Chromatin loops in gene regulation

The control of gene expression involves regulatory elements that can be very far from the genes they control. Several recent technological advances have allowed the direct detection of chromatin loops that juxtapose distant genomic sites in the nucleus. Here we review recent studies from various model organisms that have provided new insights into the functions of chromatin loops and the mechanisms that form them. We discuss the widespread impact of chromatin loops on gene activation, repression, genomic imprinting and the function of enhancers and insulators.     

Chromatin domains, insulators, and the regulation of gene expression

The DNA sequence elements called insulators have two basic kinds of properties. Barrier elements block the propagation of heterochromatic structures into adjacent euchromatin. Enhancer blocking elements interfere with interaction between an enhancer and promoter when placed between them. We have dissected a compound insulator element found at the 5' end of the chicken β-globin locus, which possesses both activities. Barrier insulation is mediated by two kinds of DNA binding proteins: USF1/USF2, a heterodimer which recruits multiple enzyme complexes capable of marking histone on adjacent nucleosomes with 'activating' marks, and Vezf1, which protects against DNA methylation. We have found that the heterochromatic region upstream of the insulator element is maintained in its silent state by a dicer-dependent mechanism, suggesting a mechanism for Vezf1 function in the insulator. Enhancer blocking function in the β-globin insulator element is conferred by a binding site for CTCF. Consistent with this property, CTCF binding was found some years ago to be essential for imprinted expression at the Igf2/H19 locus. Work in many laboratories has since demonstrated that CTCF helps stabilize long-range interactions in the nucleus. We have recently shown that in the case of the human insulin locus such an interaction, over a distance of ~300kb, can result in stimulation of a target gene which itself is important for insulin secretion. This article is part of a Special Issue entitled: Chromatin in time and space.     

A Toxoplasma gondii Superantigen: Biological effects and implications for the host-parasite interaction

Superantigens exert their biological effects by activating large families of T cells, based on expression of the variable beta chain of the T-cell receptor. As a result, the reactive cells proliferate, secrete high levels of inflammatory cytokines, and ultimately die or become anergic to further stimulation. It is now becoming clear that the intracellular protozoan Toxoplasma gondii has many of these same superantigenic properties. As discussed here by Eric Denkers, this activity may play a key role in the induction of cell-mediated immunity to the parasite, and may prove to be responsible for much of the pathology associated with the clinical manifestations of toxoplasmosis.     

Toxoplasma gondii: the model apicomplexan

Toxoplasma gondii is an obligate intracellular protozoan parasite which is a significant human and veterinary pathogen. Other members of the phylum Apicomplexa are also important pathogens including Plasmodium species (i.e. malaria), Eimeria species, Neospora, Babesia, Theileria and Cryptosporidium. Unlike most of these organisms, T. gondii is readily amenable to genetic manipulation in the laboratory. Cell biology studies are more readily performed in T. gondii due to the high efficiency of transient and stable transfection, the availability of many cell markers, and the relative ease with which the parasite can be studied using advanced microscopic techniques. Thus, for many experimental questions, T. gondii remains the best model system to study the biology of the Apicomplexa. Our understanding of the mechanisms of drug resistance, the biology of the apicoplast, and the process of host cell invasion has been advanced by studies in T. gondii. Heterologous expression of apicomplexan proteins in T. gondii has frequently facilitated further characterisation of proteins that could not be easily studied. Recent studies of Apicomplexa have been complemented by genome sequencing projects that have facilitated discovery of surprising differences in cell biology and metabolism between Apicomplexa. While results in T. gondii will not always be applicable to other Apicomplexa, T. gondii remains an important model system for understanding the biology of apicomplexan parasites.     

Toxoplasma gondii: siRNA can mediate the suppression of adenosine kinase expression

Adenosine kinase (AK) is one of the most important enzymes in the Toxoplasma gondii purine salvage pathway. Three siRNAs specific to the AK gene were designed in the present study. At 24h following electroporation, two of them (siRNA786 and siRNA1200) significantly reduced the mRNA level compared with mock electroporation (P <0.05). The ability to incorporate [3H]-adenosine in the parasites electroporated with 4 microM siRNA786 or 4 microM siRNA1200 was decreased to 39+/-11% and 39+/-7% of the mock electroporation, respectively. At the 48th hour of electroporation, the enzyme's activity was still significantly lower than that of mock electroporation. The data show the siRNAs transfected into cells can work efficiently to regulate gene expression in T. gondii. The application of siRNA in interrupting gene expression in T. gondii would be useful for elucidating gene function as a step toward development of anti-toxoplasmasis vaccines and therapeutic reagents.     

The in vivo conformation of the plastid DNA of Toxoplasma gondii: implications for replication

The Phylum Apicomplexa comprises thousands of obligate intracellular parasites, some of which cause serious disease in man and other animals. Though not photosynthetic, some of them, including the malaria parasites (Plasmodium spp.) and the causative organism of Toxoplasmosis, Toxoplasma gondii, possess a remnant plastid partially determined by a highly derived residual genome encoded in 35 kb DNA. The genetic maps of the plastid genomes of these two organisms are extremely similar in nucleotide sequence, gene function and gene order. However, a study using pulsed field gel electrophoresis and electron microscopy has shown that in contrast to the malarial version, only a minority of the plastid DNA of Toxoplasma occurs as circular 35 kb molecules. The majority consists of a precise oligomeric series of linear tandem arrays of the genome, each oligomer terminating at the same site in the genetic map, i.e. in the centre of a large inverted repeat (IR) which encodes duplicated tRNA and rRNA genes. This overall topology strongly suggests that replication occurs by a rolling circle mechanism initiating at the centre of the IR, which is also the site at which the linear tails of the rolling circles are processed to yield the oligomers. A model is proposed which accounts for the quantitative structure of the molecular population. It is relevant that a somewhat similar structure has been reported for at least three land plant chloroplast genomes.     

Cloning and expression of the dihydroorotate dehydrogenase from Toxoplasma gondii

A full-length dihydroorotate dehydrogenase (DHODase) sequence was cloned from a Toxoplasma gondii tachyzoite cDNA library. The sequence was most similar to family 2 DHODases, and had a calculated molecular mass of 65.1 kDa. The full-length and two N-terminally truncated T. gondii DHODase sequences were expressed as recombinant proteins. One of the truncated sequences complemented a DHODase-deficient bacterial host.     

Identification and characterization of Letm1 gene in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan that causes toxoplasmosis.Previous studies have shown that the perturbation of mitochondrial metabolism in T.gondii results in growth deficiency in host cells and lack of virulence in animals.Members of this Letm1 protein family are inner mitochondrial membrane proteins which play a role in potassium and hydrogen ion exchange.Letm1 has not been characterized in T.gondii.In this study,a potential TgLetm1 gene (TgGT1_288400) with Letm1-like protein domain coding sequence was identified in T.gondii.Indirect immunofluorescence assays suggested that TgLetm1 localized to the mitochondria in tachyzoites,as indicated by the colocalization with mitochondrial marker Mitotracker.TgLetm1 was found in the membrane fraction by western blot analysis.To investigate the role of TgLetm1 in T.gondii,we generated a tetracycline-inducible TgLetm1-knock-down mutant.The conditional deletion of TgLetm1 resulted in mitochondrial swelling.Functional studies showed that the conditional deletion of TgLetm1 resulted in growth inhibition,deficiency in invasion and replication,and lack of virulence in mice.     

Comparative analysis of antisense RNA,double-stranded RNA,and delta ribozyme-mediated gene regulation in Toxoplasma gondii

RNA tools, namely, antisense RNA, double-stranded RNA (dsRNA), and delta ribozyme, were comparatively analyzed for the development of effective RNA-based gene modulators. The gene encoding uracil phosphoribosyltransferase (UPRT) of Toxoplasma gondii was used as a target and a negative selectable marker. Using plasmid transformation and drug selection assays, we obtained T. gondii transformants resistant to 5-fluoro-2'-deoxyuridine (FDUR), the cytotoxic prodrug and substrate of UPRT, when the plasmids expressing dsRNA and active delta ribozyme were used. No resistant transformants were detected when the plasmids carrying the antisense RNA, the inactive delta ribozyme, or the chloramphenicol acetyltransferase (CAT) genes were used. Parasites generated using the plasmids expressing dsRNA and the delta ribozyme become resistant to FDUR with an LD50 of 50 +/- 5 microM and 25 +/- 8 microM, respectively. These values are approximately 25-fold and 12-fold higher than that of the RH parental parasite strain, indicating that UPRT activity of the transformed parasites was drastically inhibited. Using Northern and Southern blot analysis, we demonstrated that dsRNA and the delta ribozyme interrupt the expression of UPRT. These two RNA tools should, thus, be very useful for the study of gene expression.