Transcription factors act as specifying agents of cell differentiation during development by binding to DNA enhancer sequences and activating them to control developmental gene expression. Enhancer activation is typically associated with the removal of nucleosomes, which decorate eukaryotic genomes and normally wrap roughly 150 base pairs of DNA in a highly stable configuration. A persistent puzzle of developmental gene regulation is how TFs bind and activate their target enhancers when they are initially wrapped in nucleosomes, which typically inhibit TF binding. One hypothesis posits that a special class of ?pioneer factors? are able to bind their targets in the context of nucleosomal wrapping and displace the nucleosomes they bind to activate and expose the enhancer for downstream TF binding. However, it has been exceedingly difficult to confirm the presence of nucleosome binding ?pioneer activity? in vivo, leaving the developmental roles of pioneer factors in question. We recently used high-resolution epigenome profiling to identify instances of nucleosome binding by pioneer factors that were enriched at enhancers with suboptimal motif binding sequences, presenting the intriguing possibility that pioneer activity is a mechanism to ensure the fidelity of enhancer activation at sites that are vulnerable to natural fluctuations in the local chromatin environment. Pioneer factors often function in early development, which maintains high fidelity despite natural variation in chromatin structure that is sensitive to the metabolic state of the cell. Therefore, pioneer factors may play a direct role in insulating developmental transitions against metabolic variance. However, the potential roles of pioneer factors in developmental fidelity and buffering against metabolic heterogeneity have not been uncovered to date. In this proposal, I will use a controlled pioneer factor expression system to study how pioneer factor-driven developmental changes are buffered against deliberate chromatin and metabolic perturbations.
In Aim 1, I will test the hypothesis that pioneer activity facilitates developmental fidelity by observing development after genetically enforcing chromatin barriers to pioneer factor binding and inactivating the nucleosome binding pioneer activity of a specific pioneer factor.
In Aim 2, I will use a model system of metabolic control of development to understand how pioneer factor binding responds to metabolic changes, and how specific pioneer factor-enhancer activation events underlie different developmental outcomes in response.
These Aims will uncover mechanistic explanations for the disparity between variance in gene regulatory processes on the molecular level and the precision of cell fate outcomes on the developmental level, and my findings will be of direct consequence to diseases such as cancer where extreme heterogeneity overwhelms the checks and balances on cell fate. A K99/R00 Award will be instrumental in addressing these questions and furnishing me with high level training in new methods and biological theory that will prepare me to continue to pursue major research avenues related to pioneer factor and chromatin control of development in my future independent career.
This project will directly address a persistent puzzle in developmental gene regulation: how do newly active developmental transcription factors bind and activate their target enhancers, when they are tightly wrapped by nucleosomes that prevent transcription factor binding in the first place? The experiments proposed to solve this puzzle will not only leverage cutting edge molecular technologies to probe the dynamics of transcription factor- chromatin conflicts, but will also probe whether there are biological rationales for the persistence of these conflicts, such as for the maintenance of developmental fidelity across variable metabolic contexts that are commonly experienced in vivo. In summary, the proposed project will improve our understanding of the function of chromatin as a regulator of cell fate that can be applied across myriad developmental and disease contexts, and will integrate this understanding of molecular regulation with our broader understanding of cellular metabolic state.
