A Forward Genetic Screen for Suppressors of Somatic P Granules inCaenorhabditis elegans

Abstract

In Caenorhabditis elegans, germline expression programs are actively repressed in somatic tissue by components of the synMuv (synthetic multi-vulva) B chromatin remodeling complex, which include homologs of tumor suppressors Retinoblastoma (Rb/LIN-35) and Malignant Brain Tumor (MBT/LIN-61). However, the full scope of pathways that suppress germline expression in the soma is unknown. To address this, we performed a mutagenesis and screened for somatic expression of GFP-tagged PGL-1, a core P-granule nucleating protein. Eight alleles were isolated from 4000 haploid genomes. Five of these alleles exhibit a synMuv phenotype, whereas the remaining three were identified as hypomorphic alleles of known synMuv B genes, lin-13 and dpl-1. These findings suggest that most suppressors of germline programs in the soma of C. elegans are either required for viability or function through synMuv B chromatin regulation.

ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in Caenorhabditis elegans

“…To discover regulators of P-granule accumulation, EMS mutagenesis was performed on a C . elegans strain with an integrated transgene expressing the constitutive P-granule component, PGL-1, tagged with GFP ( Fig 1A ) [ 35 ]. The F2 generation was then screened for accumulation of large and more intense PGL-1::GFP granules ( Fig 1B ), similar to those previously observed with csr-1 RNAi [ 12 ].…”

“…Hawaiian Variant Mapping was used in combination with genome-wide sequencing and the Cloudmap pipeline to identify the remaining alleles on chromosomes I and IV [ 35 , 36 ]. On chromosome I, mutations were identified in three known csr-1 cofactors.…”

“…Two-thousand F1 progeny were cloned to individual plates, and F2 grandchildren were screened under a Leica M165FC fluorescence stereomicroscope for enlarged, bright PGL-1::GFP granules. This screen was done in parallel with a now published screen looking for somatic PGL-1::GFP expression [ 35 ]. To quantify P-granule size, cross-sections of the surface of ten germlines/strain were acquired with a 63X objective and fixed exposure conditions, and a fixed threshold was used to outline and measure the area of P granules using ImageJ.…”

“…CB4856 (Hawaiian) males were crossed into mutant strains. F1 cross progeny were picked to new plates, and on average 500 F2s worms with the enlarged P-granule phenotype were handpicked from each cross and pooled for whole genome sequencing [ 35 , 62 ]. Samples were sequenced on Illumina HiSeq2500 and NextSeq systems.…”

“…To assay RNAi enhancement, approximately 60 L1s were placed on each of five his-44 RNAi feeding plates for each strain, and animals arrested during larval development were scored 3 days later [ 35 , 64 ].…”