Investigation of the mechanisms by which the HSP70A promoter counteracts transcriptional gene silencing in Chlamydomonas

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Transgenic approaches in higher eukaryotes often suffer from the silencing of the introduced transgenes. We have found recently that the Chlamydomonas HSP70A promoter may counteract transcriptional silencing of other promoters when fused upstream of these. This effect was mediated by two independent sequence elements within the HSP70A promoter that, most likely, act by attracting chromatin remodeling activities. The goal of this project is to investigate in molecular detail the underlying mechanism(s) of this finding. For this, we will first determine the effects that the HSP70A promoter may have on chromatin structure. Next, we want to identify the core motifs within the two HSP70A promoter elements and to purify the binding (activator) proteins. Furthermore, we will analyse the protein complexes interacting with these activators. At last, we want to elucidate which of the Chlamydomonas genes require these activators for their proper expression. On a long term, the data obtained will be used to establish concepts for the design of synthetic promoters which are less prone to silencing and, thus, guarantee normalized transgene expression in Chlamydomonas and higher organisms. Opinion of the review panel (including one written comment): Dr. Schroda is a young researcher who started his own group little more than a year ago. The project proposed by Dr. Schroda builds upon the observation that the HSP70A promoter is able to overcome a silencing of other promoters. This work has already been published by him. Within the HSP70A promoter two domains could be identified which are important for the antagonistic effect. The project suggested here attempts to investigate these interesting findings in more detail. The working hypothesis is that the HSP70A promoter binds chromatin-remodelling components. Seven different approaches are listed that should address this hypothesis. The review panel was impressed by the enthusiasm of the investigator, by the solid basis provided for the suggested project, by his own prework and by the thorough outline of the different approaches. The only concern of the reviewers was that Dr. Schroda might be overwhelmed by directing two PhD students and all the proposed approaches right from the beginning. It was, therefore, recommended that initially only one PhD position should be granted and that the budget for consumables should be reduced to 20,000.- EUR.