Basic helix-loop-helix pioneer factors interact with the histone octamer to invade nucleosomes and generate nucleosome-depleted regions.

Nucleosomes drastically limit transcription factor (TF) occupancy, while pioneer transcription factors (PFs) somehow circumvent this nucleosome barrier. In this study, we compare nucleosome binding of two conserved S. cerevisiae basic helix-loop-helix (bHLH) TFs, Cbf1 and Pho4. A cryo-EM structure of Cbf1 in complex with the nucleosome reveals that the Cbf1 HLH region can electrostatically interact with exposed histone residues within a partially unwrapped nucleosome. Single-molecule fluorescence studies show that the Cbf1 HLH region facilitates efficient nucleosome invasion by slowing its dissociation rate relative to DNA through interactions with histones, whereas the Pho4 HLH region does not. In vivo studies show that this enhanced binding provided by the Cbf1 HLH region enables nucleosome invasion and ensuing repositioning. These structural, single-molecule, and in vivo studies reveal the mechanistic basis of dissociation rate compensation by PFs and how this translates to facilitating chromatin opening inside cells.

DNA sequence influences hexasome orientation to regulate DNA accessibility.

Nucleosomes, the fundamental organizing units of eukaryotic genomes, contain ∼146 base pairs of DNA wrapped around a histone H3-H4 tetramer and two histone H2A-H2B dimers. Converting nucleosomes into hexasomes by removal of a H2A-H2B dimer is an important regulatory event, but its regulation and functional consequences are not well-understood. To investigate the influence of hexasomes on DNA accessibility, we used the property of the Widom-601 Nucleosome Positioning Sequence (NPS) to form homogeneously oriented hexasomes in vitro. We find that DNA accessibility to transcription factors (TF) on the hexasome H2A-H2B distal side is identical to naked DNA, while the accessibility on the H2A-H2B proximal side is reduced by 2-fold, which is due to a 2-fold reduction in hexasome unwrapping probability. We then determined that a 23 bp region of the Widom-601 NPS is responsible for forming homogeneously oriented hexasomes. Analysis of published ChIP-exo data of hexasome containing genes identified two DNA sequence motifs that correlate with hexasome orientation in vivo, while ExoIII mapping studies of these sequences revealed they generate homogeneously oriented hexasomes in vitro. These results indicate that hexasome orientation, which is influenced by the underlying DNA sequence in vivo, is important for modulating DNA accessibility to regulate transcription.

Dissociation rate compensation mechanism for budding yeast pioneer transcription factors.

Nucleosomes restrict the occupancy of most transcription factors (TF) by reducing binding and accelerating dissociation, while a small group of TFs have high affinities to nucleosome-embedded sites and facilitate nucleosome displacement. To understand this process mechanistically, we investigated two Saccharomyces cerevisiae TFs, Reb1 and Cbf1. We show that these factors bind to their sites within nucleosomes with similar binding affinities as to naked DNA, trapping a partially unwrapped nucleosome without histone eviction. Both the binding and dissociation rates of Reb1 and Cbf1 are significantly slower at the nucleosomal sites relative to those for naked DNA, demonstrating that the high affinities are achieved by increasing the dwell time on nucleosomes in order to compensate for reduced binding. Reb1 also shows slow migration rate in the yeast nuclei. These properties are similar to those of human pioneer factors (PFs), suggesting that the mechanism of nucleosome targeting is conserved from yeast to humans.