III. Investigation of the mechanisms by which the HSP70A promoter activates transgene expression.

Rationale

Transgene silencing is frequently observed in eukaryotic systems and may be mediated by epigenetic mechanisms. This is particularly the case in Chlamydomonas and therefore epigenetic (trans)gene silencing is easy to study in this organism. Approaches undertaken so far aimed only at the identification of factors involved in transgene silencing, whereas no information exists on factors involved in transgene activation. We have in earlier work found that transgene expression in Chlamydomonas becomes activated when the promoter of the HSP70A gene is fused upstream of transgene-driving promoters. Hence, the mechanism underlying HSP70A promoter-mediated transgene activation might be applied to generally facilitate transgenic approaches in eukaryotes. 

Aim

To understand the molecular mechanisms by which transgene silencing in Chlamydomonas (and other organisms?) may be overcome.

Current state and Focus

We could show that the transgene activating effect of the HSP70A promoter is mediated largely by heat shock elements and heat shock transcription factor 1 (HSF1). Our most recent data suggest that HSF1 constitutively forms a scaffold at the transgenic HSP70A promoter, presumably containing mediator and TFIID, from which local chromatin remodeling and polymerase II recruitment to downstream promoters is realized. Surprisingly, an open chromatin state per se was not sufficient to activate otherwise silent promoters fused downstream of the HSP70A promoter. Next steps will aim at identifying and characterizing factors recruited by HSF1 (mediator, TFIID, chromatin remodelers) to form the postulated scaffold for recruiting polymerase II to downstream promoters.