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

The C-Module DNA-Binding Factor Mediates Expression of the Dictyostelium Aggregation-Specific Adenylyl Cyclase ACA

Aggregation of Dictyostelium discoideum amoebae into multicellular structures is organized by cyclic AMP (cAMP), which acts as a chemoattractant, as a second messenger, and as a morphogen. Aggregation of D. discoideum cells depends on the expression of adenylyl cyclase ACA, which provides extracellular cAMP for signal relay and intracellular cAMP for the induction of genes required at multicellular stages. We have identified a DNA-binding activity specific for a highly A+T-enriched motif in the upstream region of the ACA-encoding gene, acaA. The factor shows DNA-binding characteristics very similar to those of C-module-binding factor (CbfA). Although CbfA was originally identified as a putative regulator of the activity of D. discoideum retrotransposon TRE5-A, it also was found to be essential for aggregation of D. discoideum cells. The identified DNA-binding activity was absent in mutant cells depleted of CbfA, and CbfA could be precipitated using an acaA promoter fragment. We propose that CbfA binds to the acaA promoter to provide a basal transcription activity that is required for induction of ACA expression after the onset of D. discoideum development

Dictyostelium transfer RNA gene-targeting retrotransposons: Studying mobile element-host interactions in a compact genome

The model species of social amoebae, Dictyostelium discoideum, has a compact genome consisting of about two thirds protein-coding regions, with intergenic regions that are rarely larger than 1,000 bp. We hypothesize that the haploid state of D. discoideum cells provides defense against the amplification of mobile elements whose transposition activities would otherwise lead to the accumulation of heterozygous, potentially lethal mutations in diploid populations. We further speculate that complex transposon clusters found on D. discoideum chromosomes do not a priori result from integration preferences of these transposons, but that the clusters instead result from negative selection against cells harboring insertional mutations in genes. D. discoideum cells contain a fraction of retrotransposons that are found in the close vicinity of tRNA genes. Growing evidence suggests that these retrotransposons use active recognition mechanisms to determine suitable integration sites. However, the question remains whether these retrotransposons also cause insertional mutagenesis of genes, resulting in their enrichment at tRNA genes, which are relatively safe sites in euchromatic regions. Recently developed in vivo retrotransposition assays will allow a detailed, genome-wide analysis of de novo integration events in the D. discoideum genome

Protein Interactions Involved in tRNA Gene-Specific Integration of Dictyostelium discoideum Non-Long Terminal Repeat Retrotransposon TRE5-A▿

Mobile genetic elements that reside in gene-dense genomes face the problem of avoiding devastating insertional mutagenesis of genes in their host cell genomes. To meet this challenge, some Saccharomyces cerevisiae long terminal repeat (LTR) retrotransposons have evolved targeted integration at safe sites in the immediate vicinity of tRNA genes. Integration of yeast Ty3 is mediated by interactions of retrotransposon protein with the tRNA gene-specific transcription factor IIIB (TFIIIB). In the genome of the social amoeba Dictyostelium discoideum, the non-LTR retrotransposon TRE5-A integrates ∼48 bp upstream of tRNA genes, yet little is known about how the retrotransposon identifies integration sites. Here, we show direct protein interactions of the TRE5-A ORF1 protein with subunits of TFIIIB, suggesting that ORF1p is a component of the TRE5-A preintegration complex that determines integration sites. Our results demonstrate that evolution has put forth similar solutions to prevent damage of diverse, compact genomes by different classes of mobile elements

Role of RNA Polymerase III Transcription Factors in the Selection of Integration Sites by the Dictyostelium Non-Long Terminal Repeat Retrotransposon TRE5-A

In the compact Dictyostelium discoideum genome, non-long terminal repeat (non-LTR) retrotransposons known as TREs avoid accidental integration-mediated gene disruption by targeting the vicinity of tRNA genes. In this study we provide the first evidence that proteins of a non-LTR retrotransposon interact with a target-specific transcription factor to direct its integration. We applied an in vivo selection system that allows for the isolation of natural TRE5-A integrations into a known genomic location upstream of tRNA genes. TRE5-A frequently modified the integration site in a way characteristic of other non-LTR retrotransposons by adding nontemplated extra nucleotides and generating small and extended target site deletions. Mutations within the B-box promoter of the targeted tRNA genes interfered with both the in vitro binding of RNA polymerase III transcription factor TFIIIC and the ability of TRE5-A to target these genes. An isolated B box was sufficient to enhance TRE5-A integration in the absence of a surrounding tRNA gene. The RNA polymerase III-transcribed ribosomal 5S gene recruits TFIIIC in a B-box-independent manner, yet it was readily targeted by TRE5-A in our assay. These results suggest a direct role of an RNA polymerase III transcription factor in the targeting process

Tower Controller Command Prediction for Future Speech Recognition Applications

Air traffic controllers' (ATCos) workload often is a limiting factor for air traffic capacity. Thus, electronic support systems intend to reduce ATCos' workload. Automatic Speech Recognition (ASR) can extract controller command elements from verbal clearances to deliver automatic air traffic control (ATC) system input and avoiding manual input. Assistant Based Speech Recognition (ABSR) systems with high command recognition rates and low error rates have proven to dramatically reduce ATCos' workload and increase capacity as an effect. However, those ABSR systems need accurate hypotheses about expected commands to achieve the necessary performance. Based on the experience with an ATC approach hypotheses generator, a prototypic tower command hypotheses generator (TCHG) was developed to face current and future challenges in the aerodrome environment. Two human-in-the-loop multiple remote tower simulation studies were performed with 13 ATCos from Hungary and Lithuania at DLR Braunschweig. Almost 40 hours of speech with corresponding radar data were recorded for training of the TCHG prediction models in 2017/2018. More than 45 hours of speech and radar data comprising roughly 4,600 voice utterances were recorded in the second simulation campaign for the TCHG evaluation test end of 2018. The TCHG showed operational feasibility with a sufficiently low command prediction error rate of down to 7.3% and low context portion predicted having a sufficiently fast command prediction frequency of once per 120ms to timely deliver the hypotheses to a speech recognition engine. Thus, the next step is to build an integrated ABSR system for the tower environment

CbfA, the C-Module DNA-Binding Factor, Plays an Essential Role in the Initiation of Dictyostelium discoideum Development

We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum