Michael Kaufmann, Jana Schiefner, Thomas Spaller
Non-long terminal repeat (non-LTR) retrotransposons are ancient mobile elements that amplify within host cell genomes via reverse transcription of RNA intermediates. Dictyostelium discoideum is an exceptionally nice model to study the evolution of targeted integration of retrotransposons under the constraint of a very compact host cell genome. D. discoideum retrotransposons must adapt to this environment by developing mechanisms to avoid the disruption of genes upon integration. At least in the D. discoideum genome, but also in yeasts, the targeting of tRNA genes seems to be a particularly successful strategy to colonize a compact genome and maintain a population of actively retrotransposing elements without significantly harming the host cell.
The D. discoideum genome harbors a family of tRNA gene-targeted retrotransposons (TREs) that integrate ca. 48 bp upstream (TRE5 elements) or ca. 100 bp downstream (TRE3 elements) of tRNA genes. We have recently developed retrotransposition assays that allow for the analysis of tRNA gene-targeted integration of endogenous TRE5-A elements or artificial, genetically traceable elements in vivo. Experiments using a "TRE trap" revealed that both TRE5-A elements are actively retrotransposing in current D. discoideum laboratory strains. The TRE trap assay has provided some insight into how TRE5-A recognizes tRNA genes as integration targets. We have shown that the B box promoter element of an active tRNA gene is required for targeted intgration, suggesting that RNA polymerase III-specific transcription factors are involved in target site selection. Subsequent studies have demonstrated that direct protein interactions of TRE5-A-encoded ORF1 protein with TFIIIB subunits are responsible for integration site selection by TRE5-A. Currently we investigate the significance of ORF1-TFIIIB protein interaction for TRE5-A targeting in vivo. We have generated TRE5-A derivatives tagged with a selectable marker that becomes active only after a complete retrotransposition cycle. These artificial TRE5-A elements are valuable tools for the isolation of natural integration sites from the D. discoideum genome as well as for screening of D. discoideum gene replacement mutants for the involvement of host genes in the expression and retrotransposition of TRE5-A.
Sara Müller, Anika Schmith
The C-module-binding factor (CbfA) is a multidomain protein that belongs to the family of "jumonji-type transcription regulators". Carboxy-terminal jumonji domains (JmjC) are thought to contribute to the deciphering of the histone code by chromatin-remodeling factors by removing methyl groups from methyllysine or methylarginine residues in histone tails. Besides its JmjC domain, CbfA contains two zinc finger-like regions (ZF) and a distinct carboxy-terminal domain (CTD). We have performed detailed transciptome analyses on the a CbfA-underexperessing mutant using both DNA microarray and Illumina RNA sequencing technology. We found that CbfA can both enhance and suppress gene expression and about one half of all CbfA-dependent genes turned out to require only the carboxy-terminal domain of CbfA for proper expression. Thus, if the JmjC domain of CbfA acts as a demethylase on modified histones, only part of CbfA's gene-regulatory activity seems to be facilitated by chromatin modulation.
Complementation studies with CbfA mutants are currently carried out to learn more about the functions of CbfA domains in the regulation gene expression. This work is accompanied by phylogenetic studies of the domain structure of CbfA, and the work is extended by studies on the protein CbfB that is paraphyletic to CbfA but has a completey different carboxy-terminal domain. We follow the hypothesis that CbfB's carboxy-terminal domain is an autonomous domain acapabale of regulating gene expression independently of the JmjC/ZF domains of the protein, analogous to CbfA function.
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