Control of sex-specific apoptosis in C. elegans by the BarH homeodomain protein CEH-30 and the transcriptional repressor UNC-37/Groucho

Abstract

Apoptosis is essential for proper development and tissue homeostasis in metazoans. It plays a critical role in generating sexual dimorphism by eliminating structures that are not needed in a specific sex. The molecular mechanisms that regulate sexually dimorphic apoptosis are poorly understood. Here we report the identification of the ceh-30 gene as a key regulator of sex-specific apoptosis in Caenorhabditis elegans. Loss-of-function mutations in ceh-30 cause the ectopic death of male-specific CEM neurons. ceh-30 encodes a BarH homeodomain protein that acts downstream from the terminal sex determination gene tra-1, but upstream of, or in parallel to, the cell-death-initiating gene egl-1 to protect CEM neurons from undergoing apoptosis in males. The second intron of the ceh-30 gene contains two adjacent cis-elements that are binding sites for TRA-1A and a POU-type homeodomain protein UNC-86 and acts as a sensor to regulate proper specification of the CEM cell fate. Surprisingly, the N terminus of CEH-30 but not its homeodomain is critical for CEH-30's cell death inhibitory activity in CEMs and contains a conserved eh1/FIL domain that is important for the recruitment of the general transcriptional repressor UNC-37/Groucho. Our study suggests that ceh-30 defines a critical checkpoint that integrates the sex determination signal TRA-1 and the cell fate determination and survival signal UNC-86 to control the sex-specific activation of the cell death program in CEMs through the general transcription repressor UNC-37.

Figure 1.

Mapping and rescue of the sm130 mutant. (A, top panel) The genetic markers and SNPs used to map the sm130 mutation are indicated above the horizontal bar. The numbers below the bar indicate the fraction of 49 recombinants between lon-2 and sm130 that occurred between particular SNP markers. (Middle panel) Cosmids tested for rescue. Transgenic animals carrying the indicated Cosmid DNA as extrachromosomal arrays were generated and scored for rescue of the ectopic CEM death defect in the sm130 mutant as described in Materials and Methods. The number of rescued lines versus total lines generated is indicated to the right. (Bottom panel) Partial restriction map of the rescuing Cosmid C33D12 and the sm130 rescue data observed for several subclones of C33D12. ORFs in this region are indicated with boxes representing exons and lines representing intronic sequences. The position of the sm130 mutation is indicated with an arrowhead. Two deletion alleles (tm272 and tm2157) of ceh-30 and the ceh-30 regions removed by these mutations are represented below the ceh-30 ORF. (B) Alignment of the ceh-30 intron 2 DNA sequences from three related nematode species (C. elegans, C. briggsae, and C. remanei). Conserved residues are indicated by asterisks. The sm130 lesion is highlighted, and two conserved binding sites for POU-type homeodomain proteins (In2-POU) and TRA-1A (In2-TRA) are underlined.

Figure 2.

The N terminus of CEH-30 but not its homeodomain is required for its activity to protect male CEMs from cell death. Graphical presentations of five ceh-30 constructs are shown. Boxes indicate ceh-30 exons or the coding region for GFP (in green). The region encoding the homeodomain is highlighted with blue, the region encoding the SRD is highlighted with yellow, and the region encoding the eh1/FIL motif is highlighted with red. Transgenic smIs26; him-5(e1490); ceh-30(tm272) animals carrying the indicated DNA construct as extrachromosomal arrays were scored for rescue of the ectopic CEM death defect as described in Materials and Methods. For each construct, at least three independent transgenic lines were scored and found to have similar results. (+) Rescue; (−) failure to rescue.

Figure 3.

Both UNC-86 and TRA-1A bind to the intron 2 sequence of ceh-30 in vitro. (A) Binding of GST∷UNC-86 to a POU-type homeodomain-binding site (In2-POU) found in intron 2 of ceh-30 and an altered site [In2-POU(m)] derived from the sm130 mutant. Twenty-five nanograms of recombinant GST∷UNC-86 (lanes 2,4,6) or binding buffer alone (lanes 1,3,5) were incubated with labeled oligonucleotides derived from a known UNC-86-binding site (CS2; lanes 1,2), In2-POU (lanes 3,4), or In2-POU(m) (lanes 5,6) and resolved on an 8% native polyacrylamide gel. (U) The gel shift species containing the UNC-86 monomer; (U₂) the gel shift species containing the UNC-86 homodimer (Xue et al. 1993). (B) The sm130 lesion reduces the binding of GST∷UNC-86 to In2-POU. The fraction of labeled probe shifted by GST∷UNC-86 was quantified from four independent experiments as described in Materials and Methods. (C) UNC-86 stabilizes the binding of TRA-1A to the intron 2 sequence of ceh-30. Twenty-five nanograms of recombinant GST∷UNC-86 and 5 ng of TRA-1∷His₆ either alone or together were incubated with labeled CS2 (lanes 1,2), 257-bp DNA fragment containing the TRA-1A-binding site from the egl-1 gene (egl-1, lanes 3,4), or 91-bp intron 2 DNA fragment from ceh-30 containing both In2-POU and In2-TRA (ceh-30 In2, lanes 5–8). The reactions were resolved on a 4% native polyacrylamide gel. (T) The gel shift species containing the TRA-1 protein; (UT) the gel shift species containing both UNC-86 and TRA-1A.

Figure 4.

CEH-30 expression patterns in C. elegans embryos. (A) GFP expression directed from an integrated array (smIs54) containing P_ceh-30ceh-30∷gfp at ∼360 min post-first cleavage. Three different focal planes of the same embryo are shown with the anterior toward the left and posterior toward the right. (B) Expression of CEH-30∷GFP in a smIs54 XX embryo (∼420 min post-first cleavage, top panel) and a tra-1(e1099); smIs54 XX embryo at the similar stage (bottom panel). The ventral CEM neuron is indicated by a white arrowhead. (C) Frequency of CEH-30∷GFP expression in ventral CEMs of XX embryos at ∼380–430 min after first cleavage. The genotypes of XX embryos are indicated.

Figure 5.

CEH-30 and UNC-37 interact through the eh1/FIL motif. (A) Alignment of the eh1/FIL motifs in various proteins from C. elegans, Drosophila (D. Mel), mouse (M. Mus), and human (H. sap). Highly conserved amino acid residues are highlighted. (B) Yeast two-hybrid assays. (Panel 1) Yeast transformants expressing CEH-30 and UNC-37 GAL4 fusions as indicated were spotted on synthetic complete medium lacking trytophan and leucine (SC-Leu-Trp). (Panel 2) Growth on the SC-Leu-Trp-His plate with 10 mM 3AT. Growth indicates an interaction between the two tested fusion proteins. (Panel 3) Growth on the SC-Leu-Trp-Ura plate containing 0.2% 5FOA. Poor or no growth on the SC-Leu-Trp-Ura plate with 0.2% 5FOA indicates interaction between two tested fusion proteins. (Panel 4) β-Galactosidase staining was performed on a nitrocellulose membrane as described in Materials and Methods. Dark staining indicates interaction between the two tested fusion proteins. β-Galactosidase activity was also measured by the Chlorophenol red-β-D-galactopyranoside (CPRG) cleavage assay and is indicated below the staining assay. The numbers shown are folds of increase in activity over the yeast strain that expressed GAL4-AD and GAL4-DB. Fulllength CEH-30 (wild type or F42E mutant) and UNC-37 proteins were fused to either the GAL4-AD or the GAL4-DB. GAL4-DB/GAL4-AD and GAL4-DB∷E2F1/GAL4-AD∷RB were used as negative and positive controls for protein interaction, respectively.

Figure 6.

Transcriptional regulation of sex-specific CEM cell death. (A) UNC-86, a transcription activator, and TRA-1A, a transcription repressor, interact at intron 2 of the ceh-30 gene to regulate the expression of ceh-30, leading to the proper cell death specification of CEMs. XO indicates males, and XX indicates hermaphrodites. (B) Genetic pathways regulating sexually dimorphic cell deaths in C. elegans. ceh-30 is the key gene that interprets the sex determination signal transduced by tra-1 and the survival signal specified by unc-86 to turn off or on programmed cell death in CEMs along with unc-37, probably through suppressing the expression of egl-1 and/or another proapoptotic gene. In HSNs, TRA-1A directly inhibits the expression of egl-1. (C) Formation of a CEH-30/UNC-37 repressosome at the promoter of a proapoptotic target gene. A CEM-specific transcription factor that binds to the promoter may recruit the UNC-37/Groucho transcription repressor through CEH-30 via its eh1/FIL motif (in red).