The anchor cell (AC)-specific enhancer element of lin-3. (A) The wild-type, but not`G to A', form of ACEL can activateΔ pes-10::gfp transcription in the AC. A base change from guanine (G) to adenine (A) was identified in the lin-3(e1417) allele (`G to A'). The wild-type and`G to A' forms of the putative ACEL elements (59 bp) were cloned into a Δpes-10::gfp enhancer assay vector. The number in parenthesis represents the number of animals in which gfp expression in the AC was examined. (B) Conservation of the ACEL sequence between C. briggsae (CB) and C. elegans (CE). The intron sequence (3′ part of the fourth intron of lin-3) is in lower case letters and the exon sequence (5′ part of the fourth exon of lin-3) is in upper case letters (purple underline). The e1417 mutation, indicated with a dot, is located in the conserved region of the FTZ-F1 NHR binding site, underlined in green. E boxes are underlined in red. Binding sites of POU homeodomain proteins are underlined in blue. The unbroken blue line indicates the site conserved in both species; the broken blue line represents the site that exists only in C. elegans.

Deletion and site-directed mutagenesis analyses of ACEL. (A) Deletion analysis of ACEL. Deletions from the 5′ end of the ACEL were made with the endogenous lin-3 promoter (constructs 1-4). Deletions from the 5′ or the 3′ end of the ACEL were generated by PCR and the DNA fragments were fused with a Δpes-10::gfp enhancer assay vector (constructs 5-8). (B) Site-directed mutagenesis analysis of ACEL. Mutations in the ACEL were generated using PCR and the PCR products were cloned into theΔ pes-10::gfp vector. Construct 1 has no mutations and the others (2-18) have changes in the ACEL as indicated. For each construct, about 30 animals that express gfp in the tail were examined for gfp expression in the AC.

HLH-2 binds to both E-boxes in the ACEL. (A) Synthesis and purification of FLAG-Luciferase and FLAG-HLH-2 proteins. Both proteins were expressed in insect cells using a baculovirus expression system and then purified using Ni²-columns. The purified proteins were resolved on an SDS-PAGE gel for Coomassie staining and immunoblotting with anti-FLAG antibodies. (B) Probe A contains both E-boxes, probes B and C contain only one E-box, and probe D does not contain any E-boxes. The probes contain an extra 70 bp at the 3′ end of the ACEL. Thus, the first E-box (E-1) is near the 5′ end of the probe and the second E-box (E-2) is in the middle of the probe. (C) An EMSA that shows the binding of the HLH-2 protein to both E-boxes in the ACEL. The purified proteins were incubated with the wild-type and the mutated DNA probes of the ACEL (Fig. 3B). F indicates the migration of free DNA probe, B is the FLAG-HLH-2 protein/DNA probe complex, and Supershift is created by adding anti-FLAG antibodies (lanes 6 to 8).

The hlh-2 and nhr genes are involved in vulval induction and the expression of lin-3::gfp in the AC. (A-D) Defective lin-3::gfp expression in the AC was caused by soaking animals in the dsRNA solution against hlh-2. Transgenic animals that contain lin-3::gfp extrachromosomal arrays were soaked in the control dsRNA solution (RNA synthesized from blank vectors) and in the dsRNA solution against hlh-2. (B,D) GFP expression in the AC was examined in the animals that express gfp in pharynx at the early L4 stage. (E-G) Defects in vulval induction and vulval-uterine connection in animals treated with dsRNA against hlh-2. (E) Wild-type vulvae with control RNAi. (F) Defective vulval induction after hlh-2 RNAi. (G) Defective vulval-uterine connection after hlh-2 RNAi. (H-J) Defects in vulval induction and vulval-uterine connection in animals treated with dsRNA against nhr-25. (H) Defective vulval induction after nhr-25 RNAi. (I,J) Defective vulval-uterine connection after nhr-25 RNAi.

NHR-25 binds to the wild-type, but not the e1417, form of ACEL. (A) A cis-element in the ACEL, which contains the e1417 mutation site, is similar to the FTZ-F1 binding site consensus. (B) Two forms (α- and β-) of nhr-25 cDNA. DB represents a DNA binding domain and LB a ligand-binding domain. Both messages are trans-spliced with SL1 RNA. Theα -form contains an intact DNA-binding domain and the β-form partially deletes the domain. (C) Synthesis of both forms of the NHR-25 protein. The proteins were synthesized using in vitro transcription and translation (TNT) in rabbit reticulocyte lysates with ³⁵S-methionine. The proteins were visualized by autoradiography after SDS-PAGE. (D) EMSA showing the binding of NHR-25 to the wild-type ACEL DNA probe. The α- and β- NHR-25 proteins, which were synthesized using in vitro TNT with cold methionine, were incubated with the ³²P-labeled wild-type (wt) or e1417 form of ACEL DNA probes. The reaction mixtures were separated on a non-denaturing polyacrylamide gel and the radioactivity signals were detected by phosphoimager. F indicates the migration of free DNA probes, NS indicates the migration of a non-specific protein/DNA probe complex, and B is the NHR-25/DNA probe complex. An equal amount of non-specific protein/DNA probe complex (NS) was observed in all of the binding reactions, regardless of the synthesis of NHR-25, showing that equal amounts of the reticulocyte lysates were used for the binding assay.