Moyle, Mark et al. (2013) International Worm Meeting "Mechanistic Insights Into The Recruitment of the Spindle Checkpoint Protein MDF-1 To Unattached Kinetochores."

Oegema, Karen, Desai, Arshad, Moyle, Mark

[International Worm Meeting, 2013]

Correct alignment and segregation of chromosomes during cell division is essential for proper development and growth. When chromosomes are improperly attached an intracellular signaling cascade, termed the spindle checkpoint, generates a "wait anaphase" signal which delays cell cycle progression until all chromosomes are properly aligned. This signal originates from unattached kinetochores, the macromolecular spindle microtubule-binding machines built on the centromeric regions of chromosomes. Central to the "wait anaphase" signal is the protein MDF-1 (Mad1 in human), but it is still unclear how MDF-1 is recruited to and activated at unattached kinetochores. To address this question, we performed a yeast two-hybrid screen with MDF-1 and a library of kinetochore constituents. We identified an interaction between MDF-1 and BUB-1, a conserved kinase implicated in spindle checkpoint signaling. We then performed a mutagenic yeast two-hybrid screen, which identified alleles of MDF-1 that no longer interact with BUB-1. Prior work had shown that BUB-1 is required for the kinetochore localization of MDF-1. Employing the MosSCI system, we generated MDF-1 mutants that do not interact with BUB-1 and found that the mutant forms of MDF-1 are compromised in their localization to unattached kinetochores. Consistent with this, the MDF-1 mutants that do not interact with BUB-1 were defective in generating a spindle checkpoint signal. As BUB-1 is a kinase, we generated kinase-dead mutants of BUB-1 and found that these mutants no longer interact with MDF-1 in the two-hybrid assay, suggesting that the kinase activity of BUB-1 is required for this interaction. Currently, we are investigating BUB-1 kinase-dead mutants in vivo. Finally, we performed a compensatory mutagenic yeast two-hybrid screen and found a mutation in BUB-1 that restores interaction with a MDF-1 mutant, strongly suggesting that MDF-1 and BUB-1 directly interact. Overall, our findings elucidate the recruitment of MDF-1 to unattached kinetochores via the kinase BUB-1, and show that the specific interaction underlying this recruitment is required for generation of the "wait anaphase" checkpoint signal.

Moyle, Mark et al. (2015) International Worm Meeting "Towards the living connectome: generation of a 4D atlas of neurodevelopment."

Kumar, Abhishek, Shah, Pavak, Wu, Yicong, Santella, Anthony, Colon-Ramos, Daniel, Marquina, Javier, Kovacevic, Ismar, Moyle, Mark, Gutierrez, Natasha, Mohler, William, Bao, Zhirong, Shroff, Hari, Christensen, Ryan

[International Worm Meeting, 2015]

How neuronal networks develop during embryogenesis to generate a functional nervous system is a fundamental question in developmental biology and neuroscience. In C. elegans the entire wiring diagram of the adult nervous system ("the connectome") has been mapped. However, it is unclear how the nervous system is organized during embryogenesis to correctly form the connectome. Through a multidisciplinary collaboration, we have established an imaging and analysis pipeline to generate and share a 4D atlas of C. elegans neurodevelopment throughout embryogenesis. To this end, we created a light-sheet microscope capable of prolonged time-lapse imaging with subcellular resolution. The dual-view selective plane illumination microscope (diSPIM) is capable of constant volumetric 350nm isotropic imaging at speeds 10-1000x faster than conventional 4D imaging techniques. Also, we have collected existing and are generating novel C. elegans strains that label sparse subsets of neurons to facilitate segmentation and are pioneering strategies for heat-shocking/photoactivation to allow for efficient use of pan-neuronal promoters. Furthermore, we developed novel image analysis software (StarryNite) capable of systematically tracking and identifying every developing cell, as well as software for segmenting individual neurons in 4D, and linearizing the twisted C. elegans embryo. Finally, to allow for widespread dissemination of these large data sets, we created iOS and Android applications, and an Internet-based viewing program called CytoSHOW, which allows the scientific community access to all the neurodevelopmental data. We envision a dynamic workspace where any researcher can visualize, integrate, and interrelate the diverse scope of C. elegans neurological and developmental data.

David J Katz et al. (2005) International Worm Meeting "Investigating the link between NANOS and chromatin for germ cell maintenance in C. elegans"

William G Kelly, David J Katz

[International Worm Meeting, 2005]

In Drosophila, nanos is required for primordial germ cells (PGCs) to differentiate into a functional germ line. Lack of NANOS function causes defects in germ cell migration, differentiation, and quiescence and ultimately leads to sterility in both males and females. Similarly, in mice a lack of nanos3 results in the complete loss of germ cells in both sexes (Tsuda et al. 2003) and functional loss of two of the three nanos genes in C. elegans, nos1 and nos2, also results in sterility (Subramaniam and Seydoux, 1999). It has therefore been proposed that nanos is a conserved master regulator of the germ cell fate. In the C. elegans early embryo, the chromatin in all cells contain the active chromatin mark, histone H3 methylated on lysine 4 (H3meK4). However upon their birth, the primordial germ cells Z2 and Z3 dramatically lose this mark. This loss of the active H3meK4 mark has been suggested to be part of a chromatin-based transcriptional repression mechanism. Remarkably, absence of the H3meK4 mark has been shown to be dependent upon nanos activity (Schaner et al. 2003). Thus it is possible that nanos may be partially affecting its role in maintaining the undifferentiated germ cell fate through a chromatin-based mechanism. We aim to explore this potential link by further characterizing the role of C. elegans nanos in germ cell fate. To this end, we are taking both forward and reverse genetic approaches as well as conducting ectopic expression studies.

Snyder, Matthew P. et al. (2013) International Worm Meeting "Turnover of the H3K9me2 Mark During Late Spermatogenesis."

Snyder, Matthew P., Xu, Xia, Maine, Eleanor

[International Worm Meeting, 2013]

Meiotic silencing is a conserved phenomenon targeting unpaired chromosomes and chromosomal regions during prophase of meiosis I. Meiotic silencing in animals typically occurs at the chromatin level and involves accumulation of histone modifications thought to promote a closed chromatin configuration. This chromatin structure may contribute to transcriptional repression and meiotic chromosomal events such as chromosome disjunction (Bean et al 2004, Jaramillo-Lambert and Engebrecht 2010). During meiosis in C. elegans, non-synapsed chromosomes are enriched for H3K9me2 relative to synapsed chromosomes (Kelly et al. 2002; Bean et al. 2004). Such non-synapsed chromosomes include the male X, homologous chromosomes that fail to synapse due to mutation, and chromosomal translocations/duplications. The pattern of H3K9me2 accumulation during meiosis depends on activity of the small RNA machinery, which may have a role in targeting the mark to unpaired chromosomes and ensuring that the mark does not persist abnormally (Maine et al. 2005; She et al. 2009). Taking a combined biochemical/genetic approach, we are identifying additional factors important for regulating H3K9me2 distribution during meiosis. Through this approach, we are identifying genes that appear to be important for turnover of the H3K9me2 mark during late spermatogenesis.

Gyoergyi Csankovszki et al. (2002) West Coast Worm Meeting "Mapping chromosomal targets of the dosage compensation complex"

Barbara J Meyer, Gyoergyi Csankovszki

[West Coast Worm Meeting, 2002]

The worm C. elegans compensates for the difference in X-linked gene dosage between XX hermaphrodites and XO males by reducing gene expression two-fold in XX animals. Downregulation of gene expression is achieved by the dosage compensation complex (DCC), a large multiprotein complex that binds the two X chromosomes in hermaphrodites. However, the cis-acting chromosomal elements that recruit and bind the complex are not known. Using free and attached X chromosomal duplications we are attempting to identify regions of the X chromosome that are sufficient to recruit the complex. By analyzing the volume and number of DCC-bound chromosomal bodies, a large duplication of the right 30% of X, mnDp10, was shown earlier to be competent to bind the DCC (1). Using combined fluorescent in situ hybridization (to mark X chromosomal territories) and immunofluorescence (to mark chromosomes that bind the dosage compensation complex) we confirmed these results. By analyzing smaller duplications that span the entire chromosome we hope to map chromosomal targets of the DCC.

Pokhrel, Bharat et al. (2017) International Worm Meeting "Characterising C. elegans COMPASS complex in gene expression."

Chen, Ron, Appert, Alex, Pokhrel, Bharat, Clifford, Rosamund, Chen, Yannic, Ahringer, Julie, Dong, Yan, Stempor, Przemyslaw

[International Worm Meeting, 2017]

Trimethylation of histone lysine 4 (H3K4me3) is regarded as an active promoter mark across all eukaryotes. H3K4me3 is mainly deposited by an evolutionarily conserved COMPASS complex that contains two key subunits: SET-2, H3K4me3/2 methyltransferase, and CFP-1, a conserved CpG binding protein. Perturbation of this mark has been found to be associated with different diseases, development defects, and incorrect cell fate specification. However, it is unclear whether H3K4me3 play an instructive role in transcription or just a consequence of gene expression. To address this question, we phenotypically characterised and compared cfp-1(tm6369) mutants with a reported loss-of-function set-2(bn129) mutant allele. We observed dramatic loss of H3K4me3, poor fertility, slow growth and ectopic gene expression in both cfp-1 and set-2 mutants. We found that the role of H3K4me3 is to fine tune gene induction, likely by modulating other histone modifications that play an essential role in gene activation. Our results shed light on the role of H3K4me3 in chromatin regulation and function.

Sujata Bhattacharyya et al. (2008) Development & Evolution Meeting "The Role of Histone Deacetylation in the Maintenance of Transcriptional Repression in Primordial Germ Cells"

Sujata Bhattacharyya, William Kelly

[Development & Evolution Meeting, 2008]

Experimental data from a number of model organisms suggests that transcriptional quiescence is essential for safe-guarding totipotency of the embryonic germ line. In C.elegans, global repression in germline blastomeres is mediated by a maternal protein, PIE-1. Subsequent to its degradation, chromatin-based mechanisms accompanied by epigenetic erasure events suppress transcription in the primordial germ cells, Z2 and Z3. In particular, genome-wide H3K18 acetylation is deleted from Z2 and Z3 within 30 minutes of their birth.The rapidity with which this mark is lost suggests an enzymatic mode of removal. This hypothesis is corroborated by evidence which indicates that loss of H3K18Ac occurs on a normal schedule in the psa-1 mutant which is defective in nucleosome remodeling. Consequently, we are analyzing mutants in each of nine histone deacetylases to determine which in particular is responsible for this erasure event. RNA interference will be used if genetic redundancy is suspected. The functional consequences of inappropriately retaining this mark on the totipotency of the germ line will be elaborated in the appropriate loss-of-function mutant(s).

Murthy K et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "In vivo imaging of retrogradely transported synaptic vesicle proteins in C. elegans neurons"

Bhat J M, Murthy K, Koushika S P

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Axonal transport is an essential process that carries cargoes in the anterograde direction to the synapse and in the retrograde direction back to the cell body. Currently there is no in vivo method to exclusively mark retrogradely moving compartments carrying specific proteins, as opposed to anterogradely moving compartments. We have developed a method in the C. elegans model to mark retrogradely moving compartments carrying Synaptobrevin-1 in neurons. This method is based on the uptake of a fluorescently labeled anti-GFP antibody delivered in an animal expressing Synaptobrevin-1::GFP in neurons. We show that this method largely labels only retrogradely moving compartments. Very little labeling is observed if there is a block of vesicle exocytosis or if the synapse is physically separated from the cell body. The extent of labeling is also dependent on the dyenin-dynactin complex. These data support the interpretation that the labeling of Synaptobrevin-1::GFP largely occurs after vesicle fusion and the major labeling likely takes place at the synapse. This method is very general and can be readily adapted to any transmembrane protein on synaptic vesicles with a GFP tag inside the vesicle. It can also be easily extended to other model systems such as Drosophila.

Wanyuan Ao et al. (2001) International C. elegans Meeting "What is TLF doing during the C. elegans cell cycle?"

Wanyuan Ao, S E Mango

[International C. elegans Meeting, 2001]

Transcription is finely regulated during the cell cycle. In most organisms, genes are actively transcribed during interphase, but not during mitosis when DNA is condensed, nascent transcripts are released, and transcription factors are inactivated by phosphorylation and discharged from DNA. How then do cells re-establish the appropriate transcriptional repertoire in newly born daughter cells? One attractive idea is that cells ‘mark’ genes to specify those that are actively being transcribed during interphase. The molecular nature of the mark is currently unknown. We are using C. elegans embryos to investigate cell cycle regulation of RNA Polymerase II (Pol II) transcription. Like other organisms, C. elegans probably inactivates transcription at mitosis since i) Pol II in mitotic cells lacks a phosphorylated epitope normally associated with transcriptional elongation (1, 2) and ii) transcription factors are dispersed at this stage of the cell cycle (L. Kaltenbach and S.E.M., unpublished). Since embryonic blastomeres cycle very rapidly (~15-30 minutes), we envision that gene marking and rapid re-establishment of transcription after mitosis are critical for normal embryonic development. One appealing candidate for a marking protein is the TATA-binding protein (TBP) paralog TLF. TLF is required for Pol II transcription during C. elegans embryogenesis, suggesting it performs a unique function distinct from that of TBP (2, 3). TLF is required to activate a subset of Pol II promoters and facilitates the re-establishment of transcription after mitosis (2). In addition, preliminary data indicate that TLF may associate with mitotic chromosomes during the cell cycle (L. Kaltenbach and S.E.M., unpublished). This association contrasts with TBP, which is released during mitosis (L. Kaltenbach and S.E.M., unpublished). These observations suggest that TLF has a critical role during the cell cycle, possibly to mark genes for their timely reactivation during the next interphase. Our current goal is to test this hypothesis using both in viv o and in vitro approaches. Seydoux and Dunn, Development 124, 2191-2201 (1997) Kaltenbach et al., Molecular Cell, 6, 705-713 (2000). Dantonel et al., Molecular Cell 6, 714- (2000)

Seydoux GC et al. (1995) International C. elegans Meeting "GERM-CELL FATE IS LINKED TO REPRESSION OF TRANSCRIPTION IN EARLY C. ELEGANS EMBRYOS."

Fire A, Seydoux GC

[International C. elegans Meeting, 1995]

The first cleavages of the C. elegans embryo are asymmetric and mark the step-wise separation of soma and germline. We have identified several differences between somatic and germline blastomeres during early cleavages (1) 1. Germline cells protect certain maternal RNAs from a rapid degradation which occurs in somatic cells. 2. Germline cells contain clusters of poly-A+ RNAs associated with the germline-specific P granules. These clusters are not seen in somatic cells. 3. As early as the four-cell stage, somatic cells transcribe certain RNAs, which are off in germline cells. So far, no transcribed gene has been identified in early germline blastomeres. An intriguing possibility is that these cells are transcriptionally inactive.