The L-type cyclin CYL-1 and the heat-shock-factor HSF-1 are required for heat-shock-induced protein expression in Caenorhabditis elegans

Abstract

In a screen for suppressors of activated GOA-1 (Galpha(o)) under the control of the hsp-16.2 heat-shock promoter, we identified three genetic loci that affected heat-shock-induced GOA-1 expression. The cyl-1 mutants are essentially wild type in appearance, while hsf-1 and sup-45 mutants have egg-laying defects. The hsf-1 mutation also causes a temperature-sensitive developmental arrest, and hsf-1 mutants have decreased life span. Western analysis indicated that mutations in all three loci suppressed the activated GOA-1 transgene by decreasing its expression. Heat-shock-induced expression of hsp-16.2 mRNA was reduced in cyl-1 mutants and virtually eliminated in hsf-1 and sup-45 mutants, as compared to wild-type expression. The mutations could also suppress other transgenes under heat-shock control. cyl-1 and sup-45, but not hsf-1, mutations suppressed a defect caused by a transgene not under heat-shock control, suggesting a role in general transcription or a post-transcriptional aspect of gene expression. hsf-1 encodes the C. elegans homolog of the human heat-shock factor HSF1, and cyl-1 encodes a cyclin most similar to cyclin L. We believe HSF-1 acts in heat-shock-inducible transcription and CYL-1 acts more generally in gene expression.

Figure 1.—

Western blots showing reduced expression of activated GOA-1 in class II and class III suppressor mutants. (A) All strains were syIs17 background. (B) These strains contained n363, a deletion of goa-1, to eliminate endogenous goa-1 expression, in addition to syIs17. (C) Endogenous wild-type GOA-1 expression in unstressed syIs17 animals is unaffected by cyl-1(sy433).

Figure 2.—

Decreased levels of hsp-16.2 mRNA in suppressor mutants, as measured by quantitative real-time PCR. Input levels for each sample were normalized with control PCR reactions using 18S rRNA. This figure shows one of two trials, each of which gave similar results.

Figure 3.—

Sequence analysis of hsf-1. (A) Intron-exon structure of hsf-1. (B) Alignment of hsf-1 with other HSFs. CBG18699 is the C. briggsae ortholog of hsf-1 (Stein et al. 2003). Lowercase residues are not aligned. The heptad repeat HR-C is denoted by a shaded bar. Acidic residues are shown in boldface type.

Figure 4.—

Life spans of N2 and hsf-1(sy441) animals cultured at 20° (n = 42 for both; P < 0.0001, Mann-Whitney test). This figure shows one of two trials, each of which gave similar results.

Figure 5.—

Sequence analysis of cyl-1. (A) Splicing pattern of C52E4.6a as determined by sequence analysis of cDNA. The cyclin box extends from exon 4 to exon 7. A truncated splicing variant C52E4.6b has also been identified (WS110; http://www.wormbase.org) and is depicted here for comparison. (B) Sequence alignment of the cyclin boxes of cyclin L homologs from C. elegans, human, mouse, Danio, and Drosophila, and human cyclin H. Lowercase residues are not aligned. Nineteen conserved nonpolar residues in cyclin A and H α-helices are indicated by bullets; α-helices previously described in cyclin H are marked by shaded bars (Noble et al. 1997). The α1 and α4 helices of cyclin H are not conserved on an amino acid level in cyclin L; hydrophobic residues in boldface type indicate a possible location for α1 in cyclin L. Arrows mark cyl-1 mutations.