Publications

2024

1. REAL-TIME VISUALIZATION OF SPLICEOSOME ASSEMBLY REVEALS BASIC PRINCIPLES OF SPLICE SITE SELECTION

   Benjamin T Donovan, Bixuan Wang, Gloria R Garcia, Stephen M Mount, and Daniel R Larson

   Jul 2024

   Abs DOI

   The spliceosome is a megadalton protein-RNA complex which removes introns from pre-mRNA, yet the dynamic early assembly steps have not been structurally resolved. Specifically, how the spliceosome selects the correct 3’ splice site (3’SS) amongst highly similar non-functional sites is not known. Here, we develop a kinetic model of splice site selection based on single-molecule U2AF heterodimer imaging in vitro and in vivo. The model successfully predicts alternative splicing patterns and indicates that 3’SS selection occurs while U2AF is in complex with the spliceosome, not during initial binding. This finding indicates the spliceosome operates in a ‘partial’ kinetic proofreading regime, catalyzed in part by the helicase DDX42, which increases selectivity to the underlying U2AF binding site while still allowing for efficient forward progression.

2. U2AF regulates the translation and localization of nuclear-encoded mitochondrial mRNAs

   Gloria R Garcia, Murali Palangat, Josquin Moraly, Benjamin T. Donovan, Bixuan Wang, Ira Phadke, David Sturgill, Jiji Chen, Brittney Short, Gustavo A. Rivero, David M. Swoboda, Naomi Taylor, Kathy McGraw, and Daniel R Larson

   Sep 2024

   Abs DOI

   The mechanisms underlying molecular targeting to mitochondria remain enigmatic, yet this process is crucial for normal cellular function. The RNA binding proteins U2AF1/2 form a heterodimer (U2AF) that shuttles between the nucleus and cytoplasm, regulating splicing in the nucleus and translation in the cytoplasm. Our study identifies an unexpected role for U2AF in mitochondrial function. We demonstrate that U2AF interacts with nuclear-transcribed mitochondrial mRNAs and proteins, inhibits translation, localizes to the outer mitochondrial membrane, and regulates mRNA localization to mitochondria. Moreover, an oncogenic point-mutation in U2AF1(S34F) disrupts this regulation, leading to altered mitochondrial structure, increased translation, and OXPHOS-dependent metabolic rewiring, recapitulating changes observed in bone marrow progenitors from patients with myelodysplastic syndromes. These findings reveal a non-canonical role for U2AF, where it modulates multiple aspects of mitochondrial function by regulating the translation and mitochondrial targeting of nuclear-encoded mRNAs.

2023

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

   Benjamin T. Donovan, Hengye Chen, Priit Eek, Zhiyuan Meng, Caroline Jipa, Song Tan, Lu Bai, and Michael G. Poirier

   Molecular Cell, Apr 2023

   Abs DOI

   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.

2. The nucleosome unwrapping free energy landscape defines distinct regions of transcription factor accessibility and kinetics

   Benjamin T Donovan, Yi Luo, Zhiyuan Meng, and Michael G Poirier

   Nucleic Acids Research, Feb 2023

   Abs DOI

   Transcription factors (TF) require access to target sites within nucleosomes to initiate transcription. The target site position within the nucleosome significantly influences TF occupancy, but how is not quantitatively understood. Using ensemble and singlemolecule fluorescence measurements, we investigated the targeting and occupancy of the transcription factor, Gal4, at different positions within the nucleosome. We observe a dramatic decrease in TF occupancy to sites extending past 30 base pairs (bp) into the nucleosome which cannot be explained by changes in the TF dissociation rate or binding site orientation. Instead, the nucleosome unwrapping free energy landscape is the primary determinant of Gal4 occupancy by reducing the Gal4 binding rate. The unwrapping free energy landscape defines two distinct regions of accessibility and kinetics with a boundary at 30 bp into the nucleosome where the inner region is over 100-fold less accessible. The Gal4 binding rate in the inner region no longer depends on its concentration because it is limited by the nucleosome unwrapping rate, while the frequency of nucleosome rewrapping decreases because Gal4 exchanges multiple times before the nucleosome rewraps. Our findings highlight the importance of the nucleosome unwrapping free energy landscape on TF occupancy and dynamics that ultimately influences transcription initiation.

2022

1. Regulating gene expression through control of transcription factor multivalent interactions

   Benjamin T. Donovan and Daniel R. Larson

   Molecular Cell, Jun 2022

   DOI

2019

1. Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting

   Benjamin T Donovan, Anh Huynh, David A Ball, Heta P Patel, Michael G Poirier, Daniel R Larson, Matthew L Ferguson, and Tineke L Lenstra

   The EMBO Journal, Jun 2019

   Publisher: John Wiley & Sons, Ltd

   Abs DOI

   Abstract Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.

2. Dissociation rate compensation mechanism for budding yeast pioneer transcription factors

   Benjamin T. Donovan, Hengye Chen, Caroline Jipa, Lu Bai, and Michael G. Poirier

   eLife, Mar 2019

   Abs DOI

   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.

3. DNA sequence influences hexasome orientation to regulate DNA accessibility

   Matthew Brehove, Elan Shatoff, Benjamin T Donovan, Caroline M Jipa, Ralf Bundschuh, and Michael G Poirier

   Nucleic Acids Research, Jun 2019

   Abs DOI

   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.