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Posttranslational modifications, including acetylation and deacetylation of histones and other proteins, modulate hormone action. In Tribolium castaneum TcA cells, Trichostatin A, a histone deacetylase (HDAC) inhibitor, mimics juvenile hormone (JH) in inducing JH response genes (e.g., Kr-h1), suggesting that HDACs may be involved in JH action. To test this hypothesis, we identified genes coding for HDACs in T. castaneum and studied their function. Knockdown of 12 HDAC genes showed variable phenotypes; the most severe phenotype was detected in insects injected with double-stranded RNA targeting HDAC1 (dsHDAC1). The dsHDAC1-injected insects showed arrested growth and development and eventually died. Application of JH analogs hydroprene to T. castaneum larvae and JH III to TcA cells suppressed HDAC1 expression. Sequencing of RNA isolated from control and dsHDAC1-injected larvae identified 1,720 differentially expressed genes, of which 1,664 were up-regulated in dsHDAC1-treated insects. The acetylation levels of core histones were increased in TcA cells exposed to dsHDAC1 or JH III. ChIP assays performed using histone H2BK5ac antibodies showed an increase in acetylation in the Kr-h1 promoter region of cells exposed to JH III or dsHDAC1. Overexpression or knockdown of HDAC1, SIN3, or both resulted in a decrease or increase in Kr-h1 mRNA levels and its promoter activity, respectively. Overexpression of the JH receptor Methoprene tolerant (Met) was unable to induce Kr-h1 in the presence of HDAC1 or SIN3. These data suggest that epigenetic modifications influence JH action by modulating acetylation levels of histones and by affecting the recruitment of proteins involved in the regulation of JH response genes.

BackgroundJuvenile hormones (JH) and ecdysteroids control postembryonic development in insects. They serve as valuable targets for pest management. Hence, understanding the molecular mechanisms of their action is of crucial importance. CREB-binding protein (CBP) is a universal transcriptional co-regulator. It controls the expression of several genes including those from hormone signaling pathways through co-activation of many transcription factors. However, the role of CBP during postembryonic development in insects is not well understood. Therefore, we have studied the role of CBP in postembryonic development in Tribolium, a model coleopteran insect.ResultsCBP is ubiquitously expressed in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) mediated knockdown of CBP resulted in a decrease in JH induction of Kr-h1 gene expression in Tribolium larvae and led to a block in their development. Moreover, the injection of CBP double-stranded RNA (dsRNA) showed lethal phenotypes within 8 days of injection. RNA-seq and subsequent differential gene expression analysis identified CBP target genes in Tribolium. Knockdown of CBP caused a decrease in the expression of 1306 genes coding for transcription factors and other proteins associated with growth and development. Depletion of CBP impaired the expression of several JH response genes (e.g., Kr-h1, Hairy, early trypsin) and ecdysone response genes (EcR, E74, E75, and broad complex). Further, GO enrichment analyses of the downregulated genes showed enrichment in different functions including developmental processes, pigmentation, anatomical structure development, regulation of biological and cellular processes, etc.ConclusionThese data suggest diverse but crucial roles for CBP during postembryonic development in the coleopteran model insect, Tribolium. It can serve as a target for RNAi mediated pest management of this stored product pest.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4373-3) contains supplementary material, which is available to authorized users.

Post-translational modifications (PTM) such as methylation, acetylation, phosphorylation, and ubiquitination of histones and other proteins regulate expression of genes. The acetylation levels of these proteins are determined by the balance of expression of histone acetyltransferase (HATs) and histone deacetylases (HDACs). We recently reported that class I HDACs (HDAC1 and HDAC3) play important roles in juvenile hormone (JH) suppression of metamorphosis in the red flour beetle, Tribolium castaneum. Here, we report on the function of a single class IV HDAC member, HDAC11. Injection of dsRNA targeting T. castaneum HDAC11 gene into newly molted last instar larvae induced knockdown of the target gene and arrested larval development and prevented metamorphosis into the pupal stage. Dark melanized areas were detected in larvae that showed developmental arrest and mortality. Developmental expression studies showed an increase in HDAC11 mRNA levels beginning at the end of the penultimate larval stage. These higher levels were maintained during the final instar larval and pupal stages. A JH analog, hydroprene, suppressed HDAC11 expression in the larvae. Sequencing of RNA isolated from control and dsHDAC11 injected larvae identified several differentially expressed genes, including those involved in JH action, ecdysone response, and melanization. The acetylation levels of core histones showed an increase in TcA cells exposed to dsHDAC11. Also, an increase in histone H3 acetylation, specifically H3K9, H3K18 and H3K27, were detected in HDAC11 knockdown larvae. These studies report the function of HDAC11 in insects other than Drosophila for the first time and show that HDAC11 influences the acetylation levels of histones and expression of multiple genes involved in T. castaneum larval development.

The histone acetyltransferase Gcn5 is critical for gene expression and development. In Drosophila, Gcn5 is part of four complexes (SAGA, ATAC, CHAT, and ADA) that are essential for fly viability and have key roles in regulating gene expression. Here, we show that while the SAGA, ADA, and CHAT complexes play redundant roles in embryonic gene expression, the insect-specific CHAT complex uniquely regulates expression of a subset of developmental genes. We also identify a substantial decrease in histone acetylation in chiffon mutant embryos that exceeds that observed in ada2b, suggesting broader roles for Chiffon in regulating histone acetylation outside of the Gcn5 complexes. The chiffon gene encodes two independent polypeptides that nucleate formation of either the CHAT or Dbf4-dependent kinase (DDK) complexes. DDK includes the cell cycle kinase Cdc7, which is necessary for maternally-driven DNA replication in the embryo. We identify a temporal switch between the expression of these chiffon gene products during a short window during the early nuclear cycles in embryos that correlates with the onset of zygotic genome activation, suggesting a potential role for CHAT in this process.

Apoptosis has been widely studied from mammals to insects. Inhibitor of apoptosis (IAP) protein is a negative regulator of apoptosis. Recent studies suggest that iap genes could be excellent targets for RNAi-mediated control of insect pests. However, not much is known about iap genes in one of the well-known insect model species, Tribolium castaneum. The orthologues of five iap genes were identified in T. castaneum by searching its genome at NCBI (https://www.ncbi.nlm.nih.gov/) and UniProt (https://www.uniprot.org/) databases using Drosophila melanogaster and Aedes aegypti IAP protein sequences as queries. RNA interference assays were performed in T. castaneum cell line (TcA) and larvae. The knockdown of iap1 gene induced a distinct apoptotic phenotype in TcA cells and induced 91% mortality in T. castaneum larvae. Whereas, knockdown of iap5 resulted in a decrease in cell proliferation in TcA cells and developmental defects in T. castaneum larvae which led to 100% mortality. Knockdown of the other three iap genes identified did not cause a significant effect on cells or insects. These data increase our understanding of iap

Background: Hormones are chemical communication signaling molecules released into the body fluids to stimulate target cells of multicellular organisms. We recently showed that histone deacetylase 1 (HDAC1) plays an important role in juvenile hormone (JH) suppression of metamorphosis in the red flour beetle, Tribolium castaneum. Here, we investigated the function of another class I HDAC member, HDAC3, and show that it is required for the normal development of T. castaneum. Results: RNA interference-mediated knockdown of the HDAC3 gene affected development resulting in abnormally folded wings in pupae and adults. JH analog, hydroprene, suppressed the expression of HDAC3 in T. castaneum larvae. The knockdown of HDAC3 during the final instar larval stage resulted in an increase in the expression of genes coding for proteins involved in JH action. Sequencing of RNA isolated from larvae injected with dsRNA targeting malE (E. coli gene, control) or HDAC3 followed by differential gene expression analysis identified 148 and 741 differentially expressed genes based on the P-value < 0.01 and four-fold difference, and the P-value < 0.05 and twofold difference, respectively. Several genes, including those coding for myosin-I heavy chain (Myosin 22), Shaven, and nuclear receptor corepressor 1 were identified as differentially expressed genes in HDAC3 knockdown larvae. An increase in histone H3 acetylation, specifically H3K9, H3K18, and H3K27, was detected in HDAC3 knockdown insects. Conclusion: Overall, these data suggest that HDAC3 affects the acetylation levels of histones and influences the expression of genes coding for proteins involved in the regulation of growth, development, and metamorphosis.

Chiffon is the sole Drosophila ortholog of Dbf4, the regulatory subunit for the cell‐cycle kinase Cdc7 that initiates DNA replication. In Drosophila, the chiffon gene encodes two polypeptides with independent activities. Chiffon‐A contains the conserved Dbf4 motifs and interacts with Cdc7 to form the Dbf4‐dependent Kinase (DDK) complex, which is essential for a specialized form of DNA replication. In contrast, Chiffon‐B binds the histone acetyltransferase Gcn5 to form the Chiffon histone acetyltransferase (CHAT) complex, which is necessary for histone H3 acetylation and viability. Previous studies have shown that the Chiffon‐B region is only present within insects. However, it was unclear how widely the interaction between Chiffon‐B and Gcn5 was conserved among insect species. To examine this, we performed yeast two‐hybrid assays using Chiffon‐B and Gcn5 from a variety of insect species and found that Chiffon‐B and Gcn5 interact in Diptera species such as Australian sheep blowfly and yellow fever mosquito. Protein domain analysis identified that Chiffon‐B has features of acidic transcriptional activators such as Gal4 or VP16. We propose that the CHAT complex plays a critical role in a biological process that is unique to Dipterans and could therefore be a potential target for pest control strategies.