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. 2018 Apr 10;115(15):3960-3965.
doi: 10.1073/pnas.1800435115. Epub 2018 Mar 22.

Krüppel Homolog 1 Represses Insect Ecdysone Biosynthesis by Directly Inhibiting the Transcription of Steroidogenic Enzymes

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Krüppel Homolog 1 Represses Insect Ecdysone Biosynthesis by Directly Inhibiting the Transcription of Steroidogenic Enzymes

Tianlei Zhang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

In insects, juvenile hormone (JH) and the steroid hormone ecdysone have opposing effects on regulation of the larval-pupal transition. Although increasing evidence suggests that JH represses ecdysone biosynthesis during larval development, the mechanism underlying this repression is not well understood. Here, we demonstrate that the expression of the Krüppel homolog 1 (Kr-h1), a gene encoding a transcription factor that mediates JH signaling, in ecdysone-producing organ prothoracic gland (PG) represses ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes in both Drosophila and Bombyx Application of a JH mimic on ex vivo cultured PGs from Drosophila and Bombyx larvae induces Kr-h1 expression and inhibits the transcription of steroidogenic enzymes. In addition, PG-specific knockdown of Drosophila Kr-h1 promotes-while overexpression hampers-ecdysone production and pupariation. We further find that Kr-h1 inhibits the transcription of steroidogenic enzymes by directly binding to their promoters to induce promoter DNA methylation. Finally, we show that Kr-h1 does not affect DNA replication in Drosophila PG cells and that the reduction of PG size mediated by Kr-h1 overexpression can be rescued by feeding ecdysone. Taken together, our data indicate direct and conserved Kr-h1 repression of insect ecdysone biosynthesis in response to JH stimulation, providing insights into mechanisms underlying the antagonistic roles of JH and ecdysone.

Keywords: Kr-h1; direct regulation; ecdysone biosynthesis; juvenile hormone; transcriptional repression.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
JHM application delays pupariation and inhibits both ecdysone biosynthesis and transcription of steroidogenic enzyme genes in Drosophila and Bombyx. (A and A′) The application of JHM methoprene on both Drosophila and Bombyx larvae results in developmental delay. Drosophila wild-type yw larvae at 24 h AEL were fed on food supplemented with either ethanol as control (con) or JHM (A). JHM application on the body of Bombyx larvae was performed on the second day of the last larval instar (A′). (Scale bar, 1 cm.) (B and B′) Ecdysone titer changes in the whole body of Drosophila larvae and in the hemolymph of Bombyx larvae after JHM treatment. Animals treated with ethanol in Drosophila or acetone in Bombyx were used as controls. (C and C′) qRT-PCR analysis of the changes in steroidogenic enzyme mRNA levels in ex vivo cultured Drosophila brain–RG complex and Bombyx PG following JHM application. Drosophila brain–RG complex and Bombyx PG were isolated from animals at the beginning of larval–pupal transition. All experiments were performed in three biological replicates. Values are represented as the mean ± SE (error bars). For the significance test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
Fig. 2.
Fig. 2.
Level of DmKr-h1 expression in Drosophila PG affects larval development, ecdysone biosynthesis and transcription of steroidogenic enzyme genes. (A) Pupariation rate with knockdown of DmKr-h1 in Drosophila PG driven by Phm-Gal4 compared with control (white-i). (B) Measurement of both ecdysone titer in the whole body and DmE75B mRNA level in the fat body of DmKr-h1 knockdown larvae and control larvae at 96 h AEL. (C) DmKr-h1 knockdown in Drosophila PG up-regulates expression of steroidogenic enzyme genes in the brain–RG complex of larvae at 96 h AEL. (D) PG-specific overexpression of DmKr-h1 leads to developmental arrest at the first-larval (L1) instar. (Scale bar, 2.5 mm.) (E) PG-specific DmKr-h1 overexpression-mediated inhibition of ecdysone biosynthesis in the PG and DmE75B expression in the fat body at 48 h AEL. (F) Fold-change in steroidogenic enzyme gene expression in brain–RG complex overexpressing DmKr-h1 relative to control at 48 h AEL. (G) Feeding larvae with ecdysone or 20E rescued developmental L1 arrest induced by DmKr-h1 overexpression in the PG. (Scale bar, 2 mm.) Ecdysteroid feeding was started at 48 h AEL. All experiments were performed in three biological replicates. Values are represented as the mean ± SE (error bars). For the significance test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
Fig. 3.
Fig. 3.
Kr-h1 inhibits the activities of the Drosophila and Bombyx steroidogenic enzyme promoters. (A and A′) JHM application and DmKr-h1 overexpression represses the activity of steroidogenic enzyme promoters in Drosophila. Luciferase activity fold-change values were normalized to the control. (B and B′) JHM application and BmKr-h1 overexpression repress activity of steroidogenic enzyme promoters in Bombyx. (C) DmKr-h1 affects the luciferase activity driven by different truncations of the DmSpok promoter. DmKr-h1 did not show transcriptional inhibition activity when the KBS of the DmSpok promoter was truncated. (D) Effects of DmKr-h1 on the luciferase activity driven by DmSpok promoters with either a deletion or a mutation form of proximal KBS. (E) Effects of BmKr-h1 on the luciferase activity driven by BmSpo promoters with either a deletion or a mutated form of KBS. All experiments were performed in three biological replicates. Values are represented as the mean + SE (error bars). For the significance test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
Fig. 4.
Fig. 4.
Kr-h1 directly binds the KBS in DmSpok and BmSpo promoters to induce DNA methylation. (A) ChIP-PCR assay in Drosophila S2 cells with DmKr-h1 overexpression. Specific primers covering proximal KBS of the DmSpok promoter were used. (B and C) EMSA confirmed that DmKr-h1 directly binds to the proximal KBS. Recombinant DmKr-h1 protein binds to the biotinylated probes covering the proximal KBS in the DmSpok promoter in a dose-dependent manner; this binding can be competitively inhibited by unlabeled cold probes (B). Unlabeled probes with either KBS mutation or KBS deletion cannot compete for binding of recombinant DmKr-h1 protein to the biotinylated probes (C). (D) ChIP-PCR assay of the direct binding of overexpressed BmKr-h1 to the proximal KBS of the BmSpo promoter in Bombyx BmE cells. (E and F) EMSA confirms that recombinant BmKr-h1 directly binds to the proximal KBS of the BmSpo promoter. (G) Schematic diagram of methylated cytosine present in the DmSpok promoter after DmKr-h1 overexpression in S2 cells. Filled circles, methylated cytosines; empty circles, unmethylated cytosines; red rounded rectangle, proximal KBS. (H) The DNA methylation inhibitor Aza rescued the inhibitory activity of DmKr-h1. All experiments were performed in three biological replicates. Values are represented as the mean + SE (error bars). For the significance test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

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