Poster abstracts

Poster number 14 submitted by Benjamin Donovan

Determining how pioneer factors access chromatin

Ben Donovan (biophysics)

Abstract:
Packaging the eukaryotic genome into nucleosomes greatly limits binding site accessibility for transcription factors (TFs) and other DNA binding proteins [1]. Because of this, nucleosomes are precisely positioned in regulatory regions of the genome [2], [3]. For example, transcription start sites (TSSs) are typically depleted of nucleosomes and contain specific patterns of post-translational modifications (PTMs) that recruit chromatin remodelers, histone chaperones, and other PTM-reading enzymes. It is well established that these epigenetic regulatory mechanisms control gene expression by modulating the accessibility of TF binding sites within nucleosomes. Recent studies indicate a new class of TFs, called pioneer factors, are the first TFs to target a gene before activation [4], [5]. Additionally, mis-regulation of these proteins are implicated in cancers [6], [7]. Despite links to disease, how pioneer factors access binding sites within condensed chromatin is not understood. These studies focus on Reb1, a pioneer factor from S. cerevisiae involved in establishing nucleosome depleted regions (NDRs) at TSSs and installing the histone variant H2A.Z at the 5’ ends of genes [8]. Together with collaborators, we show that insertion of a single Reb1 target site into the nucleosome dyad is sufficient to establish an NDR in vivo. Furthermore, electrophoretic mobility shift assays indicate Reb1 binds to sites within nucleosomes with similar affinities as to DNA. To probe how Reb1 effects nucleosome structure, we inserted binding sites in increments of 5 basepairs (bp) throughout the entry/exit region and monitored Reb1 induced ∆FRET between Cy3-DNA and Cy5-octamer. With this approach, we show that Reb1 binds to transiently exposed binding sites on unwrapped nucleosomal DNA without evicting histones. Additionally, we show that inserting a binding site into the dyad region increases Reb1 accessibility to entry/exit sites by 2-3 fold.

References:
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Keywords: chromatin, biophysics, pioneer transcription factors