Poster abstracts

Poster number 9 submitted by Benjamin Donovan

Reb1 is a pioneer factor that dynamically regulates nucleosomal DNA accessibility

Ben Donovan (The Ohio State University), Caroline Jipa (The Ohio State University ), Chao Yan (The Pennsylvania State University), Lu Bai (The Pennsylvania State University), Michael Poirier (The Ohio State University )

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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Keywords: chromatin, pioneer factor, single molecule