Elucidation of the epigenetic mechanisms underlying transgene activation by the HSP70A promoter in Chlamydomonas reinhardtii

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Transgenic approaches in higher eukaryotes often suffer from the silencing of introduced transgenes. We have found previously that the Chlamydomonas HSP70A (A) promoter counteracts transcriptional silencing of other promoters when fused upstream from these. Our current data indicate that the effect is largely mediated by heat shock elements (HSEs) and TATA-box. Moreover, as heat shock factor 1 (HSF1) at the native A promoter mediates acetylation of histones H3 and H4 to create a particularly open chromatin structure, the anti-silencing effect at downstream promoters might be mediated by HSF1. The goal of this project is to investigate in molecular detail the mechanism(s) responsible for this anti-silencing effect. For this we ask the following questions: (i) are cis-acting elements other than HSEs involved in the anti-silencing effect? If yes, which are these? (ii) Which histone modifications serving as silencing marks at transgene-driving promoters are opposed by the A promoter? (iii) Is the anti-silencing effect indeed mediated by HSF1? (iv) Is there a cascade of different histone modifications triggered via the A promoter / HSF1 that leads to the remodeled chromatin state at transgenic promoters? We propose to address these questions by combining transgenic approaches based on various A promoter mutation/deletion constructs and an inducible artificial microRNA construct targeting HSF1 with chromatin immunoprecipitation and quantitative real-time PCR. The understanding of the epigenetic mechanisms by which the A promoter counteracts transgene silencing will open up new strategies to facilitate transgenic approaches in microalgae and potentially also in other eukaryotes.