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Analysis of different modes of TAL effector-mediated transcriptional regulation

Werner, Jeannette

The continuous development of new technologies like ZFP, TALEs and the CRISPR/Cas system to target chromosomal DNA sequences opens more efficient possibilities to edit genomic loci or to modulate the activity of targeted genes. The modular structure of recombinantTALEs allows great flexibility for target site selection. This thesiscompares this new technology to one of the best-established heterologous eukaryotic transcriptional control systems, namely the Tet system. Furthermore,it provides particular insights into the different mechanisms of TALE-mediated transcriptional repression. Creating TALEs targeting the tet operator (tetTALE), a sequence recognized by all common Tet system components, set the stage for a direct comparison in terms of efficacy, efficiency and specificity. When targeted to a region upstream of the proximal promoter sequence, tetTALE activators were demonstrated to be on par with tetR-based transcription factors. This highly efficient regulation was also reflected in trans-repression experiments where the repression of three mammalian promoters reached from distinctly decreased to total abrogation of expression. The repression was apparently not epigenetically manifested on the level of DNA methylation. Additional to silencing domain-mediated repression, TALEs were established as very effective competitive repressors. Taking advantage of the conditional nature of the Tet system, tetTALE was shown to prevail as the dominant transcription factor either when pre-bound and challenged by the tet-transactivator or when encountering a pre-occupied target sequence. This ability to efficiently compete could also be demonstrated for larger protein complexes like the transcription initiation machinery, thus substantiating a broad validity of this repression mechanism. The clear advantage of the Tet system over the new tailored DNA-targeting tools is its conditionality. However, the introduction of hormone-binding receptors to the tetTALE protein made transcriptional activation and repression dependent on the presence of a small-molecule inducer. Thereby the inducible TALE combines the best of both worlds− the virtual unlimited target site selection and the on-demand transcriptional regulation. The recently developed CRISPR/Cas-based transcription factors were subjected to a similar, albeit reduced, comparative investigation. Two single guide RNAs, targeting sequences overlapping with the tetO, were used. The analysis revealed a similar transient activation level for TALE- and Cas-based activators. Repression by dCas9-SD, however, was evinced to be inferior to tetTALE-SD, both for trans-repression and for a competitive setting. In summary, the lessons learned from the in-depth analysis of TALE capacity presented here can be used to further optimize transcriptional regulation with this and all future tools for the control of endogenous genes.