DNA methylation and regulatory elements during chicken germline stem cell differentiation

Funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.The production of germ cells in vitro would open important new avenues for stem biology and human medicine, but the mechanisms of germ cell differentiation are not well understood. The chicken, as a great model for embryology and development, was used in this study to help us explore its regulatory mechanisms. In this study, we reported a comprehensive genome-wide DNA methylation landscape in chicken germ cells, and transcriptomic dynamics was also presented. By uncovering DNA methylation patterns on individual genes, some genes accurately modulated by DNA methylation were found to be associated with cancers and virus infection, e.g., AKT1 and CTNNB1. Chicken-unique markers were also discovered for identifying male germ cells. Importantly, integrated epigenetic mechanisms were explored during male germ cell differentiation, which provides deep insight into the epigenetic processes associated with male germ cell differentiation and possibly improves treatment options to male infertility in animals and humans

Prostaglandins A1 and E1 influence gene expression in an established insect cell line (BCIRL-HzAM1 cells)

Prostaglandins (PGs) and other eicosanoids exert important physiological actions in insects and other invertebrates, including influencing ion transport and mediating cellular immune defense functions. Although these actions are very well documented, we have no information on the mechanisms of PGs actions in insect cells. Here we report on the outcomes of experiments designed to test our hypothesis that PGs modulate gene expression in an insect cell line established from pupal ovarian tissue of the moth Helicoverpa zea (BCIRL-HzAM1 cells). We treated cells with either PGA1 or PGE1 for 12 or 24 h then analyzed cell lysates by 2-D electrophoresis. Analysis of the gels by densitometry revealed substantial changes in protein expression in some of the protein spots we analyzed. These spots were processed for mass spectrometric analysis by MALDI TOF/TOF, which yielded in silico protein identities for all 34 spots. The apparent changes in three of the proteins were confirmed by semi-quantative PCR, showing that the changes in mRNA expression were reflected in changes in protein expression. The 34 proteins were sorted into six categories, protein actions, lipid metabolism, signal transduction, protection, cell functions and metabolism. The findings support the hypothesis that one mechanism of PG action in insect cells is the modulation of gene expression

Proteomic identification of PKC-mediated expression of 20E-induced protein in Drosophila melanogaster

Ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle protein (USP), are nuclear receptors that mediate the action of the insect molting hormone 20-hydroxyecdysone (20E). There is evidence that the activity of both receptors is affected by phosphorylation. Using a proteomic approach, we have shown that protein kinase C (PKC) activity is necessary for mediating 20E-induced expression of 14 specific proteins, including three previously reported 20E responsive proteins, and is also responsible for the intracellular localization of EcR and USP in larval salivary glands of Drosophila melanogaster. The 20E-dependent expression of the proteins was verified using real-time PCR and/or Western blot analysis. For some genes, inhibition of PKC activity reduced 20E-dependent transcriptional activity rapidly, raising the possibility that these are direct gene targets of EcR and USP. The data further indicate that PKC-mediated phosphorylation is also required for genes regulated indirectly by 20E-induced changes in the larval salivary gland

A Method to Generate and Analyze Modified Myristoylated Proteins

Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio-orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone-modified myristoylated Arf1 could be further labeled by fluorophore-coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid-modified proteins with additional functions before applying them to cellular systems

Transcriptomic Response of Wolf Spider, \u3cem\u3ePardosa pseudoannulata\u3c/em\u3e, to Transgenic Rice Expressing \u3cem\u3eBacillus thuringiensis\u3c/em\u3e Cry1Ab Protein

Background: Bacillum thuringiensis (Bt) toxin produced in Cry1-expressing genetically modified rice (Bt rice) is highly effective to control lepidopteran pests, which reduces the needs for synthetic insecticides. Non-target organisms can be exposed to Bt toxins through direct feeding or trophic interactions in the field. The wolf spider Pardosa pseudoannulata, one of the dominant predators in South China, plays a crucial role in the rice agroecosystem. In this study, we investigated transcriptome responses of the 5th instar spiders fed on preys maintained on Bt- and non-Bt rice. Results: Comparative transcriptome analysis resulted in 136 differentially expressed genes (DEGs) between spiderlings preying upon N. lugens fed on Bt- and non-Bt rice (Bt- and non-Bt spiderlings). Functional analysis indicated a potential impact of Bttoxin on the formation of new cuticles during molting. GO and KEGG enrichment analyses suggested that GO terms associated with chitin or cuticle, including “chitin binding”, “chitin metabolic process”, “chitin synthase activity”, “cuticle chitin biosynthetic process”, “cuticle hydrocarbon biosynthetic process”, and “structural constituent of cuticle”, and an array of amino acid metabolic pathways, including “alanine, asparatate and glutamate metabolism”, “glycine, serine and theronine metabolism”, “cysteine and methionine metabolism”, “tyrosine metabolism”, “phenylalanine metabolism and phenylalanine”, and “tyrosine and tryptophan biosynthesis” were significantly influenced in response to Cry1Ab. Conclusions: The Cry1Ab may have a negative impact on the formation of new cuticles during molting, which is contributed to the delayed development of spiderlings. To validate these transcriptomic responses, further examination at the translational level will be warranted

Transcriptomic Response of Wolf Spider, \u3cem\u3ePardosa pseudoannulata\u3c/em\u3e, to Transgenic Rice Expressing \u3cem\u3eBacillus thuringiensis\u3c/em\u3e Cry1Ab Protein

Background: Bacillum thuringiensis (Bt) toxin produced in Cry1-expressing genetically modified rice (Bt rice) is highly effective to control lepidopteran pests, which reduces the needs for synthetic insecticides. Non-target organisms can be exposed to Bt toxins through direct feeding or trophic interactions in the field. The wolf spider Pardosa pseudoannulata, one of the dominant predators in South China, plays a crucial role in the rice agroecosystem. In this study, we investigated transcriptome responses of the 5th instar spiders fed on preys maintained on Bt- and non-Bt rice. Results: Comparative transcriptome analysis resulted in 136 differentially expressed genes (DEGs) between spiderlings preying upon N. lugens fed on Bt- and non-Bt rice (Bt- and non-Bt spiderlings). Functional analysis indicated a potential impact of Bttoxin on the formation of new cuticles during molting. GO and KEGG enrichment analyses suggested that GO terms associated with chitin or cuticle, including “chitin binding”, “chitin metabolic process”, “chitin synthase activity”, “cuticle chitin biosynthetic process”, “cuticle hydrocarbon biosynthetic process”, and “structural constituent of cuticle”, and an array of amino acid metabolic pathways, including “alanine, asparatate and glutamate metabolism”, “glycine, serine and theronine metabolism”, “cysteine and methionine metabolism”, “tyrosine metabolism”, “phenylalanine metabolism and phenylalanine”, and “tyrosine and tryptophan biosynthesis” were significantly influenced in response to Cry1Ab. Conclusions: The Cry1Ab may have a negative impact on the formation of new cuticles during molting, which is contributed to the delayed development of spiderlings. To validate these transcriptomic responses, further examination at the translational level will be warranted

Expression of immune-response genes in lepidopteran host is suppressed by venom from an endoparasitoid, Pteromalus puparum

<p>Abstract</p> <p>Background</p> <p>The relationships between parasitoids and their insect hosts have attracted attention at two levels. First, the basic biology of host-parasitoid interactions is of fundamental interest. Second, parasitoids are widely used as biological control agents in sustainable agricultural programs. Females of the gregarious endoparasitoid <it>Pteromalus puparum </it>(Hymenoptera: Pteromalidae) inject venom along with eggs into their hosts. <it>P. puparum </it>does not inject polydnaviruses during oviposition. For this reason, <it>P. puparum </it>and its pupal host, the small white butterfly <it>Pieris rapae </it>(Lepidoptera: Pieridae), comprise an excellent model system for studying the influence of an endoparasitoid venom on the biology of the pupal host. <it>P. puparum </it>venom suppresses the immunity of its host, although the suppressive mechanisms are not fully understood. In this study, we tested our hypothesis that <it>P. puparum </it>venom influences host gene expression in the two main immunity-conferring tissues, hemocytes and fat body.</p> <p>Results</p> <p>At 1 h post-venom injection, we recorded significant decreases in transcript levels of 217 EST clones (revealing 113 genes identified <it>in silico</it>, including 62 unknown contigs) derived from forward subtractive libraries of host hemocytes and in transcript levels of 288 EST clones (221 genes identified <it>in silico</it>, including 123 unknown contigs) from libraries of host fat body. These genes are related to insect immune response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, stress response and transcriptional and translational regulation. We verified the reliability of the suppression subtractive hybridization (SSH) data with semi-quantitative RT-PCR analysis of a set of randomly selected genes. This analysis showed that most of the selected genes were down-regulated after venom injection.</p> <p>Conclusions</p> <p>Our findings support our hypothesis that <it>P. puparum </it>venom influences gene expression in host hemocytes and fat body. Specifically, the venom treatments led to reductions in expression of a large number of genes. Many of the down-regulated genes act in immunity, although others act in non-immune areas of host biology. We conclude that the actions of venom on host gene expression influence immunity as well as other aspects of host biology in ways that benefit the development and emergence of the next generation of parasitoids.</p

Immunization with Fc-based recombinant Epstein-Barr virus gp350 elicits potent neutralizing humoral immune response in a BALB/c mice model

Epstein-Barr virus (EBV) was the first human virus proved to be closely associated with tumor development, such as lymphoma, nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma. Despite many efforts to develop prophylactic vaccines against EBV infection and diseases, no candidates have succeeded in effectively blocking EBV infection in clinical trials. Previous investigations showed that EBV gp350 plays a pivotal role in the infection of B lymphocytes. Nevertheless, using monomeric gp350 proteins as antigens has not been effective in preventing infection. Multimeric forms of the antigen are more potently immunogenic than monomers, however the multimerization elements used in previous constructs are not approved for human clinical trials. To prepare a much-needed EBV prophylactic vaccine that is potent, safe and applicable, we constructed an Fc-based form of gp350 to serve as a dimeric antigen. Here we show that the Fc-based gp350 antigen exhibits dramatically enhanced immunogenicity compared to wild-type gp350 protein. The complete or partial gp350 ectodomain was fused with the mouse IgG2a Fc domain. Fusion with the Fc domain did not impair gp350 folding, binding to a conformation-dependent neutralizing antibody and binding to its receptor by ELISA and SPR. Specific antibody titers against gp350 were notably enhanced by immunization with gp350-Fc dimers compared to gp350 monomers. Furthermore, immunization with gp350-Fc fusion proteins elicited potent neutralizing antibodies against EBV. Our data strongly suggest that an EBV gp350 vaccine based on Fc fusion proteins may be an efficient candidate to prevent EBV infection in clinical applications. Please click Additional Files below to see the full abstract

Intragenic DNA Methylation Regulates Insect Gene Expression and Reproduction through the MBD/Tip60 Complex

DNA methylation is an important epigenetic modification. However, the regulations and functions of insect intragenic DNA methylation remain unknown. Here, we demonstrate that a regulatory mechanism involving intragenic DNA methylation controls ovarian and embryonic developmental processes in Bombyx mori. In B. mori, DNA methylation is found near the transcription start site (TSS) of ovarian genes. By promoter activity analysis, we observed that 5′ UTR methylation enhances gene expression. Moreover, methyl-DNA-binding domain protein 2/3 (MBD2/3) binds to the intragenic methyl-CpG fragment and recruits acetyltransferase Tip60 to promote histone H3K27 acetylation and gene expression. Additionally, genome-wide analyses showed that the peak of H3K27 acetylation appears near the TSS of methyl-modified genes, and DNA methylation is enriched in genes involved in protein synthesis in the B. mori ovary, with MBD2/3 knockdown resulting in decreased fecundity. These data uncover a mechanism of gene body methylation for regulating insect gene expression and reproduction

Global identification of bursicon-regulated genes in Drosophila melanogaster

Abstract Background Bursicon is a heterodimer neuropeptide responsible for regulating cuticle sclerotization and wing expansion in several insect species. Recent studies indicate that the action of bursicon is mediated by a specific G protein-coupled receptor DLGR2 and the cAMP/PKA signaling pathway. However, little is known regarding the genes that are regulated by bursicon. The identification of bursicon-regulated genes is the focus of this investigation. Results We used DNA microarray analysis to identify bursicon-regulated genes in neck-ligated flies (Drosophila melanogaster) that received recombinant bursicon (r-bursicon). Fifty four genes were found to be regulated by bursicon 1 h post r-bursicon injection, 52 being up-regulated and 2 down-regulated while 33 genes were influenced by r-bursicon 3 h post-injection (24 up-regulated and 9 down-regulated genes). Analysis of these genes by inference from the fly database http://flybase.bio.indiana.edu revealed that these genes encode proteins with diverse functions, including cell signaling, gene transcription, DNA/RNA binding, ion trafficking, proteolysis-peptidolysis, metabolism, cytoskeleton formation, immune response and cell-adhesion. Twenty eight genes randomly selected from the microarray-identified list were verified by real time PCR (qPCR) which supported the microarray data. Temporal response studies of 13 identified and verified genes by qPCR revealed that the temporal expression patterns of these genes are consistent with the microarray data. Conclusion Using r-bursicon, we identified 87 genes that are regulated by bursicon, 30 of which have no previously known function. Most importantly, all genes randomly selected from the microarray-identified list were verified by real time PCR. Temporal analysis of 13 verified genes revealed that the expression of these genes was indeed induced by bursicon and correlated well with the cuticle sclerotization process. The composite data suggest that these genes play important roles in regulating the cuticle sclerotization and wing expansion processes. The data obtained here will form the basis for future studies aimed at elucidating the exact mechanisms upstream from the secretion of bursicon and its binding to target cells