Goetsch, P. D. | Search bioRxiv

bioRxiv2026

The WormFood CURE: Screening for bioactive metabolites that antagonize the Caenorhabditis elegans Ras signaling pathway

Course-based Undergraduate Research Experiences (CUREs) provide an accessible, scalable platform for scientific discovery. Here, we present the WormFood CURE, which mines environmental bacterial isolates for bioactive secondary metabolites using Caenorhabditis elegans phenotype suppression as a functional readout. Utilizing the multivulva (Muv) phenotype, our pilot cohort interrogated 41 wild bacterial isolates for suppression of Ras/MAPK signaling. We identified one Bacillus safensis isolate BAC-08 and one Bacillus altitudinis isolate BAC-44 that significantly inhibited ectopic vulval precursor cell (VPC) induction in Muv strains when fed as a live food source. BAC-08 and BAC-44 also significantly affected wild-type nematode development and growth. Metabolic pathway reconstruction from annotated genome assemblies did not support nutritional deficiency as the potential mechanism; instead, we observed that methanol-soluble intracellular extracts from BAC-44 were sufficient to inhibit pseudovulvae growth. We concluded that the observed Muv suppression is likely driven by a secondary metabolite effect. Comparative genomic analysis further identified unique biosynthetic gene clusters (BGCs) present in both BAC-08 and BAC-44 isolates compared to the other isolated Bacillus species. Altogether, our study demonstrates that the WormFood CURE model successfully identifies novel bacterial-genetic interactions, providing a scalable platform for discovery of new natural microbial products that modulate conserved eukaryotic signaling pathways.

genetics↗

bioRxiv2019

DREAM Interrupted: Severing MuvB from DREAM’s pocket protein impairs gene repression but not DREAM assembly on chromatin

The mammalian pocket protein family, which includes the Retinoblastoma protein (pRb) and Rb-like pocket proteins p107 and p130, regulates entry into and exit from the cell cycle by repressing cell cycle gene expression. Although pRb plays a dominant role in mammalian systems, p107 and p130 are the ancestral pocket proteins. The Rb-like pocket proteins interact with the highly conserved 5-subunit MuvB complex and an E2F-DP transcription factor heterodimer, forming the DREAM (for Dp, Rb-like, E2F, and MuvB) complex. DREAM complex assembly on chromatin culminates in repression of target genes mediated by the MuvB subcomplex. Here, we examined how the Rb-like pocket protein contributes to DREAM formation and function by disrupting the interaction between the sole Caenorhabditis elegans pocket protein LIN-35 and the MuvB subunit LIN-52 using CRISPR/Cas9 targeted mutagenesis. A triple alanine substitution of LIN-52s LxCxE motif severed LIN-35-MuvB association and caused classical DREAM mutant phenotypes, including synthetic multiple vulvae, high-temperature arrest, and ectopic expression of germline genes in the soma. However, RNA-seq revealed limited upregulation of DREAM target genes when LIN-35-MuvB association was severed, as compared to gene upregulation following LIN-35 loss. Based on chromatin immunoprecipitation, disrupting LIN-35-MuvB association did not affect the chromatin localization of E2F-DP, LIN-35, or MuvB components. In a previous study we showed that in worms lacking LIN-35, E2F-DP and MuvB chromatin occupancy was reduced genome-wide. With LIN-35 present but unable to associate with MuvB, our present study suggests that the E2F-DP-LIN-35 interaction promotes E2F-DPs chromatin localization, which we hypothesize supports MuvB chromatin occupancy indirectly through DNA. Altogether, this study highlights how the pocket proteins association with MuvB supports DREAM function but is not required for DREAMs chromatin occupancy.

molecular biology↗