Nucleosome Accessibility to Multiple Transcription and Pioneer Factors
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Date
2020-05
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The Ohio State University
Abstract
DNA contains vital information for a cell to function including genes that must be transcribed into functioning proteins. Due to the limited size of a cell and to provide protection from damage, DNA is packaged into chromatin. Additionally, Chromatin serves as a regulator of gene expression, with more densely packed regions being less accessible to transcription factors and less dense regions being more transcribed. The base unit of chromatin is the Nucleosome, 147 bp of DNA wrapped around a histone octamer. Merely packaging into Nucleosomes reduces Transcription Factor binding around 100 fold. So the question arises, how do transcription factors access more dense regions of chromatin? Enter a class of transcription factors called Pioneer Factors who can bind to DNA and Nucleosomes with the same affinity. These are believed to be the first to bind dense regions of chromatin and open them up to Transcription factors. However their mechanism is currently unknown as one pioneer factor on its own cannot evict a nucleosome. Previous studies have looked at cooperativity in protein binding between Transcription Factors as a way to make nucleosomes more accessible. However a comprehensive look a Transcription Factor and Pioneer Factor cobinding has not been done and may hint at a mechanism through which Pioneer factors increase accessibility.
We used two Transcription Factors from S. Cerevisiae, Gal4 and Pho4 along with two Pioneer Factors, Reb1 and CBF1 to test the change in accessibility of multiple proteins binding to a nucleosome, both with sites across the nucleosome and with sites adjacent to one another. Binding affinity is measured through ensemble Fluorescence Resonance Energy Transfer (FRET). We found that binding across the nucleosomes tends to decrease accessibility two-fold regardless of whether the transcription factor has pioneering abilities. The only exception is two pioneer factors, where binding seems unaffected. For adjacent binding, accessibility to an internal site in the nucleosome increases by 10-fold regardless of whether it is a Pioneer Factor or transcription factor. The exception again is the case of two pioneer factors where the effect is smaller, only a 3.5 fold increase. Overall, the decrease in accessibility across the nucleosome and the increase in accessibly of an adjacent site seem to be a universal property of nucleosomes and may reflect a structural change caused by binding. Pioneer factors only seem to act differently when in contact with another pioneer factor, in which case the effect on accessibility is reduced. However the mechanism by which they would be able to detect another pioneer factor's presence requires further study.
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Keywords
Nucleosomes, Chromatin, FRET, Pioneer Factors, Cobinding