Lynch, Tina R. et al. (2015) International Worm Meeting "Visualization of regulated transcription using single-molecule FISH."

Lynch, Tina R., Lee, ChangHwan, Kimble, Judith

[International Worm Meeting, 2015]

Transcriptional regulation controls many aspects of germ cell development, including maintenance of stem cells and sex determination. Our lab is particularly interested in Notch signaling and its transcriptional regulation of downstream genes required for germline stem cell (GSC) maintenance. We recently identified two genes, lst-1 and sygl-1, that respond directly to Notch signaling and are essential for GSC maintenance, albeit redundantly (Kershner et al., 2014). Transcriptional reporters are widely used to visualize the readout of signaling. However, here we use single-molecule RNA Fluorescent In Situ Hybridization (smFISH) as a more direct readout of transcriptional regulation and compare endogenous transcription to more common transcriptional reporters. Specifically, we visualized transcripts from the endogenous sygl-1 gene and also from transcriptional reporter transgenes, Psygl-1::SYGL-1::GFP and Psygl-1::H2B::GFP. The sygl-1 transcripts were detected using both sygl-1 intron- and exon-specific probe sets whereas the reporter transcripts were seen using a gfp exon-specific probe set. Each probe set consisted of more than 30 20nt-long probes designed against the sequence of interest, and all probes within a probe set were labeled with the same fluorophore. Using these probes we saw many relatively dim cytoplasmic spots and a few bright nuclear spots in the distal germline. The cytoplasmic spots disappeared from N2 and Psygl-1::SYGL-1::GFP worms after treatment with sygl-1 siRNA, as expected for mature mRNAs. By contrast, the bright nuclear foci disappeared after alpha-amanitin treatment, as expected for primary transcripts. Importantly, the nuclear foci disappear in a glp-1(ts) mutant soon after shifting to restrictive temperature. We are currently quantifying both primary and cytoplasmic transcripts as a function of distance from the distal end for both endogenous and reporter transcripts. We propose that the smFISH method to visualize both primary and cytoplasmic Notch-regulated transcripts will provide new insights into the dynamics of the Notch signaling response and provide a glimpse of how transgenes are expressed compared to endogenous genes.Kershner, A.M., Shin, H., Hansen, T.J., and Kimble, J. (2014). Discovery of two GLP-1/Notch target genes that account for the role of GLP-1/Notch signaling in stem cell maintenance. Proc. Natl. Acad. Sci. U. S. A. 111, 3739-3744.

Dee R Denver et al. (2001) International C. elegans Meeting "Rates and Patterns of Mutation in the Nuclear and Mitochondrial Genomes of Caenorhabditis elegans"

Larissa Vassilieva, Katherine Harris, Dee R Denver, Suzanne Randall Estes, W Kelley Thomas, Krystalynne Morris, Michael Lynch

[International C. elegans Meeting, 2001]

Mutations are the ultimate source of genetic disorders and offer important data for phylogenetic, forensic and population genetic studies. We directly assayed the rates and patterns of mutation in a set of 74 Caenorhabditis elegans mutation accumulation (MA) lines, where mutations accumulate over time in an effectively neutral manner as each MA line is propagated across generations as a single, random hermaphrodite (Vassilieva and Lynch, 1999). We performed a uniquely direct assay for the underlying rates and patterns of mutation in the MA lines with large-scale DNA sequence collection and analysis from both the nuclear and mitochondrial genomes. The mitochondrial DNA assays revealed a mutation rate that is two orders of magnitude higher than previous indirect estimates, a highly biased mutational spectrum, multiple mutations affecting coding function and mutational hotspots at homopolymeric nucleotide runs (Denver et.al ., 2000). Mutational hotspots have also been identified in the nuclear DNA sequence assays. Furthermore, we have analyzed the same mitochondrial and nuclear loci in C. elegans natural isolates to evaluate the relative roles of mutation and natural selection in shaping molecular variation observed in natural populations. References: L.L. Vassilieva and M. Lynch , Genetics 151 , 119 (1999). D.R. Denver, K. Morris, M. Lynch, L.L. Vassilieva and W.K. Thomas, Science 289 , 2342 (2000).

Crittenden, Sarah L. et al. (2017) International Worm Meeting "Visualizing nuclear GLP-1/Notch NICD."

Battula, Sindhu, Lynch, Tina R., Kimble, Judith, Sorensen, Erika, Lee, ChangHwan, Seidel, Hannah S., Lei, Cecilia, Crittenden, Sarah L.

[International Worm Meeting, 2017]

Notch is a conserved regulator of stem cells and differentiation. Upon activation by ligand, the Notch receptor is cleaved, releasing its intracellular domain (NICD) for assembly of a nuclear complex and transcriptional activation of downstream targets. Our work focuses on Notch signaling in the C. elegans germline stem cell niche, where well-defined signaling and receiving cells facilitate quantitative analyses. We recently reported that the transcriptional response to GLP-1/Notch activation occurs in a steep gradient across the germline stem cell pool (Lee et al., 2016). An outstanding question is whether this transcriptional gradient reflects a corresponding gradient in GLP-1/Notch activation. We are visualizing nuclear GLP-1/Notch NICD as a readout of GLP-1/Notch activation. Our reagents include MOS-SCI transgenes encoding a tagged GLP-1 receptor that rescues a glp-1 null allele as well as CRISPR-inserted tags at the endogenous locus. To date, we have detected signal with Myc, V5, OLLAS and Halo tags. The signal is low, however, and we are working on methods to bring the signal into a quantifiable range without changing receptor abundance. Our progress will be reported at the meeting.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.

Hicks, Tara et al. (2021) International Worm Meeting "R-loop-induced irreparable DNA damage in C. elegans meiosis"

Hicks, Tara, Koury, Emily, Williams, Cameron, Smolikove, Sarit

[International Worm Meeting, 2021]

Most DNA-RNA hybrids are formed naturally during transcription and are composed of a nascent RNA strand hybridized to DNA as part of R-loops. The accumulation of these structures in S-phase can result in replication-transcription conflict, an outcome which can lead to the formation of double strand breaks (DSBs). While R-loops' role in mitotically dividing cells has been characterized, there are only a handful of studies describing the effect of R-loops in meiosis and these studies present a complex picture of the outcome of R-loop formation on germ cells. Here we show that DSBs formed by R-loops trigger an altered cellular response to DNA damage. RNase H is an enzyme responsible for degradation of the RNA strand in DNA-RNA hybrids and plays an essential role in preventing this outcome and its deleterious consequences. Using null mutants for the two Caenorhabditis elegans genes encoding for RNase H1 and RNase H2 (hereby rnh mutants), our studies explore the effects of replication stress-induced DNA-RNA hybrid accumulation on meiosis. As expected, rnh mutants exhibit an increase in R-loop formation. Consequently, an elevation of DSBs in germline nuclei is evidenced by the accumulation of RAD-51 foci. Despite no repair mechanism abrogation, rnh mutants fail to repair all DSBs generated, leading to a fragmentation of chromosomes in diakinesis oocytes. By combining our double mutant with a spo-11 null mutation, we show that although replicative defects are the main contributor to the phenotype, R-loops formed in meiosis are likely contributors as well. We present evidence that while rnh mutants accumulate DNA-RNA hybrids and subsequent DSBs may signal a degree of checkpoint activation in mitosis, some damaged nuclei prevail past the checkpoint, enter into meiosis, and remain unrepaired throughout. Moreover, we find no evidence of an increase in apoptosis, which indicates that DNA damage generated by R-loops remain undetected by an apoptotic checkpoint. This data altogether points to DSBs initiated by R-loops representing an irreparable type of DNA damage that evades cellular machineries designed for damage recognition.

Birsoy B et al. (2010) Biology of the C. elegans Male, Madison, WI "Novel Left-Right Asymmetries of Male C.elegans"

Rothman J H, Choi S, Birsoy B, Downes J C, Bloss T A

[Biology of the C. elegans Male, Madison, WI, 2010]

While the mechanism that breaks left-right (L-R) anatomical asymmetry has been described in C. elegans, we have found that at least one additional mechanism must exist to generate L-R differences in the deployment of alternative cell death pathways in the male tail, male mating behavior and patterning of the male gonad. Upon recognizing a hermaphrodite, males initiate backward locomotion and continuously scan along the hermaphrodite's body. When their tails reach either the head or tail of the hermaphrodite, males turn to maintain contact and then continue backward locomotion on the opposite side of the hermaphrodite. We found that this turning behavior shows a distinct right-handed bias: when individual males were allowed to mate with lin-2(e1309) vulvaless hermaphrodites, >70% of the turns when made over the hermaphrodites' body (away from the agar surface) and >55% of turns when made under the hermaphrodites' body (toward the agar surface) are right-handed. To determine whether this bias in turning direction correlated with L-R asymmetry in the male mating structure, which results from stochastic EGL-1-dependent loss of sensory rays, we examined the turning bias in egl-1(n1084n3082) mutants. Wild-type males lack rays more frequently on the right and egl-1 mutant males have no ray loss but still display the right-hand turning bias. To assess whether this L-R behavioral bias is dependent on anatomical asymmetry, we analyzed gpa-16(it143) mutants in which the L-R asymmetry of the internal organs is reversed as a result of the reversal in the early embryonic symmetry break. Males with reversed anatomical asymmetry showed a virtually identical right hand bias in turning, demonstrating that an asymmetry-determining system that is independent of the previously described L-R symmetry breaking event must control this behavior. During the characterization of the gonad reversals of males, we also observed a previously unreported temperature sensitive reversal of L-R asymmetry of the male gonads in N2 (Bristol) and a Hawaiian wild isolate. We will describe our recent findings on these novel L-R asymmetries of the C.elegans male anatomy and behavior.

L C Bowen et al. (2002) European Worm Meeting "The C. elegans cyclic AMP-dependent protein kinase (PK-A) catalytic subunit gene (kin-1)"

L C Bowen, M Tabish, M J Fisher, H H Rees, R A Clegg

[European Worm Meeting, 2002]

PK-A is one of the major multifunctional ser/thr protein kinases found in eukaryotic organisms. The holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. Both R and C subunits occur in multiple isoforms encoded by distinct genes. In many organisms, the genes that encode the C subunits also show alternative splicing behaviour. This generates an even broader array of C subunit heterogeneity. The diversity of C subunit polypeptides may enable this protein kinase to selectively target substrate polypeptides for phosphorylation.