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. 2018 Jan 23;8(1):1426.
doi: 10.1038/s41598-018-19667-6.

CREB-binding Protein Plays Key Roles in Juvenile Hormone Action in the Red Flour Beetle, Tribolium Castaneum

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CREB-binding Protein Plays Key Roles in Juvenile Hormone Action in the Red Flour Beetle, Tribolium Castaneum

Jingjing Xu et al. Sci Rep. .
Free PMC article

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Abstract

Juvenile hormones (JH) and ecdysteroids regulate many biological and metabolic processes. CREB-binding protein (CBP) is a transcriptional co-regulator with histone acetyltransferase (HAT) activity. Therefore, CBP is involved in activation of many transcription factors that regulate expression of genes associated with postembryonic development in insects. However, the function of CBP in JH action in insects is not well understood. Hence, we studied the role of CBP in JH action in the red flour beetle, Tribolium castaneum and the Tribolium cell line. CBP knockdown caused a decrease in JH induction of genes, Kr-h1, 4EBP and G13402 in T. castaneum larvae, adults and TcA cells whereas, Trichostatin A [TSA, a histone deacetylase (HDAC) inhibitor] induced the expression of these JH-response genes. Western blot analysis with specific antibodies revealed the requirement of CBP for the acetylation of H3K18 and H3K27 in both T. castaneum and TcA cells. Chromatin immunoprecipitation (Chip) assays showed the importance of CBP-mediated acetylation of H3K27 for JH induction of Kr-h1, 4EBP, and G13402 in TcA cells. These data suggest that CBP plays an important role in JH action in the model insect, T.castaneum.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
TcA cells respond to both juvenile hormone and 20-hydroxyecdysone. TcA cells respond to 10 µM JH III or 20E. Total RNA was isolated from 100,000 cells that were cultured in the medium containing either DMSO or JH III or 20E at a final concentration of 10 µM for 6 hr. Total RNA was converted to cDNA, and the relative levels of Kr-h1, 4EBP, G13402, HR4, E74, E75A, E75B and Ftz-f1 mRNA were determined by qRT-PCR using RP49 as a control. The data shown are the Mean + S.D. (n = 3). The numbers on the control Kr-h1 bar show the relative expression levels for this treatment.
Figure 2
Figure 2
CBP is required for induction of JH-response genes in TcA cells. (A) CBP is required for JH induced gene expression. Total RNA was isolated from 100,000 cells exposed to malE, TcCBP, TcMet and TcSRC dsRNA for 72 hr. The cells were then exposed to 10 µM JH III for 6 hr. Total RNA was converted to cDNA, and the relative Kr-h1, 4EBP, and G13402 mRNA levels were determined by qRT-PCR using RP49 as a control.  The data shown are the Mean + S.D. (n = 3). (B) CBP is not required for gene expression induced by heat shock; Total RNA was isolated from 100,000 cells cultured with malE, TcCBP dsRNA for 72 hr followed by heat shock at 42 °C for 1hr. The relative levels of hsp90 mRNA were determined by qRT-PCR using RP49 as a control. The data shown are the Mean + S.D. (n = 3).
Figure 3
Figure 3
CBP is required for JH induction of genes in final instar larva (left panel) and newly emerged adults (right panel) of T. castaneum. CBP is required for JH induction of Kr-h1, 4EBP, and G13402 in day three adults or final instar larva. The newly emerged adults or the final instar larva were injected with either malE or CBP dsRNA. Seventy-two hours after injection 0.5 µl of 10 μM hydroprene was topically applied. Six hours after application, total RNA was isolated and used to quantify mRNA levels of Kr-h1, 4EBP, and G13402 by qRT-PCR using RP49 as a control. The data shown are the Mean + S.D. (n = 4).
Figure 4
Figure 4
Trichostatin A (TSA, HDAC inhibitor, left panel) and JH III (right panel) induce Kr-h1, 4EBP and G13402 expression in a dose-dependent manner. TcA cells were cultured in the medium containing 0–5 μM TSA or 0–10 μM JH III for 6 hr. Total RNA was isolated from the treated cells and used to quantify mRNA levels of Kr-h1, 4EBP, G13402, and hsp90. The total RNA extraction and subsequent quantification of mRNA levels of Kr-h1, 4EBP, G13402, and hsp90 were performed as described in Fig. 1. The data shown are the Mean + S.D. (n = 3).
Figure 5
Figure 5
CBP is not required for expression of JH receptor Met and steroid receptor co-activator, SRC in TcA cells. Total RNA was isolated from 100,000 cells exposed to malE or TcCBP dsRNA for 72 hr. Total RNA was converted to cDNA, and the relative mRNA levels of CBP, Met, SRC and Kr-h1 were determined by qRT-PCR using RP49 as a control. The data shown are the Mean + S.D. (n = 3). (One-way ANOVA, letters represent significance at 95% CI).
Figure 6
Figure 6
CBP is involved in the acetylation of H3 in T. castaneum adults and TcA cells. 50 µg total proteins extracted from the day 5 adults and TcA cells were separated by SDS-PAGE gels, transferred to the membrane and probed with acetyl-lysine, H3K9, H3K18 and H3K27 antibodies. The same concentration of proteins in parallel blots were probed with actin and H3 antibodies are shown on the top and bottom. The blots shown are cropped from the full-length blots presented in Supplementary Figure 1.
Figure 7
Figure 7
(A) Chip assay. dsCBP exposure decreased the localization of the acetylated H3K27 to Kr-h1, 4EBP, and G13402 promoters thus affected the enrichment of the gene promoters compare to the cells exposed to dsmalE. The cells were exposed to dsCBP or dsmalE and treated with DMSO, JH III, and TSA. The chromatin was cross-linked and precipitated with H3K27 antibodies. The DNA was amplified using primers targeting Kr-h1, 4EBP,G13402 or HSP90 gene promoters. The highest expression level in the negative control experiment (assay with IgG antibody) was indicated by a solid line and marked as NC. (B–D) Boxplots showing the results of statistical analysis represented in the tabular form in Table 2. Each interaction corresponded to an individual plot.
Figure 8
Figure 8
A model for CBP and HDAC regulation of acetylation and JH action.

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