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We expressed the heat-labile enterotoxin B (LTB) subunit from enterotoxigenic Escherichia coli and the cholera toxin B (CTB) subunit from Vibrio cholerae under the control of the rice (Oryza sativa) globulin (Glb) promoter. Binding of recombinant LTB and CTB proteins was confirmed based on GM1-ganglioside binding enzyme-linked immunosorbent assays (GM1-ELISA). Real-time PCR of three generations (T3, T4, and T5) in homozygous lines (LCI-11) showed single copies of LTB, CTB, bar and Tnos. LTB and CTB proteins in rice transgenic lines were detected by Western blot analysis. Immunogenicity trials of rice-derived CTB and LTB antigens were evaluated through oral and intraperitoneal administration in mice, respectively. The results revealed that LTB- and CTB-specific IgG levels were enhanced in the sera of intraperitoneally immunized mice. Similarly, the toxin-neutralizing activity of CTB and LTB in serum of orally immunized mice was associated with elevated levels of both IgG and IgA. The results of the present study suggest that the combined expression of CTB and LTB proteins can be utilized to produce vaccines against enterotoxigenic strains of Escherichia coli and Vibrio cholera, for the prevention of diarrhea.

Studying epigenetic landscapes is important to understand the condition for gene regulation. Clustering is a useful approach to study epigenetic landscapes by grouping genes based on their epigenetic conditions. However, classical clustering approaches that often use a representative value of the signals in a fixed-sized window do not fully use the information written in the epigenetic landscapes. Clustering approaches to maximize the information of the epigenetic signals are necessary for better understanding gene regulatory environments. For effective clustering of multidimensional epigenetic signals, we developed a method called Dewer, which uses the entropy of stationary wavelet of epigenetic signals inside enriched regions for gene clustering. Interestingly, the gene expression levels were highly correlated with the entropy levels of epigenetic signals. Dewer separates genes better than a window-based approach in the assessment using gene expression and achieved a correlation coefficient above 0.9 without using any training procedure. Our results show that the changes of the epigenetic signals are useful to study gene regulation.

The aim of this study is to demonstrate the feasibility of down-regulating endogeneous prefoldin-2 root-knot nematode transcripts by expressing dsRNA with sequence identity to the nematode gene in tobacco roots under the influence of strong Arabidopsis ubiquitin (UBQ1) promoter. Root-knot nematodes (RKNs) are sedentary endoparasites infecting a wide range of plant species. They parasitise the root system, thereby disrupting water and nutrient uptake and causing major reductions in crop yields. The most reliable means of controlling RKNs is via the use of soil fumigants such as methyl bromide. With the emergence of RNA interference (RNAi) technology, which permits host-mediated nematode gene silencing, a new strategy to control plant pathogens has become available. In the present study, we investigated host-induced RNAi gene silencing of prefoldin-2 in transgenic Nicotiana benthamiana. Reductions in prefoldin-2 mRNA transcript levels were observed when nematodes were soaked in a dsRNA solution in vitro. Furthermore, nematode reproduction was suppressed in RNAi transgenic lines, as evident by reductions in the numbers of root knots (by 34–60 % in independent RNAi lines) and egg masses (by 33–58 %). Endogenous expression of prefoldin-2, analysed via real-time polymerase chain reaction and Western blotting, revealed that the gene was strongly expressed in the pre-parasitic J2 stage. Our observations demonstrate the relevance and potential importance of targeting the prefoldin gene during the nematode life cycle. The work also suggests that further improvements in silencing efficiency in economically important crops can be accomplished using RNAi directed against plant-parasitic nematodes.

Background Recent mapping of 5-hydroxymethylcytosine (5hmC) provides a genome-wide view of the distribution of this important chromatin mark. However, the role of 5hmC in specific regulatory regions is not clear, especially at enhancers. Results We found a group of distal transcription factor binding sites highly enriched for 5-hdroxymethylcytosine (5hmC), but lacking any known activating histone marks and being depleted for nascent transcripts, suggesting a repressive role for 5hmC in mouse embryonic stem cells (mESCs). 5-formylcytosine (5fC), which is known to mark poised enhancers where H3K4me1 is enriched, is also observed at these sites. Furthermore, the 5hmC levels were inversely correlated with RNA polymerase II (PolII) occupancy in mESCs as well as in fully differentiated adipocytes. Interestingly, activating H3K4me1/2 histone marks were enriched at these sites when the associated genes become activated following lineage specification. These putative enhancers were shown to be functional in embryonic stem cells when unmethylated. Together, these data suggest that 5hmC suppresses the activity of this group of enhancers, which we termed “silenced enhancers”. Conclusions Our findings indicate that 5hmC has a repressive role at specific proximal and distal regulatory regions in mESCs, and suggest that 5hmC is a new epigenetic mark for silenced enhancers. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-670) contains supplementary material, which is available to authorized users.

Cockayne syndrome is an inherited premature aging disease associated with numerous developmental and neurological defects, and mutations in the gene encoding the CSB protein account for the majority of Cockayne syndrome cases. Accumulating evidence suggests that CSB functions in transcription regulation, in addition to its roles in DNA repair, and those defects in this transcriptional activity might contribute to the clinical features of Cockayne syndrome. Transcription profiling studies have so far uncovered CSB-dependent effects on gene expression; however, the direct targets of CSB's transcriptional activity remain largely unknown. In this paper, we report the first comprehensive analysis of CSB genomic occupancy during replicative cell growth. We found that CSB occupancy sites display a high correlation to regions with epigenetic features of promoters and enhancers. Furthermore, we found that CSB occupancy is enriched at sites containing the TPA-response element. Consistent with this binding site preference, we show that CSB and the transcription factor c-Jun can be found in the same protein-DNA complex, suggesting that c-Jun can target CSB to specific genomic regions. In support of this notion, we observed decreased CSB occupancy of TPA-response elements when c-Jun levels were diminished. By modulating CSB abundance, we found that CSB can influence the expression of nearby genes and impact nucleosome positioning in the vicinity of its binding site. These results indicate that CSB can be targeted to specific genomic loci by sequence-specific transcription factors to regulate transcription and local chromatin structure. Additionally, comparison of CSB occupancy sites with the MSigDB Pathways database suggests that CSB might function in peroxisome proliferation, EGF receptor transactivation, G protein signaling and NF-κB activation, shedding new light on the possible causes and mechanisms of Cockayne syndrome., Author Summary Cockayne syndrome is a devastating inherited disease, in which patients appear to age prematurely, have sun sensitivity and suffer from profound neurological and developmental defects. Mutations in the CSB gene account for the majority of Cockayne syndrome cases. CSB is an ATP-dependent chromatin remodeler, and these proteins can use energy from ATP-hydrolysis to alter contacts between DNA and histones of a nucleosome, the basic units of chromatin structure. CSB functions in DNA repair, but accumulating evidence reveals that CSB also functions in transcription regulation. Here, we determined the genomic localization of CSB to identify its gene targets and found that CSB occupancy displays high correlation to regions with epigenetic features of promoters and enhancers. Furthermore, CSB is enriched at genomic regions containing the binding site for the c-Jun transcription factor, and we found that these two proteins interact, uncovering a new targeting mechanism for CSB. We also demonstrate that CSB can influence gene expression in the vicinity of its binding sites and alter local chromatin structure. Together, this study supports the hypothesis that defects in the regulation of gene expression and chromatin structure by CSB might contribute to the diverse clinical features of Cockayne syndrome.

Mechanisms that maintain transcriptional memory through cell division are important to maintain cell identity, and sequence-specific transcription factors that remain associated with mitotic chromatin are emerging as key players in transcriptional memory propagation. Here, we show that the major transcriptional effector of Notch signaling, RBPJ, is retained on mitotic chromatin, and that this mitotic chromatin association is mediated through the direct association of RBPJ with DNA. We further demonstrate that RBPJ binds directly to nucleosomal DNA in vitro, with a preference for sites close to the entry/exit position of the nucleosomal DNA. Genome-wide analysis in the murine embryonal-carcinoma cell line F9 revealed that roughly 60% of the sites occupied by RBPJ in asynchronous cells were also occupied in mitotic cells. Among them, we found that a fraction of RBPJ occupancy sites shifted between interphase and mitosis, suggesting that RBPJ can be retained on mitotic chromatin by sliding on DNA rather than disengaging from chromatin during mitotic chromatin condensation. We propose that RBPJ can function as a mitotic bookmark, marking genes for efficient transcriptional activation or repression upon mitotic exit. Strikingly, we found that sites of RBPJ occupancy were enriched for CTCF-binding motifs in addition to RBPJ-binding motifs, and that RBPJ and CTCF interact. Given that CTCF regulates transcription and bridges long-range chromatin interactions, our results raise the intriguing hypothesis that by collaborating with CTCF, RBPJ may participate in establishing chromatin domains and/or long-range chromatin interactions that could be propagated through cell division to maintain gene expression programs., Author Summary How does a cell remember what it should be after cell division? One mechanism that is beginning to emerge is the retention of a few key regulatory proteins on the highly condensed mitotic chromatin during cell division. These proteins are called mitotic bookmarks, as they are believed to offer critical information as to how genetic information should be read immediately after mitosis. We have found that a protein called RBPJ, which plays pivotal roles in regulating cell-fate choices, is retained on mitotic chromatin. RBPJ transmits to DNA signals elicited by the Notch pathway: a pathway that conveys information resulting from the communication between two adjacent cells. Unlike many other factors, we found that RBPJ can bind to nucleosomes, which are the basic unit of packaged DNA consisting of DNA wrapped around eight histone proteins. We also found that RBPJ interacts with and binds to DNA sites regulated by the CTCF protein, which plays important roles in regulating long-range DNA interactions. Together, our results suggest that RBPJ can function as a mitotic bookmarking factor, to help maintain genetic programs, higher-order structural information and consequently the memory of cell identity through cell division.

Non-classical results due to reactant correlation effects have been observed in the study of Monte Carlo simulations of the A2 + B2 → 2AB reaction. These simulation results, interpreted by the Pair Density Analysis method and the experimental kinetic studies, may provide an explanation of the early time anomalous kinetic behavior of the catalytic hydrogen isotope exchange reaction exhibited for surface-diffusion limited conditions on alumina supported platinum islands.

Monte Carlo simulations of the A2 + B2→ P (with P = AA, AB, and BB) reaction on various monodisperse island systems provided various models for the reaction H2 + D2→ HD on alumina supported platinum catalysts. Anomalous reaction kinetics and reaction orders due to the effects of diffusion, reactant correlation, and ensemble character of the catalyst in low dimensional systems are observed not only at early time but also at steady state. Concentration effects and spillover studies indicate that surface diffusion and hydrogen spillover may be responsible for the anomalous behaviors.

SummaryType 2 diabetes mellitus (T2DM) is a complex disease characterized by the inability of the insulin-producing β cells in the endocrine pancreas to overcome insulin resistance in peripheral tissues. To determine if microRNAs are involved in the pathogenesis of human T2DM, we sequenced the small RNAs of human islets from diabetic and nondiabetic organ donors. We identified a cluster of microRNAs in an imprinted locus on human chromosome 14q32 that is highly and specifically expressed in human β cells and dramatically downregulated in islets from T2DM organ donors. The downregulation of this locus strongly correlates with hypermethylation of its promoter. Using HITS-CLIP for the essential RISC-component Argonaute, we identified disease-relevant targets of the chromosome 14q32 microRNAs, such as IAPP and TP53INP1, that cause increased β cell apoptosis upon overexpression in human islets. Our results support a role for microRNAs and their epigenetic control by DNA methylation in the pathogenesis of T2DM.

Background Histone variants play further important roles in DNA packaging and controlling gene expression. However, our understanding about their composition and their functions is limited. Results Integrating proteomic and genomic approaches, we performed a comprehensive analysis of the epigenetic landscapes containing the four histone variants H3.1, H3.3, H2A.Z, and macroH2A. These histones were FLAG-tagged in HeLa cells and purified using chromatin immunoprecipitation (ChIP). By adopting ChIP followed by mass spectrometry (ChIP-MS), we quantified histone post-translational modifications (PTMs) and histone variant nucleosomal ratios in highly purified mononucleosomes. Subsequent ChIP followed by next-generation sequencing (ChIP-seq) was used to map the genome-wide localization of the analyzed histone variants and define their chromatin domains. Finally, we included in our study large datasets contained in the ENCODE database. We newly identified a group of regulatory regions enriched in H3.1 and the histone variant associated with repressive marks macroH2A. Systematic analysis identified both symmetric and asymmetric patterns of histone variant occupancies at intergenic regulatory regions. Strikingly, these directional patterns were associated with RNA polymerase II (PolII). These asymmetric patterns correlated with the enhancer activities measured using global run-on sequencing (GRO-seq) data. Conclusions Our studies show that H2A.Z and H3.3 delineate the orientation of transcription at enhancers as observed at promoters. We also showed that enhancers with skewed histone variant patterns well facilitate enhancer activity. Collectively, our study indicates that histone variants are deposited at regulatory regions to assist gene regulation. Electronic supplementary material The online version of this article (doi:10.1186/s13072-015-0005-9) contains supplementary material, which is available to authorized users.