Poster abstracts
Poster number 13 submitted by Ben Donovan
Reb1 is a pioneer factor that dynamically regulates nucleosomal DNA accessibility
Benjamin Donovan (Biophysics), Caroline Jipa (Department of Physics), Chao Yan (Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802 ), Lu Bai (Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802 )
Abstract:
Packaging the eukaryotic genome into nucleosomes greatly limits transcription factor (TF) occupancy by restricting binding site accessibility and accelerating TF off-rates. Because of this, nucleosomes are precisely positioned to regulate TF activation of transcription. Reb1 is a TF from S. cerevisiae that can recruit chromatin remodelers and generate nucleosome-depleted regions. Previous studies also suggest that Reb1 preferentially targets nucleosomes at the DNA entry/exit region. To probe Reb1-nucleosome interactions, we inserted a Reb1 binding site in increments of 5 bp throughout the entry/exit region and monitored Reb1 binding and Reb1-induced nucleosome structural changes. Gel shift measurements reveal Reb1 binds nucleosomes with a similar affinity to naked DNA, which is in stark contrast to other TFs such as Gal4 that binds nucleosomal binding sites with drastically lower affinities. This property is shared with eukaryotic pioneer factors Sox2 and Oct4 suggesting Reb1 functions as a pioneer factor. Ensemble FRET measurements show that Reb1 binding to its site within the entry-exit region traps nucleosomes in a partially unwrapped state without evicting histones. This indicates that Reb1 functions to expose nucleosomal DNA and reduce nucleosome stability. Single molecule fluorescence measurements show that despite similar equilibrium affinities, exchange kinetics are 50-fold slower at nucleosomal DNA sites relative to naked DNA, highlighting another distinct nucleosome binding behavior as compared with other TFs that have dramatically increased exchange kinetics at nucleosomal binding sites. From these results, we propose that Reb1 can function as a pioneer factor that induces nucleosome unwrapping and resides at nucleosomal DNA entry/exit sites for minutes to facilitate the recruitment of chromatin remodelers.
References:
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[4] A. Soufi, M. F. Garcia, A. Jaroszewicz, N. Osman, M. Pellegrini, and K. S. Zaret, «Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming», Cell, vol 161, no 3, pp 555–568, 2014.
Keywords: chromatin, pioneer factor, single molecule