Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - TFtoChromatin (Transcription factor binding as a function of chromatin)

Teaser

The development of multicellular organisms requires the maintenance of cellular states as well as their controlled differentiation. These rely on the establishment of correct transcriptional patterns and their perturbation can lead to the development of disease. The binding of...

Summary

The development of multicellular organisms requires the maintenance of cellular states as well as their controlled differentiation. These rely on the establishment of correct transcriptional patterns and their perturbation can lead to the development of disease. The binding of TFs to specific DNA sequence motifs creates specificity in gene regulation. However, most TFs only occupy a subset of their motifs and it is assumed that the presence of nucleosomes and repressive epigenetic modifications hinder TF binding. Therefore, nucleosome remodeling and open chromatin marks are thought to be required to expose a motif for TFs to bind. Indeed several chromatin features correlate with active regulatory regions, including the absence of nucleosomes and the presence of particular epigenetic marks. However, studies that investigate TF binding, nucleosome positioning and epigenetic marks generally only report co-occurrences but not if and how chromatin modulates the ability for TFs to bind their motifs in vivo. Gaining insight into the sensitivity of individual TFs to the local chromatin state would significantly advance our understanding of epigenetic regulation.
Our understanding of the effect that epigenetic information has on the regulation of TF binding is limited due to the inability to manipulate chromatin in vivo and assess the co-occurrence of chromatin state and bound TFs at the single molecule level. However, recent developments in epigenetic editing tools and single molecule footprinting provided the exciting opportunity to manipulate chromatin state in vivo and measure nucleosome and TF occupancy, and DNA methylation in parallel at the single molecule level.
The overall objectives of the “TFtoChromatin” project were to take a multifaceted approach to investigate the effects of local chromatin state on TF binding. A reductionist system was established that enables the controlled manipulation of specific chromatin features around a library of TF motifs inserted into a defined genomic locus in mESCs. This required the combination of various molecular techniques (e.g. library cloning, recombinase mediated cassette exchange, genetic and epigenetic editing, footprinting, and epigenetic mark mapping) with computational analysis. More specifically, a strategy to position nucleosomes over TF motifs and measure nucleosome and TF occupancy simultaneously by single molecule footprinting was established. Similarly, various epigenetic modifying enzymes were used to modify specific epigenetic marks around TF motifs followed by footprinting analysis. Computational dissection of the resulting data gave insights into the sensitivity of a range of TFs to nucleosome positioning and specific epigenetic marks, permitting the generation of predictive models of TF sensitivity to chromatin state.

Work performed

Initially a system was set up to perform single molecule footprinting of TF binding in the genome. This method provided a unique assay to assess TF binding in chromatin and has led to data on a large number of TF motifs about their ability to bind their motifs. Interestingly, among the motifs screened were some that have been experimentally identified but have no associated binding factor and a footprint was detected for one of these motifs. The factor binding to this motif was identified and the genomic binding and chromatin sensitivity of this factor has been characterised. This highlights the power of the reductionist approach to learn about the interplay between TF binding and chromatin.
The development of ways to manipulate nucleosome occupancy and epigenetic marks around TF motifs enabled investigation of how local changes in chromatin state effect TF binding. This has lead to the establishment of a hierarchy of TF sensitivity to chromatin, which is now being expanded to the genome to understand the importance of identified chromatin sensitivities for binding and gene regulation.
The reductionist approach lead to the discovery of a novel TF that seemed sensitive to specific epigenetic marks and has a binding profile only seen for a few other factors. We then took a genome wide approach to assessing if the chromatin sensitivities seen at a defined locus are relevant for the genome. This has confirmed that these aspects of chromatin are important for genomic function for this factor.

The results from a number of collaborative projects that were made possible by the Marie-Curie fellowship were popularised via scientific journals. In addition, the project was presented and discussed at international scientific conferences (e.g. ‘EMBL fellows meeting’, EMBL Heidelberg, Germany, and the Cold Spring Harbor Laboratory (CSHL) meeting on ‘Mechanisms of Eukaryotic Transcription’, New York, USA). Networks dedicated to science and research (e.g. ResearchGate) were also utilised to share scientific findings with the public.

Final results

In general this project has been able to define the sensitivity of a number of TFs to chromatin and how manipulating the chromatin environment that a motif sits in can influence the binding of a TF to its motif. These rules can be used to predict the response of factors in the genome but it is still unclear how relevant these chromatin sensitivities are in the genome where factors seldom act alone. Furthermore, many of the TF motifs tested did not show detectable TF binding, therefore, there are additional effects of chromatin that are not clear, these factors might need further information or work together with additional factors to bind and elicit their effects in the genome. One way to start looking at these additional factors required for binding is to look at motif combinations in different chromatin contexts.
To move beyond the reductionist approach, we have chosen a number of endogenous sites to perform detailed genomic manipulations to see how this affects transcriptional output. In this case also with a particular interest in understanding how combinatorial binding of factors might facilitate some factor to bind that are essential for the transcriptional output of the gene. The development of cell lines with modified epigenetic modification pathways will also be pursued to gain genome wide information on how TF binding and ultimately gene output is effect by specific chromatin modifications.
Understanding the factors that promote and inhibit binding of TFs to their sequence motifs in the genome are of high impact to a broad audience familiar with TF biology. Particularly in our understanding of development and disease.