Activity of heat shock genes' promoters in thermally contrasting animal species

Abstract

Heat shock gene promoters represent a highly conserved and universal system for the rapid induction of transcription after various stressful stimuli. We chose pairs of mammalian and insect species that significantly differ in their thermoresistance and constitutive levels of Hsp70 to compare hsp promoter strength under normal conditions and after heat shock (HS). The first pair includes the HSPA1 gene promoter of camel (Camelus dromedarius) and humans. It was demonstrated that the camel HSPA1A and HSPA1L promoters function normally in vitro in human cell cultures and exceed the strength of orthologous human promoters under basal conditions. We used the same in vitro assay for Drosophila melanogaster Schneider-2 (S2) cells to compare the activity of the hsp70 and hsp83 promoters of the second species pair represented by Diptera, i.e., Stratiomys singularior and D. melanogaster, which dramatically differ in thermoresistance and the pattern of Hsp70 accumulation. Promoter strength was also monitored in vivo in D. melanogaster strains transformed with constructs containing the S. singularior hsp70 ORF driven either by its own promoter or an orthologous promoter from the D. melanogaster hsp70Aa gene. Analysis revealed low S. singularior hsp70 promoter activity in vitro and in vivo under basal conditions and after HS in comparison with the endogenous promoter in D. melanogaster cells, which correlates with the absence of canonical GAGA elements in the promoters of the former species. Indeed, the insertion of GAGA elements into the S. singularior hsp70 regulatory region resulted in a dramatic increase in promoter activity in vitro but only modestly enhanced the promoter strength in the larvae of the transformed strains. In contrast with hsp70 promoters, hsp83 promoters from both of the studied Diptera species demonstrated high conservation and universality.

Fig 1. A. General arrangement of the HSPA1 cluster in mammals. B. The level of transcription activity of individual hsp70 gene promoters comprising the cluster based on the measurement of transient luciferase luminescence driven by constructs carrying different human and camel hspA1 promoters. (i)—HSPA1A, (ii)—HSPA1B and (iii)—HSPA1L. The signal levels are shown as the ratio of the intensity of the luminescence of firefly (pFF) and renilla (pRL) luciferase. C. EMSA experiments with human and camel HSPA1L promoters fragments: 1—H. sapiens promoter without 112 bp fragment specific for primates only, 2—isolated H. sapiens 112 bp fragment, 3—C. dromedarius promoter. *** and ### p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Fig 2. A. Various hsp70 promoter strengths based on the luciferase luminescence levels in S2 cells and the effects of experimental GAGA element insertion into the hsp70S3 promoter. B. The level of S. singularior and D. melanogaster hsp83 promoter activity in D. melanogaster S2 cells. *** p ≤ 0.001, ## p ≤ 0.01.

Fig 3. A. EMSA experiments with protein extracts from S2 cells and adult D. melanogaster flies exploring labeled fragments of the D. melanogaster hsp70Aa and S. singularior hsp70S3 and S4 genes. 1—Control (25°C), 2—heat shock (37°C), 3—heat shock + preimmune serum, 4—super shift with anti-HSF. The arrow indicates the position of the HSF-HSE complex. B. Recombinant D. melanogaster GAF protein effectively binds to the D. melanogaster hsp70 and S. singularior hsp70S3 promoters with the experimental insertion of three GAGA elements (lanes 1 and 4) but not with the wild-type hsp70S3 and hsp70S4 promoters (lanes 2 and 3). The arrow indicates the position of the GAF-GAGA complexes.

Fig 4. Left panel: General arrangement of constructs used in the transformation experiments.

The thick green arrows in I(S-GAGA) indicate the position of the inserted GAGA elements. HSE, GAGA elements and TATA boxes are indicated by square boxes of different color. Right panel: in situ hybridization of heat-shocked salivary gland chromosomes with the white gene fragment included in the constructs. The sites of the inserts are shown by arrows with puffs formed only in the strains (the bottom panel) containing constructs with D. melanogaster hsp70 promoters. In all panels, the heat shock puffs formed in the locations of major D. melanogaster hsp genes (i.e., 63B, 61C and 95D) that represent an internal control are indicated. 3B is the white locus that hybridizes with the labeled probe and represents an internal control for hybridization efficiency. In each strain, at least ten larvae were used for puff detection after HS.

Fig 5. The results of RT-PCR experiments using RNA isolated from adult flies (A) or the third instar larvae (B) of D. melanogaster strains transformed with constructs containing different hsp70 promoters.

A significant dichotomy in the expression of the I(S-GAGA) construct in adults and larvae is evident, while all other constructs exhibit similar strengths at both stages. *** p ≤ 0.001.