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.
