With sadness I have to report here that we decided to terminate our parMRC project, even though it was super-exciting. After a lot of efforts over multiple years, we could not convince ourselves that the parMRC complex that we chose can be harnessed to push genomic loci apart in eukaryotes.
We have no doubts that fluorescently tagged parM can form beautiful filaments in mammalian cells. We also confirmed by surface plasmon resonance assays (many thanks to Alex Fish) that parR binds as a multimeric complex to parC in vitro. Although we did observe some spots of parR-GFP in yeast carrying parC in its genome (many thanks to Jos Meeussen and Tineke Lenstra), in mammalian cells we failed to detect binding of parR to parC by ChIP-seq. Possibly the wrapping of parC into nucleosomes makes it hard for parR to bind.
Most importantly, we obtained negative results in two key functional experiments that we thought should demonstrate that parMRC can push two loci apart. First, in a very sensitive yeast assay (thank you for help: James Haber, Tibor van Welsem, Fred van Leeuwen) we tested whether parMRC could interfere with the repair of a double-strand break when parC was inserted on each side of the break. Unfortunately, there was no hint of an effect. Second, in mouse ES cells we flanked the Sox2 gene and its distal enhancer by parC sites, and carefully measured Sox2 expression in the tagged allele (thank you: Mathias Eder). There was no hint of reduced Sox2 expression, even though deletion of the enhancer causes ~90% loss of Sox2 expression. Thus either the enhancer does not need to contact the Sox2 promoter, or parMRC just did not disrupt the contacts. We favour the second interpretation, although we did not do 4C or Hi-C experiments to confirm this.
Of course, it is possible that we would eventually be able to get the system to work if we had tried many more experimental conditions and configurations. The results that I summarize above are also not always based on multiple independent experiments (this is why we probably won’t publish this work as a proper scientific paper). But every scientific project requires continuous weighing of the chances of success, and if those chances seem to become too small, it is better to drop the project. This is what we decided to do… it was a very difficult decision!
I am extremely thankful to Ezequiel Miron, the brave and wonderful postdoc who pioneered this project, knowing that it was high-risk. Also many thanks to our technician Marcel de Haas, who has done a lot of excellent (as always) work for this project. Ezequiel now has a scientific job outside academia, working in the field of microscopy, his old passion.
-Bas van Steensel