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DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It has been reported that the methyltransferase METTL4 can catalyze the methylation of 6mA; however, the function of METTL4 remains largely unknown. In this study, we aim to investigate the role of the Bombyx mori homolog METTL4 (BmMETTL4) in silkworm, a lepidopteran model insect. By using CRISPR-Cas9 system, we somatically mutated BmMETTL4 in silkworm individuates and found that disruption of BmMETTL4 caused the developmental defect of late silkworm embryo and subsequent lethality. We performed RNA-Seq and identified that there were 3192 differentially expressed genes in BmMETTL4 mutant including 1743 up-regulated and 1449 down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that genes involved in molecular structure, chitin binding, and serine hydrolase activity were significantly affected by BmMETTL4 mutation. We further found that the expression of cuticular protein genes and collagens were clearly decreased while collagenases were highly increased, which had great contributions to the abnormal embryo and decreased hatchability of silkworm. Taken together, these results demonstrated a critical role of 6mA methyltransferase BmMETTL4 in regulating embryonic development of silkworm. [ABSTRACT FROM AUTHOR]

Translationally controlled tumor protein (TCTP) is a highly conserved protein possessing numerous biological functions and molecular interactions, ranging from cell growth to immune responses. However, the molecular mechanism by which TCTP regulates immune function is largely unknown. Here, we found that knockdown of Bombyx mori translationally controlled tumor protein (BmTCTP) led to the increased susceptibility of silkworm cells to virus infection, whereas overexpression of BmTCTP significantly decreased the virus replication. We further demonstrated that BmTCTP could be modified by SUMOylation molecular BmSMT3 at the lysine 164 via the conjugating enzyme BmUBC9, and the stable SUMOylation of BmTCTP by expressing BmTCTP -BmSMT3 fusion protein exhibited strong antiviral activity, which confirmed that the SUMOylation of BmTCTP would contribute to its immune responses. Further work indicated that BmTCTP is able to physically interact with interleukin enhancer binding factor (ILF), one immune molecular, involved in antivirus, and also induce the expression of BmILF in response to virus infection, which in turn enhanced antiviral activity of BmTCTP. Altogether, our present study has provided a novel insight into defending against virus via BmTCTP SUMOylation signaling pathway and interacting with key immune molecular in silkworm. [ABSTRACT FROM AUTHOR]

Abstract Increasing knowledge of DNA methylation that occurs on the sixth position of adenine ( N 6-methyladenine, 6mA) has emerged as a novel epigenetic mark in eukaryotes and plays an important role in regulating gene transcription, DNA replication and repair, transposable activities, and others. Here, we show DNA 6mA methylation is present in Bombyx mori , a lepidopteran model insect, and identify the 6mA methyltransferase, METTL4, and 6mA demethylase, NMAD, which regulate the levels of 6mA in embryogenesis and cultured cells of B. mori. Importantly, RNAi knockdown of METTL4 and NMAD not only induce cell cycle arrest at G1 phase but also result in defects of chromosome alignments at metaphase. We further demonstrate that 6mA methylation is widely distributed across the genome of B. mori by 6mA-Seq and primarily enriched in the regulatory regions as well as gene bodies. Integrated analysis of 6mA-Seq and RNA-Seq reveals that 6mA methylation in B. mori is preferentially related with lowly expressed genes and negatively correlated with active gene transcription, which provides a novel regulatory mechanism of DNA 6mA methylation on target genes. Altogether, these data identify 6mA methylation in B. mori and demonstrate a crucial role of 6mA signaling in controlling cell cycle progression. Highlights • DNA methylation on N 6-adenine (6mA) is widely present in the silkworm genome. • 6mA is primarily enriched in regulatory regions and gene bodies in silkworm. • 6mA is negatively associated with active gene transcription in silkworm. • 6mA is dynamically regulated by METTL4 methyltransferase and NMAD demethylase. [ABSTRACT FROM AUTHOR]

Polycomb group (PcG) complexes are known to be chromatin modifiers and transcriptional repressors. In this work, we reported that the histone-modifying PcG complexes are able to participate in the repair process of ultraviolet (UV)-induced DNA lesions in the silkworm, Bombyx mori . The silkworm cells with depletion of PcG genes showed hypersensitive to UV–C irradiation and increased inhibition of cell proliferation. Interestingly, an SQ site in the silkworm-human chimeric H2A protein synthesized here was phosphorylated rapidly upon UV–C exposure, which could be used as a marker for monitoring the response to DNA damage in silkworm cells. Under these UV–C irradiated conditions, we found that PRC1-mediated ubiquitylation of H2AX, but not of H2AZ, were decreased and this deubiquitylation was independent of its phosphorylation event. In contrast, UV–C irradiation induced the increase of trimethylation of lysine 27 on histone H3 (H3K27me3), a mark of transcriptionally silent chromatin catalyzed by another PcG subcomplex, PRC2. Collectively, we provided the first evidence on chromatin remodeling in response to UV–C lesion in silkworm and revealed another layer role for PcG complexes-mediated histone modifications in contributing to creating an open chromatin structure for the efficient repair of DNA damages. [ABSTRACT FROM AUTHOR]

Simple Summary: The mechanism of the polyamine pathway in the lepidopteran silkworm is largely unknown. In the current study, we aimed to characterize the function of polyamines and polyamine pathway genes in silkworm cells as a regulator of cell cycle progression. For the first time, we identified the homologous genes of the polyamine pathway in silkworm, and analyzed their expression characteristics in different tissues and their subcellular localizations in cultured silkworm cells. We measured the abundant levels of polyamines in silkworm cells by HPLC analysis. We found that exogenous supplementation of spermidine in cells promoted DNA replication and cell cycle progression and, in contrast, treatment with polyamine biosynthesis inhibitors DFMO and MGBG prevented DNA replication and cell cycle progression. Indeed, the mechanism studies indicated that spermidine increased the expression of cell cycle-related genes, whereas this increase could be abolished by treatment with inhibitors. Taken together, our findings highlight that appropriate levels of polyamines have beneficial effects on the progression of the cell cycle by regulating cell cycle genes in silkworm. Background: Putrescine, spermidine, and spermine are polyamines that are ubiquitously distributed in prokaryotic and eukaryotic cells, which play important roles in cell proliferation and differentiation. Methods: We investigated the expression profiles of polyamine pathway genes by qRT-PCR in different tissues of the lepidopteran silkworm. The polyamine levels in cultured silkworm cells were measured by HPLC. Spermidine and polyamine biosynthetic inhibitors were used for treating the cultured silkworm cells in order to clarify their effects on cell cycle progression. Results: We identified the anabolic and catabolic enzymes that are involved in the polyamine biosynthetic pathway in silkworm. Transcriptional expression showed at least seven genes that were expressed in different silkworm tissues. Treatments of the cultured silkworm cells with spermidine or inhibitor mixtures of DFMO and MGBG induced or inhibited the expression of cell cycle-related genes, respectively, and thus led to changed progression of the cell cycle. Conclusions: The present study is the first to identify the polyamine pathway genes and to demonstrate the roles of polyamines on cell cycle progression via regulation of the expression of cell cycle genes in silkworm. [ABSTRACT FROM AUTHOR]

Polyamidoamine (PAMAM) dendrimers are emerging as intriguing nanovectors for nucleic acid delivery because of their unique well-defined architecture and high binding capacity, which have been broadly applied in DNA- and RNA-based therapeutics. The low-cost and high-efficiency of PAMAM dendrimers relative to traditional liposomal transfection reagents also promote their application in gene function analysis. In this study, we first investigated the potential use of a PAMAM system in the silkworm model insect. We determined the binding property of G5-PAMAM using dsRNA and DNA in vitro, and substantially achieved the delivery of dsRNA and DNA from culture medium to both silkworm BmN and BmE cells, thus leading to efficient knockdown and expression of target genes. Under treatments with different concentrations of G5-PAMAM, we evaluated its cellular cytotoxicity on silkworm cells, and the results show that G5-PAMAM had no obvious toxicity to cells. The presence of serum in the culture medium did not affect the delivery performance of DNA and dsRNA by G5-PAMAM, revealing its convenient use for various purposes. In conclusion, our data demonstrate that the PAMAM system provides a promising strategy for delivering dsRNA and DNA in cultured silkworm cells and promote its further application in individuals. [ABSTRACT FROM AUTHOR]

The centromere, in which kinetochore proteins are assembled, plays an important role in the accurate congression and segregation of chromosomes during cell mitosis. Although the function of the centromere and kinetochore is conserved from monocentric to holocentric, the DNA sequences of the centromere and components of the kinetochore are varied among different species. Given the lack of core centromere protein A (CENP-A) and CENP-C in the lepidopteran silkworm Bombyx mori, which possesses holocentric chromosomes, here we investigated the role of CENP-N, another important member of the centromere protein family essential for kinetochore assembly. For the first time, cellular localization and RNA interference against CENP-N have confirmed its kinetochore function in silkworms. To gain further insights into the regulation of CENP-N in the centromere, we analyzed the affinity-purified complex of CENP-N by mass spectrometry and identified 142 interacting proteins. Among these factors, we found that the chaperone protein heat shock cognate 70 (HSC70) is able to regulate the stability of CENP-N by prohibiting ubiquitin–proteasome pathway, indicating that HSC70 could control cell cycle-regulated degradation of CENP-N at centromeres. Altogether, the present work will provide a novel clue to understand the regulatory mechanism for the kinetochore activity of CENP-N during the cell cycle. [ABSTRACT FROM AUTHOR]

Abstract Fibroin modulator binding protein-1 (FMBP-1) is a novel DNA-binding protein containing a conserved score and three amino acid peptide repeat (STPR) domain. The roles of factors containing STPR domain are less known. Although multiple transcription factors are involved in the transcriptional regulation of silk protein genes during the development of silkworm, the mechanism of transcriptional repression of silk protein genes during molting remains unclear. Here, we found that FMBP-1 expression was contrary to that of fibroin heavy chain (fib-H) during the fourth molting period of Bombyx mori. FMBP-1 repressed fib-H promoter activity by directly binding to the −130 element in the fib-H promoter region. We also identified two proteins, Bmsage and Bmdimm, that interacted with FMBP-1 in the posterior silk gland of silkworm larvae, and further verified these interactions by far western blotting and microscale thermophoresis in vitro , as well as co-immunoprecipitation and bimolecular fluorescence complementation at the cellular level. The luciferase reporter assay showed that the interaction between FMBP-1 and Bmdimm antagonized the activation of Bmdimm on fib-H transcription, but did not affect FMBP-1-mediated transcriptional repression on fib-H gene. Therefore, we proposed the following mechanism of fib-H transcriptional repression by FMBP-1 during the molting of silkworm larvae: 1) FMBP-1 directly binds to the −130 element in the fib-H promoter to repress fib-H transcription; 2) FMBP-1 interacts with Bmdimm to antagonize the activation of Bmdimm on fib-H transcription. Our findings promote a better understanding of fib-H transcriptional regulation and provide novel insights into the transcriptional repression of fib-H by FMBP-1 and basic helix-loop-helix factors Bmdimm during the molting of silkworm larvae. Our study also provides valuable information regarding the biological function of factors containing STPR domain. Graphical abstract Image 1 Highlights • FMBP-1 represses fib-H gene transcription during molting of silkworm larvae. • FMBP-1 directly binds to the −130 element upstream of fib-H promoter to repress fib-H transcription. • FMBP-1 interacts with Bmdimm to antagonize the activation of Bmdimm on fib-H transcription. [ABSTRACT FROM AUTHOR]

DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant Bm DNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the Bm DNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, Bm DNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn 2+ and Mn 2+ . Our results provide clear evidence that Bm DNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1. [ABSTRACT FROM AUTHOR]

Abstract: Nuclear receptors (NRs) function as ligand-dependent transcription factors and are involved in diverse biological processes in different animals. The updated assembly of complete genome sequence of the Bombyx mori enabled a systematic analysis of the NRs in the five holometabolous insects including B. mori, Drosophila melanogaster, Anopheles gambiae, Apis mellifera, and Tribolium castaneum. As a result, nineteen NRs were identified in the B. mori genome, each of eighteen NRs has 1:1:1:1 ortholog in the other four insects. Interestingly, the average intron number of ligand-binding domain (LBD) of each NR gene in B. mori was 2.4, much higher than that in the other four insects; the genomic position of introns in LBDs of all orthologs for each NR presents more diversity. Phylogenetic trees of all NRs from the five insects were consistent or aberrant with classical phylogeny of these insect species. The characteristics in number, genomic structure and phylogeny of all NRs revealed their evolutionary conservation and divergence during insect evolution. The expression patterns of several NR genes displayed temporal specificity similar to that in D. melanogaster and may be associated with the key biological processes during silkworm metamorphosis. The RNAi of BmβFTZ-F1 resulted in abnormality in larva–pupa transition, further suggesting it is also crucial for silkworm metamorphosis. In conclusion, the present study provided new insights into the structure, evolution, expression, and functions of silkworm NRs. [Copyright &y& Elsevier]