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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00597

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The Snail-like CES-1 protein of C. elegans can block the expression of theBH3-only cell-death activator gene egl-1 by antagonizing the function of bHLH proteins

Marion Thellmann^*, Julia Hatzold^* and Barbara Conradt^,

Max-Planck-Institute of Neurobiology, Am Klopferspitz 18a, D-82152 Planegg-Martinsried, Germany

View larger version (70K): [in a new window]   Fig. 1. egl-1 is transcriptionally active in the NSM sister cells but not in the NSMs or undead NSM sister cells. (A) Merged Nomarski and epifluorescence image of the anterior bulb of the pharynx of a ced-3(n717) L1 larva carrying an integrated Pegl-1his24-gfp reporter construct (bcIs37). Pegl-1his24-gfp is expressed in the NSM sister cell but not in the NSM. The complete genotype of the animals scored was ced-3(n717); bcIs37. (B) Merged Nomarski and epifluorescence image of the anterior bulb of the pharynx of a ces-1(703gf); ced-3(n717) L1 larva carrying an integrated Pegl-1his24-gfp reporter construct (bcIs37). Pegl-1his24-gfp is not expressed in the undead NSM sister cell or the NSM. The complete genotype of the animals scored was unc-87(e1216) ces-1(n703gf); ced-3(n717); bcIs37.

View larger version (39K): [in a new window]   Fig. 2. Region B of the egl-1 locus is required for the specification of the NSM sister cell death. (A) Schematic representation of the egl-1 locus on linkage group V. pBC119, pBC13, pBC11, pBC149 and pBC165 represent subclones of the egl-1 rescuing fragment pBC08. The abilities of the fragments to rescue NSM sister cell death (see below) are summarized under `Rescue'. The arrow indicates Region B. (B) pBC08, pBC119 and pBC149 can rescue the death of the NSM sister cells in egl-1(lf) animals. Plasmids containing the fragments indicated were introduced by germline transformation into hermaphrodites of genotype egl-1(n1084 n3082) unc-76(e911); lin-15(n765) carrying an integrated Ptph-1gfp reporter construct (bcIs24) as described in the Materials and Methods. Rescue of NSM sister cell death was analysed as described in the Materials and Methods (n=100-240). For every plasmid, several independent transgenic lines were generated and characterized (n=4-7). (C) Region B of the egl-1 locus is highly conserved. Alignment of the sequence of C. elegans Region B (C. elegans sequence VF23B12L bp 2659-2792 and C. elegans cosmid F23B12 bp 1-218) with the sequence of the corresponding region of C. briggsae (C. briggsae cosmid G12D19.2 bp 10590-10248). Identical nucleotides are shaded. The four conserved Snail-binding sites are indicated by boxes.

View larger version (77K): [in a new window]   Fig. 3. CES-1 binds to the Snail-binding sites/E-boxes in Region B of the egl-1 locus in vitro. A GST-CES-1 fusion protein binds to wild-type but not mutant Region B in vitro. Increasing amounts of bacterially expressed, affinity-purified GST-CES-1 fusion protein [0 mol (lanes 1, 7 and 13), 8x10-13 mol (lanes 2, 8 and 14), 2x10-12 mol (lanes 3, 9 and 15), 4x10-12 mol (lanes 4, 10 and 16), 6x10-12 mol (lanes 5, 11 and 17), 8x10-12 mol (lanes 6, 12 and 18) were incubated with 7 ng of radioactively labeled wild-type Region B with four intact Snail-binding sites (lanes 1-6), or mutant Region B with all four Snail-binding sites mutated to Snail-/E-box- sites (lanes 7-12) or to Snail-/E-box+ sites (lanes 13-18). Electrophoretic mobility shift assays were performed as described in the Materials and Methods. Asterisks indicate protein-DNA complexes with one, two or three CES-1 molecules bound to Region B. Triangle indicates a protein-DNA complex that is most likely a bacterial contaminant bound to Region B.

View larger version (16K): [in a new window]   Fig. 4. A ces-1(lf) mutation is unable to suppress the NSM sister cell survival caused by hlh-2(RNAi) or hlh-3(RNAi). (A) A ces-1(lf) mutation is not able to suppress the NSM sister cell survival induced by hlh-2(RNAi). NSM sister cell survival was analysed as described in the Materials and Methods (n=96-142). dsRNA was delivered by feeding. hlh-6 dsRNA was used for control RNAi. The complete genotype of the animals analysed was as follows hlh-2(RNAi); bcIs25, ces-1(n703 n1434lf); hlh-6(RNAi); bcIs25, ces-1(n703 n1434lf); hlh-2(RNAi); bcIs25. (B) A ces-1(lf) mutation is not able to suppress the NSM sister cell survival induced by hlh-3(RNAi). NSM sister cell survival was analysed as described in the Materials and Methods (n=174-218). dsRNA was delivered by injection. hlh-6 dsRNA was used for control RNAi. The complete genotype of the animals analysed was as follows: hlh-3(RNAi); bcIs25, ces-1(n703 n1434lf); hlh-6(RNAi); bcIs25, ces-1(n703 n1434lf); hlh-3(RNAi); bcIs25. Error bars represent the standard deviation of the average percent survival obtained for the progeny of different injected animals.

View larger version (36K): [in a new window]   Fig. 5. HLH-2/HLH-3 binds to the Snail-binding sites/E-boxes in Region B of the egl-1 locus in vitro. (A) A HLH-2 homodimer and HLH-2/HLH-3 heterodimer bind to wild-type Region B in vitro. Increasing amounts of bacterially expressed, affinity-purified His6-tagged HLH-2 (lanes 2-6), HLH-3 (lanes 7-11) or both HLH-2 and HLH-3 (lanes 12-16) fusion proteins [0 mol (lane 1), 8x10-14 mol (lanes 2, 7 and 12), 2x10-13 mol (lanes 3, 8 and 13), 4x10-13 mol (lanes 4, 9 and 14), 8x10-13 mol (lanes 5, 10 and 15), 2x10-12 mol (lanes 6, 11 and 16)] were incubated with 7 ng of radioactively labeled wild-type Region B. Electrophoretic mobility shift assays were performed as described in the Materials and Methods. Asterisks indicate a DNA-protein complex with one or two heterodimers bound to Region B. (B) A HLH-2/HLH-3 heterodimer still binds to Snail-/E-box+ binding sites in Region B. Increasing amounts of both His6-tagged HLH-2 and HLH-3 [0 mol (lane 1, 7 and 13), 8x10-14 mol (lanes 2, 8 and 14), 2x10-13 mol (lanes 3, 9 and 15), 4x10-13 mol (lanes 4, 10 and 16), 8x10-13 mol (lanes 5, 11 and 17), 2x10-12 mol (lanes 6, 12 and 18)] were incubated with 7 ng of radioactively labeled wild-type Region B with four intact Snail-binding sites (lanes 1-6), or mutant Region B with all four Snail-binding sites mutated to Snail-/E-box- sites (lanes 7-12) or to Snail-/E-box+ sites (lanes 13-18). Electrophoretic mobility shift assays were performed as described in Materials and Methods. Asterisks indicate a DNA-protein complex with one or two heterodimers bound to Region B.

View larger version (66K): [in a new window]   Fig. 6. HLH-2 is most probably present in the NSM sister cells. (Upper panel) Anti-HLH-2 and anti-GFP immunofluorescence staining of a 1.5-fold embryo of genotype bcIs1 (Pegl-1gfp); ced-3(n717), and overlay (from left to right). The images presented are stacks of a confocal series through an entire embryo. The four double positive cells found in embryos of this genetic background are indicated by arrows. (Lower panel) Anti-HLH-2 and anti-GFP immunofluorescence staining of a 1.5-fold embryo of genotype unc-87(e1216) ces-1(n703gf); bcIs1 (Pegl-1gfp); ced-3(n717), and overlay. The images presented are stacks of a confocal series through an entire embryo. The two double positive cells found in embryos of this genetic background are indicated by arrows.

View larger version (33K): [in a new window]   Fig. 7. CES-1 and an activator of egl-1 expression function through the Snail-binding sites/E-boxes in Region B in vivo. Plasmids pBC08 (wild-type Region B) (A), pBC181 (Region B with Snail-/E-box- sites) (B) or pBC182 (Region B with Snail-/E-box+ sites) (C) were introduced by germline transformation into hermaphrodites of genotype ces-2(n732ts); bcIs25; egl-1(n1084 n3082) unc-76(e911) as described in the Materials and Methods. Transgenic lines were cultured at 15°C and 25°C. Rescue of the NSM sister cell death was analysed as described in the Materials and Methods (n=60-222). For every plasmid, several independent transgenic lines were generated and analysed (n=3-6).

View larger version (12K): [in a new window]   Fig. 8. Competition between CES-1 and HLH-2/HLH-3 for binding to the egl-1 promoter specifies the cell-death fate of the NSM sister cells. In wild-type animals, the level of CES-1 in the NSM sister cells might not be sufficient to compete with HLH-2/HLH-3 for binding to Region B, resulting in the activation of egl-1 transcription and NSM sister cell death. In ces-1(gf) or ces-2(lf) animals, elevated levels of CES-1 in the NSM sister cells might successfully compete with HLH-2/HLH-3 for binding to Region B, resulting in egl-1 repression and NSM sister cell survival.

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