Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods

Abstract

We report here the identification of a previously unknown transcription regulatory element for heat shock (HS) genes in Caenorhabditis elegans. We monitored the expression pattern of 11,917 genes from C. elegans to determine the genes that were up-regulated on HS. Twenty eight genes were observed to be consistently up-regulated in several different repetitions of the experiments. We analyzed the upstream regions of these genes using computational DNA pattern recognition methods. Two potential cis-regulatory motifs were identified in this way. One of these motifs (TTCTAGAA) was the DNA binding motif for the heat shock factor (HSF), whereas the other (GGGTGTC) was previously unreported in the literature. We determined the significance of these motifs for the HS genes using different statistical tests and parameters. Comparative sequence analysis of orthologous HS genes from C. elegans and Caenorhabditis briggsae indicated that the identified DNA regulatory motifs are conserved across related species. The role of the identified DNA sites in regulation of HS genes was tested by in vitro mutagenesis of a green fluorescent protein (GFP) reporter transgene driven by the C. elegans hsp-16-2 promoter. DNA sites corresponding to both motifs are shown to play a significant role in up-regulation of the hsp-16-2 gene on HS. This is one of the rare instances in which a novel regulatory element, identified using computational methods, is shown to be biologically active. The contributions of individual sites toward induction of transcription on HS are nonadditive, which indicates interaction and cross-talk between the sites, possibly through the transcription factors (TFs) binding to these sites.

Figure 1

Schema describing the steps for identification and validation of transcription regulatory elements from coregulated genes.

Figure 2

DNA motifs identified from upstream regions of heat shock (HS) genes. Motifs identified by pattern recognition programs from the upstream regions (−500–−1) of HS genes up-regulated genes. Information content (I.C.) in bits (log2) and sequence logos for the DNA motifs are given. DNA logos were generated according to Schneider and Stephens (1990).

Figure 3

Comparison of the orthologous Caenorhabditis elegans and Caenorhabditis briggsae genes. (A) Structures of two Hsp70 protein family genes from C. elegans and corresponding orthologs from C. briggsae. The two top genes (lighter gray) are from C. elegans and the bottom two (dark gray) are from C. briggsae. (B) Sequence similarity comparison of C. elegans and C. briggsae genes shown in A using the VISTA alignment method. The percentage of sequence conservation over every 100 nt window is indicated. Note large sequence similarity decreases in noncoding regions. (C) Heat shock element (HSE) and heat shock associated site (HSAS) DNA site positions and strengths in the promoter regions of the divergently expressed Hsp70 genes shown in A. Vertical bars represent the DNA sites and horizontal lines the DNA sequences. Height of the vertical bars are proportional to the scores calculated by the Patser program. (D) Sequence alignment of promoter regions of Hsp70 genes shown in A, using the GLASS alignment algorithm. HSEs are indicated in red and HSAS elements in green. Also highlighted in blue is a well-conserved sequence also found in the hsp-16–2/−41 promoter region (see Fig. 4A).

Figure 4

Summary of mutated promoter construct experiments. (A) The hsp-16–2/−41 promoter sequence in pPD122.18. Candidate promoter elements identified in bold font; sequences above the promoter elements indicate sequences introduced in mutated derivatives. TATA boxes underlined, transcriptional starts (Candido et al. 1989) indicated with grey nucleotides. Nucleotides in outlined font represent conserved site also found in F44E5.4/5 promoter region (see Fig. 3D). (B) Schematic of promoter element mutants and corresponding expression in transgenic animals. Transgenic F1 roller animals were scored as “strong GFP” (green fluorescence protein) if >50 GFP⁺ nuclei were observed. The two non-GFP animals observed in the pPD122.18 and H1 mutated construct injections likely result from rare events when the reporter construct was not incorporated in the Rol marker-containing array. Transgenic animals were scored as “any GFP” if at least one GFP positive nucleus could be observed. Footnotes: a, GFP⁺ animals from these injections contained an average of 5.5 (predominantly neuronal) GFP positive nuclei; b, one GFP positive animal was observed with 10 GFP⁺ head neurons; c, one head neuron in one animal was GFP positive.

Figure 5

Effect of HSAS element on reporter transgene expression. First generation (F1) transgenic animals containing a GFP reporter construct with either both HSE2 and HSAS mutated (HSE1 only) or HSE2 mutated (HSE1 + HSAS) were heat shocked at 35°C, returned to 20°C for 18 hr, then imaged for GFP expression. This digitally merged differential interference contrast/epifluorescence image shows the requirement for the HSAS element for strong transgene expression when the HSE is mutated. Size bar = 50 μm.