DPL-1 DP, LIN-35 Rb and EFL-1 E2F act with the MCD-1 zinc-finger protein to promote programmed cell death in Caenorhabditis elegans

Abstract

The genes egl-1, ced-9, ced-4, and ced-3 play major roles in programmed cell death in Caenorhabditis elegans. To identify genes that have more subtle activities, we sought mutations that confer strong cell-death defects in a genetically sensitized mutant background. Specifically, we screened for mutations that enhance the cell-death defects caused by a partial loss-of-function allele of the ced-3 caspase gene. We identified mutations in two genes not previously known to affect cell death, dpl-1 and mcd-1 (modifier of cell death). dpl-1 encodes the C. elegans homolog of DP, the human E2F-heterodimerization partner. By testing genes known to interact with dpl-1, we identified roles in cell death for four additional genes: efl-1 E2F, lin-35 Rb, lin-37 Mip40, and lin-52 dLin52. mcd-1 encodes a novel protein that contains one zinc finger and that is synthetically required with lin-35 Rb for animal viability. dpl-1 and mcd-1 act with efl-1 E2F and lin-35 Rb to promote programmed cell death and do so by regulating the killing process rather than by affecting the decision between survival and death. We propose that the DPL-1 DP, MCD-1 zinc finger, EFL-1 E2F, LIN-35 Rb, LIN-37 Mip40, and LIN-52 dLin52 proteins act together in transcriptional regulation to promote programmed cell death.

Figure 1.—

The mcd-1 Zn finger and dpl-1 DP genes were identified as cell death-promoting genes from a genetic screen. (A) Ventral cord cell lineage diagrams. W, W blast cell. P, P blast cell. Arrows, cells that fail to die and that express P_lin-11 gfp. In the wild type, P3–8.aap (a, anterior daughter; p, posterior daughter) survive and express P_lin-11 gfp. W.ap, P1–2.aap, and P9–12.aap normally die (Sulston and Horvitz 1977). In ced-3(lf) animals, no cell death occurs and W.ap, P1–2.aap, and P9–12.aap survive and express P_lin-11 gfp (Reddien et al. 2001). The large green midbody region reflects GFP expression in the vulva from the lin-11 promoter. (B) Schematic of the ced-3(n2427) enhancer screen (see text for details). (C) Results from the ced-3 enhancer screen. From ∼13,000 mutagenized haploid genomes, 37 mutations were isolated. (D and E) Distribution of percentages of animals with zero to five extra cells in the ventral cord. The five cells P2.aap and P9–12.aap were scored using the assay described. At least 50 young adult animals of each genotype were scored. nIs106, P_lin-11 gfp reporter (Reddien et al. 2001). (F) n3380 is located in the region on LGII between the genes rol-6 and unc-4. n3376 is located between two polymorphisms, nP89 and nP91, on LGII, a region of ∼75 kb. See materials and methods for details. (G) Protein structure of DPL-1 DP. The blue box indicates the putative DNA-binding region of DPL-1 and the yellow box the putative E2F-binding region, as previously described (Ceol and Horvitz 2001). The n3316 mutation is a deletion following the third codon, and n2994 is a splice-site mutation predicted to alter protein structure following amino acid 227 (Ceol and Horvitz 2001). n3380 is a C-to-T nonsense mutation that is at Q486. (H) The gene Y51H1A.6 is mcd-1 (see text for details). The 5′ end carries an SL1 trans-spliced leader sequence (see text), and the 3′-UTR is ∼750 bp. We isolated a deletion allele, n4005, that removes part of intron two and part of exon three of Y51H1A.6 (see materials and methods). The red line labeled “Zn” depicts the zinc-finger-encoding region of mcd-1. The MCD-1 zinc-finger region amino acid sequence is shown below the gene structure diagram. Red stars indicate the residues that define the C2H2 domain, and blue stars indicate other residues conserved with the canonical C2H2 zinc finger. n3376 is a C-to-T mutation resulting in an H277Y substitution in the MCD-1 protein and is indicated by a red arrowhead.

Figure 2.—

Abnormalities in the cell-death process in dpl-1(n3380) and mcd-1(n4005) animals. (A) P9.aap in a dpl-1(n3380); nIs106 animal was condensed 2 hr 10 min after its generation. By 4 hr 10 min after its generation, P9.aap had recovered and appeared morphologically normal; 36 hr later, P9.aap expressed P_lin-11gfp. P9.aap is indicated by an arrow. Anterior, left. Posterior, right. Dorsal, top. Ventral, bottom. In the 4-hr 10-min image anterior is right and posterior is left. (B) P11.aap in an mcd-1(n4005); nIs106 animal condensed 3 hr 22 min after its generation. This cell had first displayed attributes of a dying cell 1 hr 35 min after its generation. Three hours 27 min after generation P11.aap recovered, and P11.aaap had normal nuclear morphology. Three hours 42 min after generation, P11.aap condensed, and P11.aaap was condensed as well. Three hours 47 min after generation, P11.aap recovered. P11.aap was observed until 5 hr 25 min after generation without any further obvious attempts at death. Thirty-six hours later, no GFP fluorescence was detected in the P11 region, indicating that this cell either failed to express lin-11 or ultimately died. P11.aap is indicated by a black arrow and P11.aaap is indicated by a red arrow. Anterior, left. Posterior, right. Dorsal, bottom. Ventral, top. (C) Model for the effects of dpl-1 and mcd-1 on cell killing. Top, in a cell specified to die in wild-type animals, the CED-3 caspase is activated and the MCD-1 Zn finger, DPL-1 DP, LIN-35 Rb, EFL-1 E2F, LIN-37 Mip40, and LIN-52 dLin52 proteins mediate transcriptional regulation of unknown targets to allow cell death to occur. Middle, in the absence of the CED-3 caspase, cells fail to display the morphological alterations characteristic of cell death. Bottom, in the absence of mcd-1 or dpl-1, CED-3 is still activated and initiates the cell-death process. The execution of cell death occasionally fails, and cells can survive and differentiate. (D) Multiple activities function independently and additively to promote cell death. The MCD-1 Zn finger, DPL-1 DP, LIN-35 Rb, EFL-1 E2F, LIN-37 Mip40, and LIN-52 dLin52 proteins define a transcriptional regulatory activity that promotes cell death. This activity functions in an additive manner with the cell-killing activity of the CED-9 Bcl-2 and CED-8 XK proteins, as well as with the genes that control the process of engulfment to promote cell death. Multiple other activities could exist and act in a similar additive manner to control cell-death execution.