Mechanisms for DNA Recognition, Scanning and Nucleosome Mechanical Actions by Pioneer Transcription Factors and their Role in Cell Fate Decisions

University Of California, Merced, Merced CA

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Abstract

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 DNA binding domain of Wor1 (WOPR) as model for high-resolution biophysical analysis. 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. Based on our previous and preliminary studies on another PTF, the Engrailed DNA binding domain (enHD), we have formulated the 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 also 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) Examine the Conformational Transitions and DNA Reshaping Dynamics of WOPR upon DNA Binding, 2) Resolve the Dynamic Scanning of WOPR with Imperfect-Motif Clustered DNA, and 3) Dissect the Mechanics of the Interactions of WOPR with Nucleosome DNA. The proposed studies will detail at the molecular level the DNA recognition processes used by Wor1 that lead to cell fate decisions and cell reprograming in the context of the White-Opaque cell switch of Candida albicans.