Diverse chromatin remodeling genes antagonize the Rb-involved SynMuv pathways in C. elegans

Abstract

In Caenorhabditis elegans, vulval cell-fate specification involves the activities of multiple signal transduction and regulatory pathways that include a receptor tyrosine kinase/Ras/mitogen-activated protein kinase pathway and synthetic multivulva (SynMuv) pathways. Many genes in the SynMuv pathways encode transcription factors including the homologs of mammalian Rb, E2F, and components of the nucleosome-remodeling deacetylase complex. To further elucidate the functions of the SynMuv genes, we performed a genome-wide RNA interference (RNAi) screen to search for genes that antagonize the SynMuv gene activities. Among those that displayed a varying degree of suppression of the SynMuv phenotype, 32 genes are potentially involved in chromatin remodeling (called SynMuv suppressor genes herein). Genetic mutations of two representative genes (zfp-1 and mes-4) were used to further characterize their positive roles in vulval induction and relationships with Ras function. Our analysis revealed antagonistic roles of the SynMuv suppressor genes and the SynMuv B genes in germline-soma distinction, RNAi, somatic transgene silencing, and tissue specific expression of pgl-1 and the lag-2/Delta genes. The opposite roles of these SynMuv B and SynMuv suppressor genes on transcriptional regulation were confirmed in somatic transgene silencing. We also report the identifications of ten new genes in the RNAi pathway and six new genes in germline silencing. Among the ten new RNAi genes, three encode homologs of proteins involved in both protein degradation and chromatin remodeling. Our findings suggest that multiple chromatin remodeling complexes are involved in regulating the expression of specific genes that play critical roles in developmental decisions.

Figure 1. The C. elegans zfp-1 Gene

(A) Schematic illustration of the transcripts and proteins of zfp-1 (http://www.wormbase.org). In zfp-1(ok554) mutant, the fifth and sixth exons were deleted and 2 nucleotides (CG) were inserted. Therefore, the short isoform, ZFP-1S, is unlikely to produce any proteins and the long isoform is expected to generate a truncated protein that lacks the OM-LZ motifs in the mutant. (B) Diagram of the functional motifs of the long isoform of ZFP-1 and the sequence alignment of the OM and LZ motifs. C. elegans ZFP-1 and its homologs from human (hAF10) and Drosophila (Alhambra) are compared.

Figure 2. The SynMuv Suppressor Genes Antagonize the SynMuv B Genes on Germline-Soma Distinction and pgl-1 Expression

(A) The early larval arrest phenotype associated with the SynMuv B mutation mep-1(lf) was rescued by RNAi of mrg-1. The growth-arrested L1 larvae of mep-1(lf) (arrowheads) and the rescued adults of mep-1(lf) treated with mrg-1(RNAi) (arrow) are indicated. RNAi of 11 other SynMuv suppressor genes displayed a varying degree of rescue (Table 3). (B) Quantification of relative mRNA levels of the germline specific gene pgl-1 in animals with the genotype indicated. Only three strong larval arrest rescuers, mes-4, mrg-1, and isw-1, were assayed here. rpl-26 was used as the internal reference. Mean values and ranges of the pgl-1/rpl-26 ratios based on three qRT-PCR trials are shown. (C–K) Immunostaining using an anti–PGL-1 antibody shows that mep-1(q660) and mrg-1(RNAi) have opposite effects on ectopic expression of pgl-1 in soma. (C–E) Immunofluoresence micrographs of L1 larvae stained with an anti-PGL antibody (red). The genotype of each animal is indicated at the bottom of the corresponding column. (F–H) DAPI staining (blue) of the same animals to indicate nuclei. (I–K) The merged micrographs. White arrowheads indicate germline cells. Arrows indicate the hypodermal cells. The mep-1 mutation (D, G, and J) as well as several other SynMuv B genes (not shown, see text) caused ectopic staining of PGL-1 in somatic cells such as hypodermal cells. This ectopic staining was suppressed by mrg-1(RNAi) (E, H, and K) and RNAi of two other SynMuv suppressor genes (isw-1 and mes-4)(not shown, text). Bar: 100 μm for A, 10 μm for C-K.

Figure 3. Identification of Genes Required for Robust RNAi

WT (N2) young adult hermaphrodites were co-injected with 300 ng/μl of each candidate dsRNA and 75 ng/μl of mom-2 dsRNA. rde-1(RNAi) was used as a positive control, while unc-15(RNAi), unc-22(RNAi), and lin-1(RNAi) were used as negative controls. The horizontal line indicates the cut-off for viability (>30%) that was used to define the positive genes reported in Table 3. All positive genes displayed significant differences from the negative controls with p < 0.01 (t test). The positive candidates include three genes (gfl-1, mes-4, and zfp-1) that were previously identified by Dudley et al. using the same approach [50]. Error bars represent standard error.

Figure 4. Somatic Trangene Silencing by tam-1(lf) Is Desilenced by RNAi of the SynMuv Suppressor Genes and Dicer

(A–B) myo-3 promoter driving-GFP reporters (myo-3::Ngfp-lacZ, myo-3::Mtgfp) displayed significant transgene silencing in the somatic tissues (mainly the muscles) in the tam-1(cc567) mutant (B), but not in the WT animals (A) [11]. (C–F) RNAi of the SynMuv suppressors, mes-4 (C), mrg-1 (D), and isw-1 (E) as well as a non-SynMuv suppressor gene, dcr-1 (F), restored GFP expression in the tam-1(cc567) mutant. dcr-1 encodes the C. elegans Dicer protein that is required for RNAi [81,82]. Bar: 100 μm. (G–H) The relative levels of gfp/lacZ of a transgene ccIs4251 (myo-3::Ngfp-lacZ, myo-3::Mtgfp) [11] were measured by qRT-PCR in various genetic background indicated. The level of ama-1 mRNA, encoding an RNA PolII, was used as the internal reference. Mean values and ranges of the lacZ/ama-1 ratios based on three qRT-PCR trials are shown.

Figure 5. The SynMuv Suppressor Genes Are Required for Germline Transgene Silencing in WT Animals and for the lag-2::gfp Ectopic Expression in lin-15B(n744)

(A–D) DIC (A) and (C) and GFP fluorescence (B) and (D) images of hermaphrodite germline from transgenic strain PD7271 that contains the multicopy let-858::gfp reporters [75]. Brackets indicate the region of germ cell nuclei. The transgene was silenced in WT, but desilenced in animals treated with mrg-1(RNAi). RNAi of seven other SynMuv suppressors also displayed a similar effect (Table 3). Bar: 10 μm. (E–H) GFP fluorescence images of mid-L4 larvae carrying the lag-2::gfp transgene. (E) WT animals display a strong expression of the transgene in distal tip cells (arrowheads) and the vulva (asterisks). (F–G) a strong ectopic expression of the transgene in the intestine (arrows) is seen in two SynMuv mutants. (H) RNAi of isw-1 suppressed the ectopic expression of lag-2::gfp in the intestine of the lin-15B(n744) mutant, but not its expression in distal tip cells and vulval cells. RNAi of 14 other SynMuv suppressors also displayed a similar effect (Table 3). Bar: 100 μm.