The Carboxy-Terminal Domain of Dictyostelium C-Module-Binding Factor Is an Independent Gene Regulatory Entity

The C-module-binding factor (CbfA) is a multidomain protein that belongs to the family of jumonji-type (JmjC) transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF) motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD). An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo

eIF2α Kinases Regulate Development through the BzpR Transcription Factor in Dictyostelium discoideum

A major mechanism of translational regulation in response to a variety of stresses is mediated by phosphorylation of eIF2α to reduce delivery of initiator tRNAs to scanning ribosomes. For some mRNAs, often encoding a bZIP transcription factor, eIF2α phosphorylation leads to enhanced translation due to delayed reinitiation at upstream open reading frames. Dictyostelium cells possess at least three eIF2α kinases that regulate various portions of the starvation-induced developmental program. Cells possessing an eIF2α that cannot be phosphorylated (BS167) show abnormalities in growth and development. We sought to identify a bZIP protein in Dictyostelium whose production is controlled by the eIF2α regulatory system.Cells disrupted in the bzpR gene had similar developmental defects as BS167 cells, including small entities, stalk defects, and reduced spore viability. β-galactosidase production was used to examine translation from mRNA containing the bzpR 5' UTR. While protein production was readily apparent and regulated temporally and spatially in wild type cells, essentially no β-galactosidase was produced in developing BS167 cells even though the lacZ mRNA levels were the same as those in wild type cells. Also, no protein production was observed in strains lacking IfkA or IfkB eIF2α kinases. GFP fusions, with appropriate internal controls, were used to directly demonstrate that the bzpR 5' UTR, possessing 7 uORFs, suppressed translation by 12 fold. Suppression occurred even when all but one uORF was deleted, and translational suppression was removed when the ATG of the single uORF was mutated.The findings indicate that BzpR regulates aspects of the development program in Dictyostelium, serving as a downstream effector of eIF2α phosphorylation. Its production is temporally and spatially regulated by eIF2α phosphorylation by IfkA and IfkB and through the use of uORFs within the bzpR 5' UTR

Cell-type specific RNA-Seq reveals novel roles and regulatory programs for terminally differentiated Dictyostelium cells

Abstract Background A major hallmark of multicellular evolution is increasing complexity by the evolution of new specialized cell types. During Dictyostelid evolution novel specialization occurred within taxon group 4. We here aim to retrace the nature and ancestry of the novel “cup” cells by comparing their transcriptome to that of other cell types. Results RNA-Seq was performed on purified mature spore, stalk and cup cells and on vegetative amoebas. Clustering and phylogenetic analyses showed that cup cells were most similar to stalk cells, suggesting that they share a common ancestor. The affinity between cup and stalk cells was also evident from promoter-reporter studies of newly identified cell-type genes, which revealed late expression in cups of many stalk genes. However, GO enrichment analysis reveal the unexpected prominence of GTPase mediated signalling in cup cells, in contrast to enrichment of autophagy and cell wall synthesis related transcripts in stalk cells. Combining the cell type RNA-Seq data with developmental expression profiles revealed complex expression dynamics in each cell type as well as genes exclusively expressed during terminal differentiation. Most notable were nine related hssA-like genes that were highly and exclusively expressed in cup cells. Conclusions This study reveals the unique transcriptomes of the mature cup, stalk and spore cells of D. discoideum and provides insight into the ancestry of cup cells and roles in signalling that were not previously realized. The data presented in this study will serve as an important resource for future studies into the regulation and evolution of cell type specialization

Genomic mining of prokaryotic repressors for orthogonal logic gates

Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >10[superscript 54] circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.United States. Air Force Office of Scientific Research (Award FA9550-11-C-0028)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a)United States. Defense Advanced Research Projects Agency. Chronical of Lineage Indicative of Origins (N66001-12-C-4016)United States. Office of Naval Research (N00014-13-1-0074)National Institutes of Health (U.S.) (GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SA5284-11210

Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-α, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with 99mTc or 111In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform ‘evidence-based biological therapy’ of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for therapy decision-making and follow-up

Identification of a protein factor binding to the 5'-flanking region of a tRNA gene and being involved in modulation of tRNA gene transcription in vivo in Saccharomyces cerevisiae.

Control mechanisms of tRNA gene transcription were studied in vivo in Saccharomyces cerevisiae. In order to be able to monitor in vivo transcription products of an individual tRNA gene, a 'tester gene' was used which is readily transcribed in vivo in yeast but does not cross-hybridize with any cellular yeast tRNA. A series of insertion mutants were constructed, modifying thereby the immediate and further distant 5'-flanking region of the 'tester tRNA gene'. Small linker molecules of different length and different sequence were inserted at positions -3 and -56 on the non-coding strand. Resulting tRNA gene variants were transformed into yeast cells and in vivo synthesized products were monitored by primer extension analysis. From the experimental data we suggest that a few essential nucleotides within the flanking region are able to determine the in vivo transcription activity of the 'tester tRNA gene'. Our results are rationalized on a biochemical level by protein binding assays: At least one protein binds to the 5'-flanking region of the 'tester tRNA gene' and different protein complexes are sequestered on active or less active tRNA gene variants

Genomic organization of the transposable element Tdd-3 from Dictyostelium discoideum.

The transposable element Tdd-3 from D. discoideum has been described originally in 1984 (Poole and Firtel, 1984). Additional copies of this element were discovered in the course of a recent study on tRNA gene organization in D. discoideum. Five out of 24 independently isolated tRNA genes proved to be associated with Tdd-3 elements. The surprising observation that all the elements always occurred within the 3'-flanking sequences of the Dictyostelium tRNA genes suggested the possibility of a general position specific integration of Tdd-3 elements upon transposition. Therefore we isolated additional Tdd-3 elements from various genomic D. discoideum libraries in order to test this hypothesis. Several new Tdd-3 elements were found associated with various tRNA genes. Additionally we identified Tdd-3 elements organized in tandem array or in association with RED (Repetitive Element of Dictyostelium), another repetitive element recently identified by our laboratory. In all cases a B-box equivalent of the eukaryotic gene-internal RNA polymerase III promoter was identified upstream of all Tdd-3 elements

The minimum intragenic sequences required for promotion of eukaryotic tRNA gene transcription.

Transcription of eukaryotic tRNA genes is controlled by two intragenic regions, the D-control region (which in the tRNA codes for the D-stem and -loop) and the T-control region (which in the tRNA codes for the T psi C loop). To determine whether these sequences alone are sufficient to promote tRNA gene transcription in vitro, the two control regions of a Drosophila tRNAArg gene were cloned separately from the context of the parental DNA (these constructions are called tRNA minigenes). The tRNA minigene that contains both intragenic control regions supports in vitro RNA synthesis in Xenopus laevis oocyte and HeLa cell transcription systems. The mutant which has deletions to nucleotide 7 within the mature tRNA coding region, pArg5.7, and minigenes derived from it do not support RNA synthesis in a Drosophila Kc cell transcription system. Xenopus and Hela extracts transcribe pArg5.7 albeit at reduced levels compared to the wild-type gene. The tRNA minigene that contained only the D-control region was not able to support RNA synthesis in any of these three transcription systems. A mutant tRNA gene comprising the 3' half of the tRNAArg gene similarly was not able to support RNA synthesis. These experiments show that the DNA sequence from nucleotides 7-58, which contains both intragenic control regions of the tRNA gene, possesses sufficient information to initiate specific transcription by RNA polymerase III in Xenopus and HeLa systems. The transcription efficiency of this tRNA minigene however is reduced to about 20% the transcription level of the wild type tRNA gene. This lowered level of transcriptional efficiency results from deleting the ends of the native tRNA gene and its adjacent flanking sequences. The affects of deleting 5' sequences are most pronounced in the Drosophila transcription system