Defining the Rules for Pioneer Factor Nucleosome Engagement

DNA binding to chromosomal DNA is essential for many fundamental biological processes including: transcriptional regulation, DNA replication and repair, recombination, and chromosome segregation. There is a fundamental gap in understanding how transcription factors (TF) bind regulatory regions located in compacted, high-order chromatin. Therefore, there is a fundamental need to determine the mechanistic rules defining TF binding to chromatin. Our long-term goal is to define the biological rules dictating TF binding to chromosomal DNA in a cell. All TFs, even pioneer factors, are not able to bind all their targets throughout the genome, indicating that there are binding constraints imposed on all binding events. Multiple factors appear to impact how TFs identify TF binding sites (TFBSs) including: TFBS positioning within a nucleosome, TFBS variants, sequence of the nucleosome, cooperativity, histone modifications, DNA methylation, and chromatin remodeling. To accomplish our goal, we will combine in vitro with in-cell assays to allow the systematic dissection of factors required for initial and sustained/functional TF binding to chromatin. At the nucleosome level, we will use our in vitro Pioneer-seq assay, which allows the direct comparison of TFBSs located in all possible nucleosome positions, with differing nucleosome sequences, across many TFBS variants, with modified histones. At the regulatory region level, we will ectopically express pioneer TFs in comprehensively characterized cell lines, with cell models of disease, to uncover the chromatin characteristics, chromatin remodelers, or cooperativity required for TF binding. With this MIRA our lab will close these knowledge gaps by answering three fundamental questions: 1) How do TFs recognize their binding sites within a nucleosome? 2) How do histone modifications and nucleosome positioning regulate TF binding? 3) How does chromatin organization regulate TF binding? Our results are expected to have an important positive impact on our mechanistic understanding of how all TFs regulate transcription and direct gene expression during normal development and disease. Our findings will likely be transformative by providing quantitative models for how proteins bind to their targets within or near nucleosomes.