The RING finger/B-box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans

Abstract

Context-dependent gene silencing is used by many organisms to stably modulate gene activity for large chromosomal regions. We have used tandem array transgenes as a model substrate in a screen for Caenorhabditis elegans mutants that affect context-dependent gene silencing in somatic tissues. This screen yielded multiple alleles of a previously uncharacterized gene, designated tam-1 (for tandem-array-modifier). Loss-of-function mutations in tam-1 led to a dramatic reduction in the activity of numerous highly repeated transgenes. These effects were apparently context dependent, as nonrepetitive transgenes retained activity in a tam-1 mutant background. In addition to the dramatic alterations in transgene activity, tam-1 mutants showed modest alterations in expression of a subset of endogenous cellular genes. These effects include genetic interactions that place tam-1 into a group called the class B synMuv genes (for a Synthetic Multivulva phenotype); this family plays a negative role in the regulation of RAS pathway activity in C. elegans. Loss-of-function mutants in other members of the class-B synMuv family, including lin-35, which encodes a protein similar to the tumor suppressor Rb, exhibit a hypersilencing in somatic transgenes similar to that of tam-1 mutants. Molecular analysis reveals that tam-1 encodes a broadly expressed nuclear protein with RING finger and B-box motifs.

Figure 1

tam-1 effects on a repetitive myo-3::gfp transgene. An integrated myo-3::gfp transgene was examined in wild-type and tam-1(cc567) mutant backgrounds. (A) Wild-type and (B) tam-1(cc567) animals photographed under fluorescent illumination showing GFP in bodywall muscles (the array produces GFP in both nuclear and mitochondrial compartments). Scale bars, 55 μm. Quantitative measurements of GFP fluorescence in wild-type and tam-1(cc567) mutant backgrounds. Differences in fluorescence level were most striking in the central region of the animal; thus quantitations were carried out in bodywall muscle nuclei in this region. Eight animals were analyzed for each genetic background. Fluorescence was quantitated for six nuclei in each animal with a Nikon U-III multipoint sensor system. Linearity of fluorescence measurements in the range of assay was confirmed by a series of neutral density filters. (wild-type mean= 140.9, wild-type (s.e.m.)= 5.9; tam-1 mean = 7.2, tam-1 s.e.m. = 0.4). A second measure of tam-1 effects on the expression of a repeated transgene is observed with a lin-3 transgene (syIs1). The average induction of vulval precursor cells (VPCs) caused by the expression of syIs1 in wild type is 5.8 VPCs induced (n = 20), however, in tam-1(sy272), there were 3.2 VPCs induced (n = 1 0) (data not shown).

Figure 2

Assessment of tam-1 effects in a nonrepetitive context. A nonrepetitive transgene array (ccEx6172) was compared in wild-type and tam-1(cc567) mutant backgrounds. (A) Wild-type and tam-1(cc567) animals photographed under fluorescent illumination showing GFP in bodywall muscles (the array produces GFP in nuclei). Scale bar, 55 μm. (B) Quantitation of fluorescence was carried out in six nuclei in the central region of the body for each of eight animals as in Fig. 1B. (wild-type mean = 15.2, wild-type s.e.m. = 1.4; tam-1 mean = 15.6, tam-1 s.e.m. = 1.7). (C) Analysis of repetitive (simple) and nonrepetitive (complex) transgene activity in wild-type, tam-1(cc567) and lin-35(n745) backgrounds. Transgenes are as described in Materials and Methods.

Figure 3

tam-1 has properties of a class-B synMuv gene. (A) lin-15A(n767); tam-1(cc567) double mutants are Muv (arrowheads indicate the position of vulvae). Scale bar, 27.5 μm. Other class-B synMuv mutants have decreased transgene expression. (B) The expression of ccIs4251(myo-3::gfp) is examined in wild-type and homozygous lin-15B(n744) animals. Scale bar, 87.5 μm. (C) The expression of the same transgene is also examined in wild-type and homozygous lin-9(n112) animals. Scale bar, 87.5 μm.

Figure 4

Molecular identification of tam-1. (A) Genetic mapping placed the tam-1 locus to the left of the gene unc-46. (B) Pools of cosmids were assayed for rescue of the reduced-transgene-expression phenotype of tam-1(cc567). Cosmid F26G5 was found to rescue the decreased-GFP phenotype of tam-1(cc567); ccIs4251(myo-3::gfp). Rescue was also observed for two other tam-1 alleles, cc587 and sy272. (C) Deletion analysis of cosmid F26G5 led to the identification of an 8.6-kb fragment (L127) with rescuing activity. Analysis of the sequence of this fragment had predicted a single coding region (C. elegans Sequencing Consortium 1998). Creation of a frameshift in a unique AatII site within this coding region abolished rescue. (D) A full-length cDNA was obtained from the Kohara laboratory. Sequencing of this cDNA confirmed that the tam-1 mRNA sequence contains seven exons and six introns. (E) Amino acid sequence of TAM-1. Underlined are the RING finger (solid line) and B-box (broken line). Sequencing of tam-1 alleles (cc567, sy272, and cc587) revealed in each case the conversion of a glutamine codon (CAA) to a nonsense codon (TAA). DNA from tam-1 mutants was amplified by PCR; this was done in several segments covering a region from 288-bp upstream of the ATG to 440-bp downstream of the deduced tam-1 translational stop. PCR products were directly sequenced, with mutations confirmed by sequencing products from at least two different PCR reactions. The three distinct mutations are indicated by arrowheads.

Figure 4

Molecular identification of tam-1. (A) Genetic mapping placed the tam-1 locus to the left of the gene unc-46. (B) Pools of cosmids were assayed for rescue of the reduced-transgene-expression phenotype of tam-1(cc567). Cosmid F26G5 was found to rescue the decreased-GFP phenotype of tam-1(cc567); ccIs4251(myo-3::gfp). Rescue was also observed for two other tam-1 alleles, cc587 and sy272. (C) Deletion analysis of cosmid F26G5 led to the identification of an 8.6-kb fragment (L127) with rescuing activity. Analysis of the sequence of this fragment had predicted a single coding region (C. elegans Sequencing Consortium 1998). Creation of a frameshift in a unique AatII site within this coding region abolished rescue. (D) A full-length cDNA was obtained from the Kohara laboratory. Sequencing of this cDNA confirmed that the tam-1 mRNA sequence contains seven exons and six introns. (E) Amino acid sequence of TAM-1. Underlined are the RING finger (solid line) and B-box (broken line). Sequencing of tam-1 alleles (cc567, sy272, and cc587) revealed in each case the conversion of a glutamine codon (CAA) to a nonsense codon (TAA). DNA from tam-1 mutants was amplified by PCR; this was done in several segments covering a region from 288-bp upstream of the ATG to 440-bp downstream of the deduced tam-1 translational stop. PCR products were directly sequenced, with mutations confirmed by sequencing products from at least two different PCR reactions. The three distinct mutations are indicated by arrowheads.

Figure 5

Sequence alignments of RING finger and B-box motifs of TAM-1 with related segments of other proteins. Conserved cysteine and histidine residues are underlined. A limited number of members of each family were chosen arbitrarily. GenBank contains >100 entries with RING finger motifs. Alignment of RING finger motifs: Numbering below each conserved cysteine refers to zinc binding ligands cysteine 1-cysteine 7 (Saurin et al. 1996) . Percent identity refers to just the RING finger domain. Alignment of B-box motifs: Sources for RING fingers and B-Box motifs used in this comparison are RAD18 (Jones et al. 1988); RPT-1 (Patarca et al. 1988); PSC, SU(Z)2 (Brunk et al. 1991); SS-A/Ro (Chan et al. 1991); XNF7 (Reddy et al. 1991); RAD16 (Bang et al. 1992); PML (Kastner et al. 1992); PAF-1 (Shimozawa et al. 1992); PwA33 (Bellini et al. 1993); MEL-18 (Ishida et al. 1993); PAR-2 (Levitan et al. 1994); BRCA1 (Miki et al. 1994); HT2A (Fridell et al. 1995); T18 (Miki et al. 1991; LeDoyerin et al. 1995); RFP (Cao et al. 1997); RING1 (Satijn et al. 1997); BMI-1 (Hemenway et al. 1998); and XL43, XL75 (Perrin and Lacroix 1998).

Figure 6

Immunofluorescence detection of TAM-1 protein in nuclei. (A) Embryo population and (C) an adult stained with mouse anti-TAM-1 and visualized with a secondary goat anti-Cy3 antibody. (B,D) Corresponding images of DNA localization, visualized with DAPI. Photos shown are from antisera to TAM-1 residues 594–712. Similar results were seen with antisera to TAM-1 residues 778–856. Signal in C has been augmented by electronic contrast enhancement. Scale bars, 27.5 μm.