2. Investigation of the mechanisms by which the HSP70A promoter activates transgene expression
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 from transgene-driving promoters. Hence, the mechanism underlying HSP70A promoter-mediated transgene activation might be applied to generally facilitate transgenic approaches in eukaryotes.
To understand the molecular mechanisms by which the HSP70A promoter activates transgene expression.
We could show that the transgene activating effect is mediated largely by heat shock elements, apparently via heat shock transcription factors (HSFs), as outlined in the following Figure:
Click the image to see a larger version with description.
Currently addressed questions and approaches
- Which factors(s) recruited by HSF carry out chromatin remodelling?
→ We apply QUICK-X, which by a combination of RNAi, stable isotope labelling, immunoprecipitation, and quantitative mass spectrometry allows for the identification of protein-protein interactions at high sensitivity.
- Which epigenetic marks are associated with transgene activation by the HSP70A promoter?
→ We use Chromatin Immunoprecipitation (ChIP), for which we have established a robust protocol for Chlamydomonas