Epigenetic regulation of post-embryonic development

Abstract

Modifications to DNA and core histones influence chromatin organization and expression of the genome. DNA methylation plays a significant role in the regulation of multiple biological processes that regulate behavior and caste differentiation in social insects. Histone modifications play significant roles in the regulation of development and reproduction in other insects. Genes coding for acetyltransferases, deacetylases, methyltransferases, and demethylases that modify core histones have been identified in genomes of multiple insects. Studies on the function and mechanisms of action of some of these enzymes uncovered their contribution to post-embryonic development. The results from studies on epigenetic modifiers could help in the identification of inhibitors of epigenetic modifiers that could be developed to control pests and disease vectors.

Introduction

Epigenetics includes the study of heritable phenotype changes caused by nucleic acid and protein modifications that do not involve DNA sequence changes [1]. Modifications to DNA (e.g., methylation) and histones (e. g., acetylation) influence chromatin organization and expression of genes that regulate many cellular and developmental processes. Recent studies in epigenetics identified key epigenetic modifiers, their mechanisms of action, and the target proteins involved. Several excellent reviews on this subject, including some that focused on epigenetic modifications in insects, were published recently [2–8]. However, not much has been covered on the role of epigenetics in hormonal regulation of post-embryonic development in insects, which is the focus of this review. After a brief introduction to epigenetics, recent progress in epigenetic modifications that influence hormonal regulation of post-embryonic development and metamorphosis will be reviewed.

In eukaryotic cells, DNA is organized into two major forms of chromatin, euchromatin (transcriptionally active) and heterochromatin (transcriptionally inactive). The building blocks of chromatin, the nucleosomes, are formed by two copies of each of four histone proteins and a 146 base pair DNA. The interactions between nucleic acids and histone proteins play key roles in the formation of euchromatin and heterochromatin. Modifications to both DNA and histones influence the organization of chromatin. Also, the non-coding RNAs also play important roles in the organization of these two forms of chromatin. Small interference RNAs and microRNAs regulate many developmental and physiological processes, including X chromosome inactivation [9]. The modifications that influence chromatin organization contribute to flexible and reversible genome expression without altering the genetic makeup of an organism. DNA and histone modifications and microRNAs regulate gene activity and expression patterns.

Modifications to DNA, especially methylation to form 5-methylcytosine (5mC) repress gene expression [10]. The enzymes responsible for DNA methylation, DNA methyltransferases have been identified, and the effect of this epigenetic modification on cellular, physiological, and developmental processes has been studied. Oxidation of 5mc by ten-eleven translocation proteins to 5-Hydroxymethylcytosine is also important in epigenetics because this modification is shown to regulate gene expression [11]. Also, aberrations in epigenetic modifications and enzymes involved in these modifications could result in growth and developmental defects leading to human diseases such as cancer [12]. Therefore, epigenetic modifiers are being explored as targets for the discovery of drugs to treat human diseases [13].

The N-terminal tails of histones mediate interactions between nucleosomes and DNA and influence the formation of chromatin structure. The histone tails undergo posttranslational modifications, including acetylation, phosphorylation, methylation, and ubiquitination, that play important roles in the organization of euchromatin and heterochromatin [14]. In eukaryotes, lysine acetyltransferases (KATs or Histone acetyltransferases HATs) and lysine deacetylases (KDACs or histone deacetylases HDACs) [15] regulate acetylation levels of histones and other proteins. The access of transcription factors to promoters is regulated by modifications to DNA and core histones. One major modification is the modulation of the positive charge density of core histones by the addition or removal of acetyl groups [16]. Lysine acetylation is a reversible posttranscriptional modification of proteins and plays a key role in the organization of multiprotein complexes involved in gene regulation in many cellular and developmental processes [17].

Methylation of histones, H3 (lysines 4, 9, 27, and 36) and H4 (lysine 20) also play key roles in the organization of chromatin [18]. Studies in the fruit fly, Drosophila melanogaster identified the suppressors of position effect variegation (PEV) including the Su(var)3–9, the polycomb-group protein Enhancer of zeste, and the trithorax-group protein Trithorax that contains a conserved SET domain [19] which share sequence similarity with SET domain-containing methyltransferases identified in plants [20]. Later human and yeast homologs of PEV proteins were identified and shown to function as histone lysine methyltransferases [21].

Epigenetic modifications in eusocial insects

DNA methylation

Initial studies in eusocial insects, especially honey bees, indicated that DNA methylation plays an important role in the regulation of caste differentiation [22,23], memory processing [24], queen larval development [25], metabolism [26] and other behaviors [8]. DNA methyltransferase 3(Dnmt3)gene was identified in honey bees, and knockdown of this gene results in reduced genome-wide methylation levels and altered RNA splicing pattern of genes suggesting an important role for Dnmt3 and DNA methylation in honeybee queen cast differentiation [26,27].In contrast to mammals, in eusocial insects, an increase in DNA methylation was detected in gene bodies of actively expressed genes [6]. The role of DNA methylation in caste differentiation has been reported in the termite, Zootermopsis nevadensis, in which queen and worker larvae have significantly different methylation patterns [28]. In ants, Camponotus floridanus and Harpegnathos saltator, a connection between DNA methylation and RNA splicing was suggested [29]. Differentially methylated genes between castes have been identified in the bumblebee, Bombus terrestris [30].

Histone modifications:

Royal jelly, which contains up to 5% HDAC inhibitor, facilitates queen cast differentiation in honey bees, suggesting that inhibition of HDAC activity might function in queen cast differentiation [31]. Differences in histone H3K27 acetylation levels that cause differential expression of genes coding for proteins involved in the development of nervous and olfactory systems between castes in Camponotus floridanus have been reported [32,33]. An HDAC, Sir2, influences lifespan in honey bees [34]. Recent studies in eusocial insects showed that histone modifications are required for caste differentiation in ants [33]. The caste-based behaviors (workers and foragers) are regulated by the balance of HATs and HDACs [33]. The acetylation of histone by HATs results in more open chromatin by neutralization of lysines, increasing accessibility to promoters and results in enhanced gene expression.

Epigenetic modifications in other insects

DNA methylation

The role of DNA methylation in the regulation of developmental and physiological processes in other insects, including the fruit fly D. melanogaster, is not clearly defined. The Dnmt3 methyltransferase known to be involved in de novo DNA methylation has not been identified in the genomes of flies, mosquitoes, the red flour beetle, and the silkworm [35]. However, low levels of DNA methylation have been observed in these insects, and the function of DNA methylation in genome expression, especially during embryonic development, has been suggested [35]. It is possible that the other two epigenetic mechanisms, i.e., modification of histones and RNAs (siRNA miRNA and ncRNA), may play important roles in epigenetic regulation of post-embryonic development in these insects.

Histone modifications

Acetylation

In D. melanogaster, many acetylation sites were identified in the proteome using high-resolution mass spectrometry [36]. Interestingly, the sites of these modifications are highly conserved between humans and fruit flies. Also, a comparison of lysine acetylation sites in the fruit fly, human, nematode, and zebrafish showed significantly more conservation in acetylated lysines than the non-acetylated lysines [36]. Genome-wide studies detected H3K9acS10ph and H3K27acS28ph as a general feature of enhancers and promoters in D. melanogaster [37]. The acetylation levels of histones are maintained by the activity of HATs and HDACs. Studies on acetylation of H3 and H4 in D. melanogaster cells by depletion of HATs and HDACs revealed alterations in histone acetylation levels [38].

Histone acetyltransferases

About 30 genes coding for HAT domain-containing proteins were identified in D. melanogaster [39]. The orthologs of most of these genes are identified in T. castaneum and Ae. aegypti genomes (George, Chereddy and Palli, unpublished). The D. melanogaster HAT domain-containing proteins were shown to function in the regulation of stem cell homeostasis[40], cell death and JAK/STAT pathway [41,42], oogenesis [43], neural development [44], autophagy and lysosome biogenesis [45], metamorphosis [46], embryogenesis [47], dosage compensation by a transcriptional upregulation in the male X chromosome [49], courtship [50], learning and memory [51]. The CREB-binding protein (CBP) is a global epigenetic and transcriptional regulator of growth and development. The D. melanogaster homolog of CBP, nejire, is one of the most studied HAT domain-containing proteins. It acts as a coactivator to a large number of transcription factors and epigenetically regulates the expression of many genes by acetylating histones [52]. It acts as a central switch, unwinding chromatin to facilitate transcription complex access to promoters. CBP serves as a transcriptional regulatory hub in transcriptional networks and interacts with more than 800 transcription factors [52]. The CBP was shown to regulate anterior-posterior polarity of embryonic segments through Hedgehog and Wingless signaling pathways, and dorsal-ventral patterning through the Transforming growth factor β (TGF-β) signaling pathway [53,54]. The CBP is also involved in the regulation of post-embryonic developmental processes such as dendrite pruning, circadian rhythm, and caste differentiation [55,56].

CBP’s role in hormone regulation of developmental pathways has been studied in the hemimetabolous insect, the German cockroach, Blattella germanica [57] and the holometabolous insects, the red flour beetle, T. castaneum [58–60], the yellow fever mosquito, Aedes aegypti [61] and the fruit fly, D. melanogaster [55]. The HAT activity of CBP is required for sox14 expression, which is a key ecdysone response gene mediated by acetylation of H3K27 [55]. For regulation of Eip74EF and Eip75B, the 20E induced transcription factors, CPB functions through acetylation of H3K23 [62]. In T. castaneum larvae, adults and TcA cells, the knockdown of CBP caused a decrease in juvenile hormone (JH) induction of genes, Kr-h1, 4EBP and G13402. Western blot analysis revealed the requirement of CBP for the acetylation of H3K18 and H3K27. ChIP assays showed the importance of CBP for JH induction of Kr-h1, 4EBP, and G13402 [59]. CBP is ubiquitously expressed in T. castaneum. The knockdown of CBP in T. castaneum reduced JH induction of Kr-h1 and led to the developmental arrest. RNA sequencing and differential gene expression analysis identified CBP target genes in T. castaneum [58,59]. Studies in Ae. aegypti showed that CBP is an essential player in JH and 20E regulation of metamorphosis and compound eye development [61]. The role of HATs in epigenetic reprogramming during metamorphosis, wounding and infection in Gallaria mellonella have also been reported [63]. As shown in the model (Fig. 1), CBP is an essential player in JH and 20E regulation of metamorphosis.

Fig. 1.

A proposed model for CBP function in hormonal regulation of metamorphosis. CBP is required for the expression of E75A. Knockdown of CPB in Ae. aegypti results in a decrease in expression of E75A and an increase in expression of EcRA. In the absence of JH and presence of 20E, EcR/UAP binds to E93/BR-C promoter and induces their expression. The BR-C and E93 promote metamorphosis. CBP is also required for the expression of Kr-h1. In the presence of JH, CBP is involved in the acetylation of core histones and the recruitment of Met and SRC to Kr-h1 and other proteins involved in Kr-h1 expression. Kr-h1 prevents EcR and USP binding to the E93 promoter resulting in suppression of E93 expression and metamorphosis.

Histone deacetylases

Homologs of twelve histone deacetylases belonging to four families have been identified in D. melanogaster, T. castaneum and Ae. aegypti [39,64]. In D. melanogaster HDACs have been shown function in regulation of apoptosis [65], synaptic plasticity and memory [66], aging [67] embryogenesis, muscle development, circadian function [68], energy balance [69] memory [70] and metabolism [71].In T. castaneum TcA cells, Trichostatin A, HDAC inhibitor, mimics JH suggesting that HDACs may be involved in JH action. Genes coding for 12 HDACs were identified in T. castaneum [64]. RNA interference (RNAi)-mediated knockdown of HDAC1 in T. castaneum showed arrest in development during the last larval and pupal stages. Treatment of T. castaneum larvae with JH analog, hydroprene or TcA cells with JH III, suppressed HDAC1 gene expression. ChIP assays showed localization of acetylated histones near the Kr-h1 promoter in TcA cells treated with 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. These data suggest that epigenetic modifications influence JH action by modulating acetylation levels of histones and affecting the recruitment of proteins involved in the regulation of JH response genes [64] (Fig. 2). HDAC3 knockdown affected development resulting in abnormally folded wings in pupae and adults. HDAC3 affected the acetylation levels of histones and influences the expression of genes coding for proteins involved in the regulation of growth, development, and metamorphosis [72]. Knockdown of HDAC 11 arrested larval development and prevented metamorphosis into the pupal stage by affecting acetylation of histones and acting at the promoter of JH-response genes [73].

Fig. 2. The function of HATs and HDACs in JH Psignaling.

In the absence of JH, HDAC-SIN3 corepressor complex is recruited to the Kr-h1 promoter. HDAC deacetylates histones and causes transcriptional repression of Kr-h1. In the presence of JH, HATs such as CBP are localized to Kr-h1 promoter and help in acetylation of core histones and recruitment of Met, SRC and other proteins resulting in expression of Kr-h1.

Methylation

Histone methyltransferases

Methylation of histones affects chromatin organization and gene expression. Proteins containing histone arginine methyltransferase and lysine methyltransferase domains transfer methyl groups to arginine and lysine residues in Histones H3 and H4. About 30 genes coding for proteins containing histone arginine methyltransferase and lysine methyltransferase domains have been identified in the genomes of D. melanogaster, T. castaneum and Ae. aegypti [74,75] (Yao and Palli, unpublished). In D. melanogaster, arginine methyltransferase 1 methylates the arginine residue of histone H4 and disruption of the gene coding for this enzyme affected metamorphosis and survival during the pupal stage and this effect is caused through repression of the ecdysone receptor activity in a ligand-dependent manner [76]. In D. melanogaster histone lysine methyltransferases contribute to stem cell division, oogenesis, female fate maintenance of germ cells and wing disc development[77–79]. Comparative genomicanalysis of SET domain family proteins in 147 sequenced arthropod genomes revealed an arthropod– specific gene family, SmydA [80]. In Smyd proteins SET domain is interrupted by MYND (Myeloid translocation protein, Nervy, Deaf, a protein-protein interaction domain involved in recruiting of co-factors). The histone lysine methyltransferases function of these proteins is maintained in arthropods suggesting that these proteins may be involved in regulating essential physiological and developmental pathways in insects and other arthropods [80].

Histone demethylases

Histone demethylases remove methyl groups from histones. A dozen genes coding for proteins containing histone demethylase domains were identified in the genomes of D. melanogaster, T. castaneum and Ae. aegypti [81,82] (Yao and Palli, unpublished). In D. melanogaster, histone methyltransferases, especially little imaginal discs, function in the regulation of growth, circadian rhythm, stress resistance, hematopoiesis, behavior, development and fertility [83–86].

Conclusions and future perspectives

Epigenetic modifications, including DNA methylation in social insects and histone modifications in other insects, have been studied during the past few years. Multiple enzymes responsible for these modifications have been identified in the fruit fly and a few other insects such as the yellow fever mosquito and the red flour beetle. The function of some of the enzymes and their mechanisms of action have been studied in the fruit fly. Future research focused on the function of epigenetic modifiers and their mechanism of action in other insects could uncover some interesting target sites for controlling pests and disease vectors. Many inhibitors of epigenetic modifiers were discovered for the treatment of human diseases such as cancer. There is an opportunity to use the knowledge gained from studies on epigenetic modifiers in pests and disease vectors to repurpose some of these compounds for the control of pests and disease vectors.

Highlights.

• Modifications of DNA influence the expression of genes required for insect post-embryonic development.

• Histone modifications including acetylation and methylations play vital roles in insect post-embryonic development.

• Multiple genes coding for epigenetic modifying enzymes have been identified in insects.

• Studies on the mechanisms of actions of epigenetic modifiers are underway.