Investigating how transcription factors cooperate and overcome the enhancer nucleosome barrier during embryonic patterning - Project Summary This misregulation of gene expression underlies several human diseases, including many cancers, diabetes, obesity, and multiple developmental disorders. Genome-wide studies and next-generation sequencing have revealed that sequence variants in enhancers, cis-regulatory DNA sequences that control spaciotemporal gene expression programs, contribute to the development of these diseases. These mutations often affect enhancer activity, which must be tightly controlled since enhancers drive tissue and cell-type specific gene expression patterns. One way that enhancer activity is controlled is through the regulation of enhancer accessibility by the nucleosome: the structural unit of chromatin comprised of 147 bp of DNA and a histone octamer. Enhancers are characterized by an intrinsically strong nucleosome barrier that prevents the binding of transcription factors (TFs), the proteins that activate enhancers, until the proper context for activation is reached, at which point TFs must overcome the nucleosome barrier and bind to the DNA. While nucleosome depletion is a key early step in enhancer activation, we do not yet understand how the nucleosome barrier is overcome and how enhancers are made accessible for gene activation, despite accessibility being a major regulator of enhancer activity. Current models suggest that specialized TFs called pioneer factors can access their motifs in the presence of nucleosomes and foment nucleosome depletion through cooperativity with additional TFs. Even still, how pioneer and non-pioneer TFs cooperate to generate chromatin accessibility at enhancers is not yet known. Furthermore, how pioneer factors perturb the nucleosomal landscape to facilitate chromatin accessibility and cooperative TF binding is unclear. This study seeks to identify how TFs overcome the nucleosome barrier at enhancers using high-resolution experimental and computational genomics techniques to map TF binding, chromatin accessibility, and nucleosome positioning. Aim 1 will characterize how pioneer and non-pioneer TFs cooperate for binding to the DNA and for establishing chromatin accessibility. This aim will combine high-resolution TF binding (ChIP- nexus) and temporally resolved chromatin accessibility (time-course ATAC-seq) information with deep learning models (BPNet) that will reveal the sequences and sequence constraints that are important for and predictive of TF cooperativity. Aim 2 will profile genome-wide nucleosome positional changes over developmental time at unprecedented resolution, using a chemical mapping of nucleosome centers approach. This aim will uncover how the nucleosome state at enhancers is altered over time to generate accessibility and how nucleosomes are positioned with respect to the underlying regulatory DNA sequences. Taken together, these aims will illuminate how TFs pioneer the chromatin landscape for enhancer activation, thereby deepening the field’s understanding of the mechanisms of gene regulation and how misregulation contributes to human disease.
