confocal microscopy. Our studies have the potential to demonstrate how histone variants function to directly show chromosome compaction and segregation are an essential component of fertility in C. elegans, providing insight into reproductive failure in humans.

Rohner, Sabine et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Activation of heat-shock - inducible genes: interaction with nuclear envelope components"

Gasser, Susan M, Meister, Peter, Rohner, Sabine

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Several stress-induced genes in different organisms have been shown to be sequestered to the nuclear periphery upon activation. These interact with the nuclear pore complexes (NPC), the gateway between the cytoplasm and the nuclear interior. Recent data in Caenorhabditis elegans (modENCODE, Jason Lieb) from ChIP on chip experiments on NPP-13 (Nuclear Pore Protein 13, homologous to the Nup93 subunit in vertebrates) indicates an interaction between the heat shock gene hsp-16.2 and the NPC. Using fluorescence in situ hybridisation in embryos, we show that the genomic hsp-16.2 locus is preferentially located at the nuclear periphery and that its peripheral localisation increases upon heat shock treatment . Moreover, the hsp-16.2 promoter on its own is able to direct peripheral localisation of small integrated bombarded transgenes. Upon HS treatment, the proportion of transgenes located at the nuclear rim increases. We are now examining the physiological importance of the peripheral localization of this gene. To do this we are identifying which components of the nuclear pore and pore-associated transcription and which sequences in the promoter are important for its peripheral localisation.

Robert Barstead et al. (2005) International Worm Meeting "The C. elegans Gene Knockout Consortium"

Angela Fisher, Candice Navaroli, Allison Hay, Robert Barstead, Anna Rankin, Lucy Liu, Joanne Lau, Nadereh Rezania, Mark Edgley, Owen Dadivas

[International Worm Meeting, 2005]

The C. elegans Gene Knockout Consortium (http://www.celeganskoconsortium.omrf.org/) pursues systematic targeted gene disruption in the nematode, both upon request for the research community and in the larger context of the whole set of genes for which human orthologs exist (about 7,000 genes). This work is currently based on a reverse-genetics PCR method, generating single-gene deletions. All deletions are stabilized as homozygous or balanced heterozygous strains, the deletion breakpoints are sequenced, and the strains and data are placed immediately into the public domain through our collaborators at the Caenorhabditis Genetics Center (CGC: http://biosci.umn.edu/CGC/CGChomepage.htm) and WormBase (http://www.wormbase.org/). To date the Consortium has generated deletions in more than 1800 genes, and more than 1300 of these have been stabilized and archived at the CGC. Distribution of Consortium strains accounts for an increasing proportion of distributions from the CGC every year, and stood at 20% for the calendar year 2004.The reverse genetics PCR method using UV/TMP as the mutagen has evolved in our hands into a robust system for efficient generation of deletion mutations. However, some target genes have been refractory to this method, and we therefore continue to evaluate other isolation/detection methods (for example, see abstracts on array CGH and resequencing at this meeting). We will report on our latest progress using all methods.Additional authors for whom inadequate space was allowed by abstract submission system: Peter Huang (UBC and BC Genome Sciences Centre), Jason Maydan (UBC), Sheldon McKay (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Lukas J. Neukomm et al. (2006) European Worm Meeting "Identification and Characterization of mtm-1, A Novel Member and Negative Regulator of the Engulfment Signaling Pathway in C. elegans"

Jason M. Kinchen, Lukas J. Neukomm, Michael O. Hengartner

[European Worm Meeting, 2006]

Lukas J. Neukomm1, Jason M. Kinchen1,2 and Michael O. Hengartner1. The proper removal of apoptotic cells is critical during embryonic development and in tissue homeostasis to prevent inflammation and autoimmunity. However, the molecular details of the engulfment machinery are not fully understood. The powerful genetics of the small nematode C. elegans has been used to identify genes involved in the programmed clearance of apoptotic cells during development and in the adult germ line. Nine genes were isolated which act in two partially redundant pathways. One group is composed five genes: the small GTPase MIG-2/RhoG and its nucleotide exchange factor UNC-73/TRIO, as well as the adaptor protein CED-2/CrkII and the bipartite RacGEF complex CED-5/Dock180 and CED-12/ELMO. In the second pathway, CED-1/CD91/LRP/SREC/EATER functions as a transmembrane receptor which might recognize the apoptotic cell. The CED-7/ABCA1 transporter is likely important for membrane dynamics, and plays an additional critical role in dying cells. CED-6 and its human homologue GULP encode a signaling adaptor molecule whose function is evolutionary conserved and which physically interact with CED-1/CD91/LRP/SREC/EATER. Both pathways converge at the level of CED-10/Rac. However, the signaling pathway between CED-6 and CED-10, as well as events downstream of CED-10 have yet to be identified.. To identify negative regulators of engulfment, we used the vital dye Acridine Orange (AO) to identify mutations that suppress the engulfment defect of ced-6(n1813) mutants. We identified 13 potential suppressors that restore partial engulfment of germ cell corpses and also show a decreased number of persistent cell corpses during embryonic development. We found that the strongest suppressor, op309, is a hypomorphic allele of mtm-1, a phosphatidyl-inositol-3-phosphatase. mtm-1(309) is a recessive mutation and acts zygotically. Furthermore kinetic 4D lineage reveals that the engulfment efficiency in mtm-1; ced-6 animals is restored compared to ced-6. Epistatic analysis suggest that mtm-1 acts upstream of the ternary complex ced-2, ced-5 and ced-12, in parallel to mig-2. Interestingly, we found that mig-2(gf) phenocopies mtm-1(lf) and also partially suppresses persistent cell corpses in ced-1, ced-6, ced-7 and ced-10 embryos. Additionally actin polymerisation around germ cell corpses is partially restored in mtm-1; ced-10 animals. We will show evidence that in mtm-1(op309) animals engulfment signaling is increased. This "promotion" of engulfment might be a prerequisite for therapeutics designed to induce the clearance of unwanted cells.