Periodic Reporting for period 3 - DynaMech (Linking Transcription Factor Binding Dynamics to Promoter Output)
Reporting period: 2019-02-01 to 2020-07-31
For the off-rate technique this has included cell growth, induction of transcription factor removal, microscopy to monitor nuclear depletion, quantification of microscopy, cross-linking, quenching of the reaction, cell disruption, chromatin extraction, chromatin fragmentation, clean-up, immunoprecipitation, quantitative (small-scale) analysis of DNA binding at specific loci (for optimisation) and DNA sequencing library preparation through multiplexing. The major result so far is that we have individual, optimized protocols for each of the substeps. The sensitivity of our measurements has improved vastly due to these efforts, with in some cases more than 100-fold improvement in our signal to noise. We have also learnt that not all transcription factors will perform equally well with these techniques. We are currently testing the entire off-rate procedure (all steps in conjunction) for a limited set of transcription factors in a full-blown experiment. These results will soon be analyzed in detail to evaluate whether the methodology is working as envisaged.
With regard to the on-rate, since this is a genomic adaptation of a protocol published by another research group, its adaptation was started later. We have run into several technical hurdles that were unforseen. This is particularly surprising given that this is a published protocol in a reputable scientific journal. We are currently working on these issues and hope that this methodology will be as advanced as the off-rate protocol within a few months, also given the fact that many of the substeps are similar to the protocol that we have first focused on.
The project is high risk-high gain. There is therefore a possibility that the techniques being set up will not work as envisaged. This is in part indicated by the problems encountered with the previously published protocol, that is not yet working as described. As described in the first scientific report, steps have been taken to circumvent any ensuing problems. This includes extra personnel, made available by the host institute. In addition, a back-up plan, focusing on single cell approaches (as suggested by the reviewers of the proposal) is also being worked on. With regard to results in the form of publications, since the project is mainly technology development, it is too early as yet to have scientific publications linked to the core of the project. The same is true for other dissemination activities. This will happen in the later stages.