Goetsch, Paul et al. (2015) International Worm Meeting "Identifying components of the germ cell transcriptional regulatory network activated upon loss of the Dp, Rb, and MuvB (DRM) complex."

Goetsch, Paul, Strome, Susan

[International Worm Meeting, 2015]

Cellular specification during embryogenesis relies on coordinated transcriptional regulatory networks that transition cells from old to new developmental programs. We seek to understand how developing somatic cells avoid expressing the germ cell program. C. elegans Synthetic Multivulval class B genes, which include genes that encode the 8 subunits of the Dp, Rb, and MuvB (DRM) transcriptional repressor complex, are known to protect somatic cells from activating germ-like regulatory networks. Mutations that compromise DRM complex activity, including loss of the Retinoblastoma-like pocket protein LIN-35, result in expression of germline genes in somatic cells. Importantly, many promoters of ectopically expressed germline genes (e.g., pgl-1) are not directly targeted by the DRM complex. We hypothesize the DRM complex represses germline genes in somatic cells through direct repression of transcription factor(s) required for germline gene expression. DRM binds to the promoters of 311 predicted transcription factor-encoding genes. Using a lin-35(n745) Ppgl-1::pgl-1::GFP reporter strain that displays somatic pgl-1::GFP expression, we tested for suppression of ectopic GFP expression following RNA interference of 243 of these candidate trans-acting factors, as compared to empty vector control. This qualitative screen identified 51 candidate trans-acting factors that potentially activate germline genes in somatic cells following loss of DRM. We also evaluated changes in chromatin accessibility, which can identify transcription factor binding sites, following loss of LIN-35. Chromatin accessibility can be measured by sensitivity of genomic regions to DNase I digestion. Changes in DNase I sensitivity as measured by quantitative PCR indicate an increase in chromatin accessibility at the pgl-1 promoter in lin-35(n745) as compared to wild type. This analysis will be linked to high throughput sequencing of DNase I hypersensitive fragments between lin-35(n745) and wild type to reveal novel transcription factor binding events enriched upon loss of DRM activity. Together, these complementary approaches will identify transcription factors that drive germline gene expression in somatic cells following loss of DRM complex activity. This study provides a strong foundation for the identification and analysis of potential novel components of the germ cell transcriptional regulatory network required for in vivo germ cell specification.

Mains PE (1994) Worm Breeder's Gazette "Interactions Between mei-1 and the unc-116 Kinesin"

Mains PE

[Worm Breeder's Gazette, 1994]

Interactions Between mei-l and the unc-116 Kinesin Paul E. Mains, Dept. of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada

Wissmann AK et al. (1994) Worm Breeder's Gazette "Characterization of the let-502 Gene"

Mains PE, Wissmann AK, McGhee JD

[Worm Breeder's Gazette, 1994]

Characterization of the let-502 gene Andreas Wissmann, James D. McGhee and Paul E. Mains, Dept. of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1

Sommer RJ et al. (1994) Worm Breeder's Gazette "Evolution of Vulva-Formation: Part II: Species With a Central Vulva"

Sommer RJ, Sternberg PW

[Worm Breeder's Gazette, 1994]

Evolution of vulva-formation: Part II: Species with a central vulva Ralf J. Sommer & Paul W. Sternberg, California Institute of Technology, Division of Biology 156-29, Pasadena, CA 91125

Li WQ et al. (1992) Worm Breeder's Gazette "Characterization of the Axonal Guidance and Outgrowth gene Unc-33"

Shaw JE, Li WQ, Herman RK

[Worm Breeder's Gazette, 1992]

Characterization of the axonal guidance and outgrowth gene unc-33 W. Li, R. K. Herman and J. E. Shaw Department of Genetics and Cell biology, University of Minnesota, St Paul, MN 55108

Washeleski, Emily et al. (2021) International Worm Meeting "Auxin-Inducible Degradation of DREAM Proteins, LIN-9 and LIN-54, in Caenorhabditis elegans"

Chosa, Karli, Goetsch, Paul, Washeleski, Emily

[International Worm Meeting, 2021]

The highly conserved Dp, Retinoblastoma-like, E2F, and MuvB (DREAM) transcriptional repressor complex is best known as a negative regulator of cell cycle progression. We previously established that the 5-subunit MuvB complex innately functions as the mediator of DRM target gene repression in Caenorhabditis elegans. How the DREAM complex mediates transcriptional repression remains an outstanding question, primarily because MuvB complex activity is required for viability, limiting the utility of mutational analyses. To overcome such limitations, we are utilizing the Auxin-Inducible Degron (AID) system to precisely and rapidly degrade MuvB subcomplex components in C. elegans. Using CRISPR/Cas9-mediated genomic editing, we degron-tagged two key MuvB components, lin-9 and lin-54, and crossed each into a transgenic strain expressing the Arabidopsis thaliana TIR1 E3 ubiquitin ligase in somatic cells. We expected that auxin-induced degradation of either LIN-9, which we hypothesize functions as a core structural component of MuvB, or LIN-54, which acts as the sole DNA-binding component in MuvB, would lead to upregulation of DREAM target genes. Interestingly, we observed that degradation of the LIN-54 but not LIN-9 caused upregulation of DREAM target genes within a 6-hour auxin treatment timecourse in L1 larvae. Our results suggest that MuvB chromatin localization mediated by LIN-54 is critical for DREAM target gene repression. These studies lay the foundation for future degron-mediated analyses assessing DREAM complex function.

Holmstrom, Christian et al. (2021) International Worm Meeting "Screening bacterial isolates for novel therapeutics: a CURE approach"

Holmstrom, Christian, Washeleski, Emily, Goetsch, Paul

[International Worm Meeting, 2021]

Natural products, also called secondary metabolites, are historically the most effective source for therapeutics. The microbial branch of the tree of life remains the largest untapped resource of secondary metabolites, and Caenorhabditis elegans is an ideal platform to screen through bacterial strains for useful drugs. Variation of C. elegans bacterial food sources have uncovered fitness benefits and costs beyond what can be explained by just changes in nutritional content. We hypothesize that altering bacterial food sources will uncover strains that harbor unique secondary metabolites that suppress conserved cell signaling and regulation pathways. To test our hypothesis, the spring semester cohort of the genetics teaching lab at Michigan Tech University piloted a Course-Based Undergraduate Research Experience (CURE) aimed towards isolating and screening wild bacterial species for enhancement or suppression of the C. elegans Sythetic Multivulval (SynMuv) phenotype. The SynMuv phenotype occurs when a SynMuv class A gene mutation is paired with a a SynMuv class B gene mutation. Each gene class redundantly antagonize RTK/Ras/MAPK signaling that directs vulval cell differentiation during development. In this 3-month-long course, we harvested environmental samples from soil, snow, ice, and compost. After isolating bacterial colonies, we performed gram-staining and 16S sequencing to identify 1 isolate per student. We next tested bacterial toxicity and food preference compared to OP50 E. coli control. We then assessed whether feeding wild bacterial isolates increased penetrance of the multivulva phenotype in the SynMuv A strain lin-8(n2731) or the SynMuv B strain lin-52(bn151). In parallel, we assessed whether feeding wild bacterial isolates decreased penetrance of the multivulva phenotype in the double mutant lin-8(n731); lin-52(b151). Enhancement or suppression of the single or double mutant strains would suggest that the bacterial isolate harbors a secondary metabolite that suppress the conserved RTK/Ras/MAPK signaling pathway or its negative regulators. Altogether, our CURE component of the genetics teaching lab provides students with the experience needed to establish their path towards their own individual undergraduate research journey.

Sommer RJ et al. (1994) Worm Breeder's Gazette "Evolution of Vulva Formation: Part IV: Variation in AC Position Can Cause a Shift of Vulva Formation Towards P(4-6).p."

Sternberg PW, Sommer RJ

[Worm Breeder's Gazette, 1994]

Evolution of vulva formation: Part IV: Variation in AC position can cause a shift of vulva formation towards p(4- 6).p Ralf J. Sommer & Paul W. Sternberg, HHMI & California Institute of Technology, Division of Biology 156-29, Pasadena, CA

Snider, Spencer et al. (2021) International Worm Meeting "DREAM interrupted: Using CRISPR/Cas9-targeted mutagenesis to assess DREAM complex formation and function"

Hurt, Garret, Richards, Alex, Snider, Spencer, Goetsch, Paul

[International Worm Meeting, 2021]

Formation of the highly conserved 8-subunit DREAM (Dp, Retinoblastoma-like, E2F, and MuvB) complex culminates in direct repression of cell cycle and developmental genes. We are interested in what determinants of DREAM complex formation are essential for transcriptional repression of target genes. Structural studies on mammalian proteins identified key interaction interfaces between the 3 major components of DREAM, the E2F-DP transcription factor heterodimer (EFL-1 and DPL-1), the Retinoblastoma-like pocket protein (LIN-35), and the 5-subunit MuvB subcomplex (LIN-9, LIN-37, LIN-52, LIN-53, and LIN-54). For example, MuvB interacts with LIN-35 via an LxCxE interacting motif on LIN-52. LIN-35 also interacts directly with the EFL-1 transactivation domain via its pocket domain. DNA-binding by the E2F-DP transcription factor heterodimer and the MuvB subunit LIN-54 coordinate DREAM complex localization to chromatin. We hypothesize that MuvB chromatin occupancy, aided and stabilized by its association with E2F-DP and the pocket protein, establishes and maintains target gene repression. Using CRISPR/Cas9-targeted mutagenesis, we disrupted the interaction between MuvB and the C. elegans pocket protein LIN-35. We expected that severing MuvB from the complex would destabilize DREAM component assembly on chromatin and its repression of target genes. Instead, we observed that disrupting LIN-35-MuvB association did not affect DREAM chromatin occupancy, but we did observe upregulation of DREAM target genes. To continue our evaluation of our model of DREAM complex formation, we identified the conserved amino acid residues that likely mediate the protein-protein and protein-DNA associations for C. elegans DREAM. This analysis lays the groundwork for future application of our CRISPR/Cas9 functional genomics pipeline to establish how the molecular events that drive DREAM complex function contribute to target gene repression.

Paul Sternberg (2002) Worm Breeder's Gazette "Position at Caltech, CA"

Paul Sternberg

[Worm Breeder's Gazette, 2002]

CURATOR. Will annotate gene functions in C. elegans , using Gene Ontology (Nature Genetics [2000], vol. 25, pp. 25-29). Duties include: analysing gene functions in the primary literature; judging the optimal description of these functions in Gene Ontology, inventing new terms for Gene Ontology where necessary; and incorporating these descriptions into Wormbase. A Ph.D. in some area of biology and substantial C. elegans experience are required. The successful job candidate will have broad scientific erudition, verbal articulacy, and creative intelligence as well as patience and a willingness to work hard. Computer literacy in UNIX or Linux is a plus, but is not required. Direct inquiries to Paul Sternberg (pws@its.caltech.edu).