Article

# Single-Molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells

[more]
(Impact Factor: 33.12). 03/2014; 156(6):1274-85. DOI: 10.1016/j.cell.2014.01.062
Source: PubMed

ABSTRACT Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.

3 Followers
·
121 Views
• Source
##### Article: Reliable scaling of Position Weight Matrices for binding strength comparisons between transcription factors
[Hide abstract]
ABSTRACT: Scoring DNA sequences against Position Weight Matrices (PWMs) is a widely adopted method to identify putative transcription factor binding sites. While common bioinformatics tools produce scores that can reflect the binding strength between a specific transcription factor and the DNA, these scores are not directly comparable between different transcription factors. Here, we provide two different ways to find the scaling parameter $\lambda$ that allows us to infer binding energy from a PWM score. The first approach uses a PWM and background genomic sequence as input to estimate $\lambda$ for a specific transcription factor, which we applied to show that $\lambda$ distributions for different transcription factor families correspond with their DNA binding properties. Our second method can reliably convert $\lambda$ between different PWMs of the same transcription factor, which allows us to directly compare PWMs that were generated by different approaches. These two approaches provide consistent and computationally efficient ways to scale PWMs scores and estimate transcription factor binding sites strength.
• ##### Article: Regulation of MYB and bHLH Transcription Factors: A Glance at the Protein Level
[Hide abstract]
ABSTRACT: In complex, constantly changing environments, plants have developed astonishing survival strategies. These elaborated strategies rely on rapid and precise gene regulation mediated by transcription factors (TFs). TFs represent a large fraction of plant genomes and among them, MYBs and basic helix-loop-helix (bHLHs) have unique inherent properties specific to plants. Proteins of these two TF families can act as homo- or heterodimers, associate with proteins from other protein families, or form MYB/bHLH complexes to regulate distinct cellular processes. The ability of MYBs and bHLHs to interact with multiple protein partners has evolved to keep up with the increased metabolic complexity of multi-cellular organisms. Association and disassociation of dynamic TF complexes in response to developmental and environmental cues are controlled through a plethora of regulatory mechanisms specifically modulating TF activity. Regulation of TFs at the protein level is critical for efficient and precise control of their activity, and thus provides the mechanistic basis for a rapid on-and-off switch of TF activity. In this review, examples of post-translational modifications, protein-protein interactions, and subcellular mobilization of TFs are discussed with regard to the relevance of these regulatory mechanisms for the specific activation of MYBs and bHLHs in response to a given environmental stimulus. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.
Molecular Plant 12/2014; DOI:10.1016/j.molp.2014.11.022 · 6.61 Impact Factor
• ##### Article: A general method to improve fluorophores for live-cell and single-molecule microscopy
[Hide abstract]
ABSTRACT: Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.
Nature Methods 01/2015; DOI:10.1038/nmeth.3256 · 25.95 Impact Factor