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First published online May 30, 2007
doi: 10.1242/10.1242/dev.02855

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EEL-1, a Hect E3 ubiquitin ligase, controls asymmetry and persistence of the SKN-1 transcription factor in the early C. elegans embryo

¹ Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
² Howard Hughes Medical Institute, Seattle, WA 98109, USA.
³ Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.
⁴ Children's Hospital and Regional Medical Center, Department of Hematology and Oncology, Seattle, WA 98105, USA.
⁵ Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA.
⁶ Committee on Developmental Biology, University of Chicago, Chicago, IL 60637, USA.

View larger version (90K): [in this window] [in a new window]   Fig. 1. Loss of eel-1 enhances efl-1(se1) to a Mex phenotype. The top row contains Nomarski images of terminally developed C. elegans embryos from eel-1(RNAi), efl-1(se1) and efl-1(se1);eel-1(RNAi) mothers incubated at 20°C. Note that eel-1(RNAi) and efl-1(se1) embryos form enclosed pretzel-shaped embryos and appear as wild type. By contrast, efl-1(se1);eel-1(RNAi) embryos do not enclose and fail to elongate. The center row shows comparable embryos stained for pharyngeal muscle to demonstrate that efl-1;eel-1(RNAi) embryos produce an excess of this tissue in comparison with embryos from either single mutant mother. The bottom row contains images of the MED-1:GFP expression pattern at the 15-cell stage. This expression pattern is wild-type in eel-1(RNAi) (n=25 of 25) and efl-1(se1) (n=22 of 23) single-mutant embryos; MED-1:GFP is expressed only in a subset of descendants of the posterior cell of the 2-cell embryo. In efl-1(se1);eel-1(RNAi) embryos (n=25 of 30), MED-1:GFP is ectopically expressed in descendants of the anterior blastomere of the 2-cell embryo. Embryos are oriented with anterior to the left. Scale bar: 10 µm.

View larger version (8K): [in this window] [in a new window]   Fig. 2. Similarities between C. elegans EEL-1 and the human MULE E3 ligases. This diagram illustrates multiple domains shared between EEL-1 and human MULE. Between each domain of similarity, percentages of identity (I) and similarity (S) are noted. The location at which zu462 is predicted to disrupt EEL-1 is indicated (zig-zag line).

View larger version (113K): [in this window] [in a new window]   Fig. 3. Loss of eel-1 disrupts SKN-1 spatial and temporal regulation. Each pair of rows shows SKN-1 staining (above) and corresponding nuclear staining (DAPI, below) of C. elegans embryos at a given stage (2-cell, 4-cell, 12-cell and 28-cell) from a hermaphrodite of indicated genotype incubated at 20°C. Embryos are oriented with anterior to the left. Scale bar: 10 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 4. SKN-1 asymmetry is disrupted in eel-1(-) and efl-1(-);eel-1(-) C. elegans embryos. (A,B) The posterior:anterior ratio (Post/Ant) of nuclear SKN-1 staining was calculated for individual embryos of the given genotype at the 2-cell (A) or 4-cell (B) stage. (C,D) Nuclear SKN-1 staining was quantitated in equivalent blastomeres for pairs of eel-1(RNAi) and wild-type embryos, and the eel-1(-) to wild-type ratio (eel-1/WT) was calculated for the anterior and posterior blastomere(s). Variability was minimized by performing individual comparisons between pairs of embryos from a single slide: 2-cell (C) and 4-cell (D) stage. The mean±s.e.m. is shown.

View larger version (58K): [in this window] [in a new window]   Fig. 5. EEL-1 interacts with SKN-1 in the yeast two-hybrid assay. (A) The level of two-hybrid interactions between C. elegans EEL-1 and SKN-1. The left column indicates the amino acids of EEL-1 fused to the GAL4 DNA-binding domain, and the central column indicates the amino acids of SKN-1 fused to the GAL4 activation domain. The domain structure of EEL-1 is shown in Fig. 2. SKN-1 has 623 amino acid residues, and its DNA-binding domain comprises residues 549-614. The right column shows the level of growth on selective medium when yeast are transformed with the corresponding plasmids. (B) Two-hybrid interaction between EEL-1 and SKN-1.

View larger version (76K): [in this window] [in a new window]   Fig. 6. The C-terminus of SKN-1 targets GFP for degradation in an EEL-1-dependent manner. Wild-type (left) and eel-1(RNAi) (right) 15-cell C. elegans embryos expressing GFP (top row) or GFP:SKN-1(298-623) fusion protein (bottom row) under control of the pie-1 promoter and pie-1 3'UTR. Embryos are oriented with anterior to the left. Scale bar: 10 µm.

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