Eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells

Background: Gene therapy has attracted attention for its potential to specifically and efficiently target cancer cells with minimal toxicity to normal cells. At present, it offers a promising direction for the treatment of cancer patients. Numerous vectors have been engineered for the sole purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Many plant proteins have anticancer properties; consequently, genes encoding some of these proteins are being used to design constructs for the inhibition of multiplying cancer cells. Results: Data addressing the function of vectors harbouring genes specifically encoding ricin, saporin, lunasin, linamarase, and tomato thymidine kinase 1 under the control of different promoters are summarised here. Constructs employing genes to encode cytotoxic proteins as well as constructs employing genes of enzymes that convert a nontoxic prodrug into a toxic drug are considered here. Conclusion: Generation of eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells may permit the broadening of cancer gene therapy strategy, particularly because of the specific mode of action of anticancer plant proteins.     

1A2 Insulator can interact with promoter of hsp70 gene in D. melanogaster

Insulators are regulatory DNA elements that participate in the modulation of the interactions between enhancers and promoters. Depending on the situation, insulators can either stabilize or destroy the contacts between enhancers and promoters. A possible explanation for the activity of insulators is their ability to directly interact with gene promoters. In the present study, it was demonstrated that, in model systems, a 1A2 insulator could interact with the core sequence of an hsp70 promoter. In this case, the insulator protein CP190 is found on the hsp70 promoter, which depends on the presence of an insulator in the transgene. The data obtained are consistent with the model, which implies that direct contacts between insulators and promoters make a considerable contribution to the modulation of the interactions between insulators and promoters.     

The alpha-fetoprotein enhancer region activates the albumin and alpha-fetoprotein promoters during liver development

The four members of the albumin gene family encode the serum transport proteins albumin, α-fetoprotein, α-albumin, and vitamin D-binding protein. These genes are transcribed primarily in the liver with each having a different pattern of developmental expression. The tight linkage of these genes, particularly that of albumin, α-fetoprotein and α-albumin, and their liver-specific expression, has led to the suggestion that these genes share common regulatory elements. To directly examine whether the α-fetoprotein enhancer region could regulate the albumin gene family, expression of these genes was monitored in mice in which this region was deleted by homologous recombination. Our data indicate that this enhancer region is required for α-fetoprotein and albumin activation early in liver development and α-fetoprotein reactivation during liver regeneration, but that albumin, α-albumin, and vitamin D-binding protein expression later in hepatic development is not affected by the absence of these enhancers. We also demonstrate that RNA polymerase II loading on the α-fetoprotein and albumin promoters is reduced in the absence of this enhancer region, indicating a direct role for these enhancers in the assembly of the RNA Polymerase II complex during liver development.     

Factor analysis of interactions between alfalfa nodule bacteria (Sinorhizobium meliloti) genes that regulate symbiotic nitrogen fixation

Factor analysis has been conducted for the data on the interaction between the genes of the root nodule bacteria (rhizobia), which influence the efficiency of symbiosis with leguminous plants, including dctA (encoding succinate permease), dctBD (activating the dctA gene due to binding its enhancer in the presence of succinate), rpoN (activating the promoters of dctA and nitrogenase genes nifHDK), and nifA (activating the nitrogenase genes due to binding their enhancers). The analysis of the alfalfa rhizobia (Sinorhizobium meliloti) recombinants that contain additional copies of these genes suggested the antagonistic (epistatic) interaction between nifA and rpoN. It may be associated either with the competition for C compounds imported into the nodules between the energy production and nitrogen assimilation processes or with the competition for redox potentials between the oxidative phosphorylation and nitrogen fixation processes. Since the phenotypic effects of the studied genes depend on the activity of nitrogen export into the aerial parts of plants, we suppose that its accumulation in bacteroids impairs the activation of the nifHDK genes by the NifA protein due to its interaction with the GlnB protein (the nitrogen metabolism regulator) or with the FixLJ and ActSR proteins (the redox potential regulators).     

Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures

Enhancers are important regulators of gene expression in eukaryotes. Enhancers function independently of their distance and orientation to the promoters of target genes. Thus, enhancers have been difficult to identify. Only a few enhancers, especially distant intergenic enhancers, have been identified in plants. We developed an enhancer prediction system based exclusively on the DNase I hypersensitive sites (DHSs) in the Arabidopsis thaliana genome. A set of 10,044 DHSs located in intergenic regions, which are away from any gene promoters, were predicted to be putative enhancers. We examined the functions of 14 predicted enhancers using the β-glucuronidase gene reporter. Ten of the 14 (71%) candidates were validated by the reporter assay. We also designed 10 constructs using intergenic sequences that are not associated with DHSs, and none of these constructs showed enhancer activities in reporter assays. In addition, the tissue specificity of the putative enhancers can be precisely predicted based on DNase I hypersensitivity data sets developed from different plant tissues. These results suggest that the open chromatin signature-based enhancer prediction system developed in Arabidopsis may serve as a universal system for enhancer identification in plants.     

Inducible CRISPRa screen identifies putative enhancers

Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates. However, the identification of enhancers in native cellular contexts still remains a challenge. Here, we develop an inducible CRISPR activation (CRISPRa) system by transgenic expression of doxycycline (Dox)-inducible dCas9-VPR in mouse embryonic stem cells (iVPR ESC). With this line, a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible, reversible, and Dox concentration-dependent manner. Taking advantage of this system, we induce tiled CRISPRa across genomic regions (105 kilobases) surrounding T (Brachyury), one of the key mesodermal development regulator genes. Moreover, we identify several CRISPRa-responsive elements with chromatin features of putative enhancers, including a region the homologous sequence in which humans harbors a body height risk variant. Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation. Therefore, our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.