Juvenile hormone action through a defined enhancer motif to modulate ecdysteroid-activation of natural core promoters

Abstract

We have established a model system of hormone action, in an Sf9 cell transfection system, using defined enhancer motifs and natural core promoters of metamorphosis-associated genes. The DR1 enhancer, that is an established DNA binding site for the ecdysone receptor/ultraspiracle heterodimer, was necessary for transcriptional activation by 20-OH ecdysone. For this activated transcription, a natural sequence closely 5' to the TATA box is necessary. Cotreatment with juvenile hormone III strongly suppressed the steroid activation of transcription. However, in the absence of the sequence located closely 5' to the TATA box, cotreatment with juvenile hormone instead increased transcription over that occurring due to 20-hydroxy-ecdysone alone. This sensitivity to activation by cotreatment with juvenile hormone could be transferred to a related, but otherwise unresponsive, hexamerin core promoter simply by transferring to the unresponsive promoter the five base transcription start site (ACAGT) from the responsive hexamerin gene. These are the first reports that the direction of JH action on 20-OH ecdysone-activated transcription can be reversed by removal of a sequence at the core promoter, and that modulatory action of juvenile hormone can be transferred to a different gene by transferring the transcription start site motif.

Figure 1

Alignment of the regions of the four hexamerin genes of Trichoplusia ni, from the TATA box (bold, underlined) to the first base (A) of the ATG translation start codon. The transcription start point (italicized A) and the four subsequent bases are underlined. A region of similarity of three genes immediately 5′ to the TATA box is in italics, whereas a region of similarly on the 3′ side is shown in bold only. A motif common to the three genes that is between the transcription and translation start points is shown in underline only. Shown above (AJHSP1) and below (BJHSP1) the specific wild type bases are the mutations to those bases that were prepared in certain experiments.

Figure 2

Transcriptional activities of the pGL2 basic vector (“pGL2”, luciferase reporter), AJHSP1 core promoter (“AJHSP1core”), BJHSP1 core promoter (“BJHSP1core”) and AJHSP1core with DR1 enhancer (two tandem copies) placed immediately 5′ to the AJHSP1core (“DR1AJHSP1”). Shown are means and standard error of transcriptional activities, 24–48 h after hormone treatment, relative to the ethanol carrier control (“EtOH, which is shown as value of 1.0). Experimental treatments were 1 μM 20-hydroxy ecdysone (“20E”), 100 μM juvenile hormone III (“JH III”), or a combined treatment of the two hormones at these concentrations (“20E/JH”). For each of the four constructs in panels A–D, none of the three hormone regimens exerted a significant effect on transcription different than the EtOH control (ANOVA, p > 0.05, for each panel).

Figure 3

Panels A and B) Transcriptional activities of the BJHSP1 core promoter with DR1 enhancer (four tandem copies, “DR1g”, 24 h hormone treatment, or a single copy, “DR1a,” placed immediately 5′ to the BJHSP1core. Panel C) Transcriptional activities of the ARYL core promoter with a DR1 enhancer (two tandem copies) placed immediately 5′ to the core promoter (24 h hormone treatment). Shown are means and standard error of reported transcriptional activities relative to ethanol (“EtOH”) carrier control treatment, which is shown as value of 1.0. Experimental treatments are 1 μM 20-hydroxy ecdysone (“20E”), 100 μM juvenile hormone III (“JH III”), or a combined treatment of the two hormones at these concentrations (“20E/JH”). For either DR1-enhanced BJHSP1 promoter construct, ANOVA was significant (p < 0.05); 20E significantly induced transcriptional activity (p< 0.01 for both DR1aBJHSP1 and DR1gBJHSP1); JH III alone had no significant effect (p>0.05), but the cotreatment of JH III with 20E significantly suppressed the reported activity below that of 20E alone (p<0.01 DR1aBJHSP1; p<0.02 DR1gBJHSP1). The DR1ARYL construct also showed a significant treatment effect (ANOVA, p < 0.05). However, in contrast to the DR1-enhanced BJHSP1 constructs, the 20E-induced activity of the DR1-enhanced ARYL core promoter (p < 0.01) was not significantly affected by cotreatment with JH III (p> 0.05). Panel D) Over a 24 h time course, the DR1BJHSP1 construct exhibited more than a 100 fold increase in transcriptional activity in response to 1 μM 20E, in comparison to the lack of response to 20E of the DR1AJHSP1 construct (note the much different scales for the two Y-axes in panel D).

Figure 4

DR1g-enhanced transcriptional activities of the wild type and several mutants of the BJHSP1 core promoter after 24 h of hormone treatment. Panel A) Normalized transcriptional activity of the indicated mutations (see Fig. 1). Panel B) Transcriptional activity of Panel A expressed instead as relative to EtOH control (= value of 1). Shown are means and standard error of the experimental treatments of 1 μM 20-hydroxy ecdysone (“20E”), 100 μM juvenile hormone III (“JH III”), or a combined treatment of the two hormones at these concentrations (“20E/JH”). Each of the single motif mutations retained a significant effect of cotreatment of JH III with 20E compared to the transcription induction with 20E only (Panel B, p < 0.01 each).

Figure 5

Normalized transcriptional activities of the wild type (WT) AJHSP1and BJHSP1 core promoter, each trimmed to include only the region from −41 to the first base of the translation start codon, and enhanced (or not, as indicated) by two copies of a 5′-placed DR1 motif. Also as indicated, the initiator of AJHSP1 was replaced with the five bases of the BJHSP1 initiator (DR1A1>B1INIT), and the initiator for the BJHSP1 was changed to be that of AJHSP1 (DR1B1>A1INIT). Shown relative to the EtOH control are means and standard error of the experimental treatments of 48 h exposure to 1 μM 20-hydroxy ecdysone (“20E”), 100 μM juvenile hormone III (“JH III”), or a combined treatment of the two hormones at these concentrations (“20E/JH”). Experiment-wise ANOVA was significant at p < 0.01. The wild type AJHSP1 core promoter, enhanced by DR1 (DR1AJHSP1WT), was not significantly affected by hormone treatment (p > 0.05), while the DR1BJHSP1WT activity was significantly induced by cotreatment with 20E and JH III, as compared to treatment with only 20E (p<0.05). However, when the initiator motif of the two promoters were exchanged (INIT), the DR1BJHSP1 promoter lost hormone responsiveness (p>0.05), while the DR1AJHSP1 promoter showed significantly more activity by cotreatment with 20E and JH III, than the treatment with 20E alone (p < 0.05).