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Linking Transcription Factor Binding Dynamics to Promoter Output

Periodic Reporting for period 3 - DynaMech (Linking Transcription Factor Binding Dynamics to Promoter Output)

Reporting period: 2019-02-01 to 2020-07-31

"Humans have approximately 20 000 protein-coding genes in our DNA. The proteins encoded by genes perform a plethora of different cellular tasks and need to be produced (""expressed"") at appropriate levels. These levels differ for different proteins, are dependent on cell types and the appropriate levels also depend on developmental stage or other circumstance that a cell may find itself in (e.g. damaged, under viral attack, under certain nutritional or hormonal conditions etc.). Appropriate control of gene expression is therefore essential for all living organisms to function properly. Disruption of gene expression is associated with many different types of disease, including cancer, immune disorders, metabolic disorders etc. In order to understand how life works in health and in disease, it is essential to understand how control of gene expression is achieved. This has already been the subject of intense research since the discovery that DNA is the carrier of genetic information. It is well-established that control is exerted by a range of different types of transcription factors, specific proteins that can bind to DNA, thereby exerting (local) control on which genes are actively transcribed or not. An important aspect of the mechanism of gene expression control is how transcription factors bind to DNA. The aim of this project is to develop technologies that will allow us to analyse DNA binding dynamics of DNA binding factors across entire genomes in living organisms. Such methods would allow us to answer how different DNA binding dynamics of different transcription factors on different genes affects control of gene expression. This will be fundamental to understanding how life works in health and in disease."
The work has focused mainly on setting up two technologies aimed at measuring transcription factor binding dynamics across entire genomes in living organisms. One technique is for measuring the off-rate, the other the on-rate. The off-rate technique is completely new, and therefore the most novel and has been more intensely focused on. The off-rate technique is a (large) adaptation of a previously published method and should be easier to set up. At the start of the project a team was assembled/recruited. Next a selection has been made for which transcription factors should initially be employed for setting up the methodology. The appropriate strains for these factors were then engineered. Each technique consists of a large number of substeps and these have each been optimized. This has first been done individually and then in conjunction.

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.
We anticipate applying the new technologies to our studies of gene expression soon. The expected results are the technologies themselves as well as fundamental insights into how different transcription factor binding dynamics affects control of gene expression. The publications included in our reporting so far all deal with previous work from the group and is not core to the work but has been included for the sake of completion.