Schlank orchestrates insect developmental transition by switching H3K27 acetylation to trimethylation in the prothoracic gland

Abstract

Insect developmental transitions are precisely coordinated by ecdysone and juvenile hormone (JH). We previously revealed that accumulated H3K27 trimethylation (H3K27me3) at the locus encoding JH signal transducer Hairy is involved in the larval-pupal transition in insects, but the underlying mechanism remains to be fully defined. Here, we show in Drosophila and Bombyx that Rpd3-mediated H3K27 deacetylation in the prothoracic gland during the last larval instar promotes ecdysone biosynthesis and the larval-pupal transition by enabling H3K27me3 accumulation at the Hairy locus to induce its transcriptional repression. Importantly, we find that the homeodomain transcription factor Schlank acts to switch active H3K27 acetylation (H3K27ac) to repressive H3K27me3 at the Hairy locus by directly binding to the Hairy promoter and then recruiting the histone deacetylase Rpd3 and the histone methyltransferase PRC2 component Su(z)12 through physical interactions. Moreover, Schlank inhibits Hairy transcription to facilitate the larval-pupal transition, and the Schlank signaling cascade is suppressed by JH but regulated in a positive feedback manner by ecdysone. Together, our data uncover that Schlank mediates epigenetic reprogramming of H3K27 modifications in hormone actions during insect developmental transition.

Keywords: H3K27 modifications; Schlank; developmental transition; epigenetic switch; hormone actions.

Fig. 1.

Increasing H3K27ac levels in the PG of Drosophila and Bombyx disturb larval–pupal transition by impairing ecdysone biosynthesis. (A and B) Immunostaining for the dynamics of H3K27ac levels in the PG of Drosophila and Bombyx during the last larval instar. AEL, after egg laying. L5D1, the first day of the fifth larval instar; L5D3, the third day of the fifth larval instar; L5D5, the fifth day of the fifth larval instar; W0, just wandering (the beginning of the prepupal stage). (C) Larval–pupal transition arrest caused by PG-specific knockdown of Drosophila Rpd3 using a VDRC RNAi line (V30600). (D) Larval–pupal transition delay caused by the application of TSA to Bombyx larvae at L5D5. (E and F) EIA measurement of the change in the ecdysteroid titer in whole Drosophila larvae at 120 h AEL following PG-specific Rpd3 knockdown and in the hemolymph of Bombyx larvae at 24 h after TSA application at L5D5. (G and H) RT-qPCR analysis of the change in the expression of steroidogenic enzymes in the PG of Drosophila larvae at 120 h AEL following PG-specific Rpd3 knockdown or Bombyx larvae at 24 h after TSA application at L5D5. (I and J) Rescue effect of 20E feeding on developmental defects induced by PG-specific Rpd3 knockdown in Drosophila or TSA application in Bombyx. Dm, Drosophila; Bm, Bombyx. [Scale bars, 20 μm (A), 200 μm (B), 1 mm (C and I), and 1 cm (D and J).] Statistical values are presented as the mean ± SE (error bars) for three biological replicates (E–H). For the significance test, *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control.

Fig. 2.

A switch of H3K27ac to H3K27me3 at the Hairy locus in the PG is required for the repression of Hairy transcription to orchestrate developmental transition. (A and B) Changes in the H3K27me3 level in the PG of Drosophila larvae at 120 h AEL following PG-specific Rpd3 knockdown and Bombyx larvae at 24 h after TSA application at L5D5. (C and D) ChIP-qPCR analysis of changes in H3K27ac and H3K27me3 enrichment at the Hairy promoter in the PG-containing brain–RG complex of Drosophila larvae at 120 h AEL following PG-specific Rpd3 knockdown. (E and F) RT-qPCR analysis of the Hairy expression change in the PG-containing brain–RG complex of Drosophila larvae at 120 h AEL following PG-specific Rpd3 knockdown and in the PG of Bombyx larvae at 24 h after TSA application at L5D5. (G and H) Hairy knockdown-mediated rescue of developmental arrest and decreased ecdysone production caused by Rpd3 knockdown in the Drosophila PG. The ecdysteroid titer in the whole Drosophila larvae at 120 h AEL was measured. AEL, after egg laying. L5D5, the fifth day of the fifth larval instar. [Scale bars, 20 μm (A), 200 μm (B), and 1 mm (G).] Statistical values are presented as the mean ± SE (error bars) for three biological replicates. For the significance test, *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control.

Fig. 3.

The transcription factor Schlank mediates the H3K27ac-to-H3K27me3 switch at the Hairy locus in the PG. (A) Volcano plot of genes differentially expressed in the Bombyx PG at L5D1 and W0. (B) GO enrichment analysis of up-regulated DEGs in the Bombyx PG at W0 compared with L5D1. DEGs, differentially expressed genes. (C) Venn diagram showing the overlap between the up-regulated transcription factors in the Bombyx PG at W0 and previously reported genes whose PG-specific knockdown can arrest the larval–pupal transition in Drosophila. TF, transcription factor. (D and E) Immunostaining analysis of changes in H3K27ac and H3K27me3 levels in the PG of Drosophila larvae at 120 h AEL following PG-specific Schlank knockdown using a VDRC RNAi line (V109418). (F and G) ChIP-qPCR analysis of the changes in H3K27ac and H3K27me3 enrichment at the Hairy promoter in the PG-containing brain–RG complex of Drosophila larvae at 120 h AEL following PG-specific Schlank knockdown. (H and I) Changes in H3K27ac and H3K27me3 levels in the PG of Drosophila larvae at 108 h AEL following PG-specific overexpression of Schlank WT, NLS2 variant, or H215D variant. WT, wild type; NLS2 variant, Schlank with two point mutations (R78A and R124A); H215D variant, Schlank with the H215D mutation. L5D1, the first day of the fifth larval instar; W0, just wandering. AEL, after egg laying. [Scale bars, 20 μm (D, E, H, and I).] Statistical values are presented as the mean ± SE (error bars) for three biological replicates. For the significance test, *P < 0.05 and **P < 0.01 versus the control.

Fig. 4.

Schlank promotes ecdysone biosynthesis and the larval–pupal transition by repressing Hairy transcription. (A) Larval–pupal transition arrest caused by PG-specific knockdown of Drosophila Schlank using a VDRC RNAi line (V109418). (B) EIA measurement of the change in the ecdysteroid titer in the whole body of Drosophila larvae at 120 h AEL following PG-specific Schlank knockdown. (C) Steroidogenic enzyme expression change in the PG-containing brain–RG complex of Drosophila larvae at 120 h AEL following PG-specific Schlank knockdown. (D) Rescue effect of 20E feeding on developmental arrest caused by PG-specific Schlank knockdown in Drosophila. (E) Effects of overexpression of Schlank WT or variants in the PG on the Drosophila larval–pupal transition. (F) Ecdysteroid titer changes in the whole body of Drosophila larvae at 108 h AEL following PG-specific overexpression of Schlank WT or variants. (G and H) Hairy expression changes in the PG-containing brain–RG complex of Drosophila larvae at 120 h AEL following PG-specific Schlank knockdown and at 108 h AEL following PG-specific overexpression of Schlank WT or variants. (I and J) Hairy knockdown-mediated rescue of developmental arrest and decreased ecdysone production caused by PG-specific Schlank knockdown in Drosophila. (K and L) ChIP-PCR assays of direct binding of overexpressed Flag-tagged Schlank to the Hairy promoter in Drosophila S2 cells and Bombyx BmE cells. WT, wild type; NLS2 variant, Schlank with two point mutations (R78A and R124A); H215D variant, Schlank with the H215D mutation. Dm, Drosophila; Bm, Bombyx. AEL, after egg laying. [Scale bars, 1 mm (A, D, and I).] Statistical values are presented as the mean ± SE (error bars) for three biological replicates. For the significance test, *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control.

Fig. 5.

Schlank recruits Rpd3 and Su(z)12 to the Hairy promoter through physical interaction. (A and B) Co-IP assay of the physical interaction between exogenously overexpressed Flag-tagged Schlank and HA-tagged Rpd3 or V5-tagged Su(z)12 in Drosophila S2 cells and Bombyx BmE cells. (C) Co-IP assay of the physical interaction between endogenous Rpd3 and Schlank or Su(z)12 in Drosophila brain–RG complexes containing the PG and Bombyx PGs. (D and E) ChIP-qPCR analysis of the changes in the enrichment of Rpd3 and Su(z)12 at the Hairy promoter in the PG-containing brain–RG complex of Drosophila larvae at 120 h AEL following PG-specific Schlank knockdown. Dm, Drosophila; Bm, Bombyx. AEL, after egg laying. WCL, whole cell lysate. Statistical values are presented as the mean ± SE (error bars) for three biological replicates. For the significance test, *P < 0.05 and **P < 0.01 versus the control.

Fig. 6.

JH represses Schlank expression and the H3K27ac-to-H3K27me3 switch at the Hairy locus in the PG. (A and B) RT-qPCR analysis of changes in Schlank expression in ex vivo cultured PG-containing brain–RG complexes from Drosophila larvae at 108 h AEL and PGs from Bombyx larvae at L5D6 following treatment with the JHM methoprene for 24 h. (C and D) Change in Schlank expression in the PG-containing brain–RG complex of Drosophila larvae at 108 h AEL following PG-specific double knockdown of both Met and Gce. (E and F) Immunostaining analysis of JHM-induced changes in the H3K27ac level in ex vivo cultured PGs from Drosophila and Bombyx. (G and H) Changes in the levels of H3K27ac and H3K27me3 in the PG of Drosophila larvae at 108 h AEL following PG-specific knockdown of both Met and Gce. (I–L) ChIP-qPCR analysis of reciprocal changes in the enrichment of H3K27ac and H3K27me3 at the Hairy locus in ex vivo cultured PG-containing brain–RG complexes from Drosophila larvae at 108 h AEL and PGs from Bombyx larvae at L5D6 following JHM treatment for 24 h. JHM, JH mimic. AEL, after egg laying. L5D6, the sixth day of the fifth larval instar. [Scale bars, 20 μm (D, E, G, and H) and 200 μm (F).] Statistical values are presented as the mean ± SE (error bars) for three biological replicates. For the significance test, *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control.