Mechanisms for DNA Recognition, Scanning and Nucleosome Mechanical Actions by Pioneer Transcription Factors and their Role in Cell Fate Decisions - Project Summary/Abstract Cell fate decisions occur during embryonic development, morphogenesis, and cell reprogramming. These wide- spread cellular changes include launching global gene expression programs that are controlled by pioneer transcription factors (PTFs) which serve as the master regulators of cell fate. Manipulating PTFs is a fundamental building block for regenerative medicine, while the dysregulation of PTFs is linked to the development of cancer. PTFs are special since they can recognize their cognate DNA sequence motifs on both naked DNA and the DNA wrapped into the nucleosomes that form chromatin. The ability of PTFs to bind DNA sequences within chromatin is thought to facilitate nucleosome unwrapping, either directly or by recruiting additional factors, to start the transcription programs. The detailed processes that permit PTFs to recognize cognate sequence motifs on naked DNA or nucleosomes as well as the mechanics of PTFs once bound to nucleosomes are largely unknown. Perhaps more importantly, PTFs operate on very long genomic regions containing clusters of imperfect sequence recognition motifs. Removing these clusters destabilizes cell fates in vivo. However, the function(s) of these imperfect recognition motifs remains a puzzle. We propose to examine the mechanics of PTF recognition progressions using the Engrailed and Wor1 as models for high-resolution biophysical analysis. Engrailed is present in all higher order organisms and plays an essential role in body patterning. Wor1 drives the White-to-Opaque cell switch of Candida albicans, the agent of common human invasive fungal infections. The White-to-Opaque cell switch, which affects virulence and niche selection in clinical candidiasis, is controlled by an easily manipulated genetic circuit in vivo. Our previous and preliminary studies demonstrate that the Engrailed DNA binding domain (enHD) is highly promiscuous in the recognition of cognate DNA sequences. Moreover, enHD appears to be a fast but heterogeneous DNA scanner and undergoes a significant conformational transition when it binds to a high-affinity cognate motif. It is our overarching hypothesis that imperfect-motif clusters organize regions in active DNA and chromatin where PTFs home in on via heterogeneous scanning and promiscuous recognition, and ultimately act on via binding-induced conformational transitions. We have implemented advanced single-molecule detection systems to resolve PTF-DNA dynamic interactions with µsec resolution at nm precision. These techniques will be used to address the following Specific Aims: 1) Dissect the EnHD and WOPR sequence recognition code for DNA and nucleosomes, 2) Resolve the dynamic scanning and mechanical interaction between EnHD and WOPR with imperfect-motif clustered DNA, and 3) Examine Wor1 interaction dynamics with the White-to-Opaque master regulatory element of C. albicans in vitro and in vivo. The proposed studies will detail the DNA recognition processes used by PTFs that lead to cell fate decisions involved in tissue patterning, morphogenesis, cancer, and cellular reprogramming.
