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First published online 14 June 2006
doi: 10.1242/dev.02444

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C. elegans ISWI and NURF301 antagonize an Rb-like pathway in the determination of multiple cell fates

Erik C. Andersen, Xiaowei Lu^* and H. Robert Horvitz

Howard Hughes Medical Institute, Department of Biology, Room 68-425, MIT, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

View larger version (55K): [in a new window]   Fig. 1. Loss-of-function mutations in isw-1 suppress the synMuv phenotype of lin-53; lin-15A mutants. (A) Bright-field micrographs of C. elegans 24 hours after the fourth larval stage. Vulvae and psuedovulvae are marked with white triangles and black triangles, respectively. (Top) The wild-type strain has no pseudovulvae. (Middle) The synMuv mutant lin-53(n833); lin-15A(n767) has three pseudovulvae. (Bottom) The lin-53(n833); isw-1(n3294); lin-15A(n767) triple mutant has no pseudovulvae. Bacteria, embryos or bubbles were removed from the images. Scale bar: 100 µm. (B) The genomic structure of isw-1. Exons are indicated by black boxes; 5' and 3' untranslated regions are indicated by white boxes. Predicted translation initiation and termination codons and the polyadenylation site are shown. The locations of the missense mutations and deletion allele are indicated. (C) A representation of the domain structure of the ISW-1 protein. ISW-1 is similar to Drosophila ISWI and other family members, especially in the named domains (see text). The amino acid substitutions of the two missense alleles and the locations of these mutations and the deletion allele are indicated. An alignment of ISW-1 with S. cerevisiae SWI2/SNF2 and Drosophila ISWI is in Fig. S1.

View larger version (28K): [in a new window]   Fig. 2. The nurf-1 locus is predicted to encode multiple proteins. Each protein shares domains with Drosophila NURF301. Only the isoform containing the HMGA domain is required for antagonism of the synMuv genes. (A) Genomic structures of the nurf-1 isoforms. Exons are indicated by black boxes; 5' and 3' untranslated regions are indicated by white boxes. The predicted translation initiation and termination codons and the polyadenylation sites are indicated. The locations of the two deletion alleles are shown. (B) A representation of the NURF-1 protein and domain structure. (Top) The Drosophila NURF301 protein. (Bottom) The predicted protein products of the nurf-1 gene. An alignment of the predicted functional domains of NURF-1 with those of Drosophila NURF301 is presented in Fig. S3. (C) Reduction of nurf-1a function suppresses the synMuv phenotype of lin-15AB mutants. RNAi of the nurf-1a isoform (pEA147) but not of the nurf-1b, nurf-1c, nurf-1d, nurf-1e isoforms (pEA30) caused suppression of the lin-15AB synMuv phenotype. In addition, deletion of the nurf-1a isoform (n4293) but not of the nurf-1b, nurf-1c, nurf-1d, nurf-1e isoforms (n4295) caused suppression of the lin-15AB synMuv phenotype. M+ denotes progeny of heterozygous mutant hermaphrodites, such progeny might retain maternally inherited nurf-1a gene products.

View larger version (46K): [in a new window]   Fig. 3. Loss of isw-1 function suppresses multiple cell-fate defects caused by mutations in class B synMuv genes. (A) isw-1(n3294) suppresses the ectopic expression of PGL-1 in the soma of L1 larvae in the class B synMuv mutant lin-15B(n744). Anti-PGL-1 staining is shown in green and nuclei (4,6-diamidino-2-phenylindole (DAPI) staining) are shown in blue. Scale bars: 10 µm. (B) Loss of isw-1 function suppresses the Tam phenotype of lin-15B animals and represses normal transgene expression. The ccIs4251 reporter is a simple repetitive transgene that expresses GFP in the nuclei of body-wall muscles. From left to right, ccIs4251 was expressed in the following backgrounds: wild type, isw-1(RNAi), lin-15B(n744) and isw-1(RNAi); lin-15B(n744). Scale bars:100 µm. A quantification of the Tam experiments is found in Fig. S4. (C) isw-1 is required for the RNAi sensitivity of the class B synMuv mutant lin-15B(n744). After exposure of animals to cel-1 RNAi, the number of arrested L2 larvae was scored in at least three independent experiments. The average percent of L2 arrested larvae is shown. Error bars indicate standard deviations. (D) The activity of isw-1 and nurf-1 are required for the larval-lethal phenotypes of mep-1(q660) and let-418(n3536) mutants. The percent of sterile adults present at 25°C reflects the suppression of larval lethality. Error bars indicate standard deviations.