Dynamic transcription regulation at the single-molecule level

Wang, Zuhui; Deng, Wulan

doi:10.1016/j.ydbio.2021.11.004

Cited by 14 publications

(11 citation statements)

References 125 publications

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“…This kinetic profile for a transcription factor is not unique to p53. Rapid on/off kinetics for transcription factors binding to chromatin in live cells has been widely observed in single particle tracking experiments, with time scales similar to what we measured here 15 . Hence single molecule approaches that enable real-time kinetic measurements (in cells and in vitro) have led to an evolution of the traditional regulatory models of stable binding between transcription factors and their REs.…”

Section: Discussionsupporting

confidence: 82%

Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements

Suwita

Ly²,

Goodrich

et al. 2022

Preprint

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The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Critical for understanding how p53 regulates gene expression is unraveling the fundamental mechanisms by which it binds to REs. Toward this goal we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of tetrameric p53 to different REs in real time under equilibrium conditions. For each RE tested we measured a single rate constant for association and two rate constants for dissociation, showing two kinetically distinguishable populations of tetrameric/RE complexes. Moreover, p53/DNA interactions were more dynamic as the RE sequence became closer to the consensus sequence. We propose a model in which p53 and its RE consensus sequence evolved to favor dynamic binding.

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Section: Discussionsupporting

confidence: 82%

Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements

Suwita

Ly²,

Goodrich

et al. 2022

Preprint

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“…Single-molecule imaging has become a powerful method for understanding the dynamics and distribution of biomolecules in living cells [1, 2], owing to technological advances such as bright fluorophores [3] and specialized illumination [4]. Researchers can obtain various biological insights from heterogeneous single-molecule behaviors using methods developed for trajectory and localization analyses, including physical modeling [5], spatial statistics [6], and machine learning [7].…”

Section: Motivation and Significancementioning

confidence: 99%

Slitflow: a Python framework for single-molecule dynamics and localization analysis

Ito

Hirose

Tokunaga

2023

Preprint

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Single-molecule imaging is a promising method for direct quantification of the dynamics and distribution of biomolecules in living cells. Although numerous methods have been developed to gain biological insights into molecular behavior, the high diversity of microscopes and single-molecule dynamics can result in incomplete reproducibility of analyses. Here, we present Slitflow, an open-source framework for a single-molecule analysis workflow that includes image processing, dynamics analysis, and figure creation. We demonstrated the integrity and flexibility of the workflow using 1) a cherry-picked tracking method combining popular tools and 2) various state-of-the-art analyses in a single pipeline. The software accommodates a large variety of data and methods, paving the way for integrative analyses.Code metadata

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“…Second, a thin layer of laser needs to illuminate the focus plane within a cell nucleus in order to lower background fluorescence from the out-offocus plane, for instance, as used for the selective plane illumination microscopy. [74][75] Oblique illumination (or highly inclined and laminated optical sheet, HILO) [76] is one of such approaches that creates a thin light sheet illumination by modifying a total internal reflection (TIR) illuminator unit that is commercially available (e.g., Figure 2C).…”

Section: Single-nucleosome Imaging With Oblique Illumination Microscopymentioning

confidence: 99%

Chromatin behavior in living cells: Lessons from single‐nucleosome imaging and tracking

2022

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Eukaryotic genome DNA is wrapped around core histones and forms a nucleosome structure. Together with associated proteins and RNAs, these nucleosomes are organized three‐dimensionally in the cell as chromatin. Emerging evidence demonstrates that chromatin consists of rather irregular and variable nucleosome arrangements without the regular fiber structure and that its dynamic behavior plays a critical role in regulating various genome functions. Single‐nucleosome imaging is a promising method to investigate chromatin behavior in living cells. It reveals local chromatin motion, which reflects chromatin organization not observed in chemically fixed cells. The motion data is like a gold mine. Data analyses from many aspects bring us more and more information that contributes to better understanding of genome functions. In this review article, we describe imaging of single‐nucleosomes and their tracked behavior through oblique illumination microscopy. We also discuss applications of this technique, especially in elucidating nucleolar organization in living cells.

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Dynamic transcription regulation at the single-molecule level

Cited by 14 publications

References 125 publications

Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements

Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements

Slitflow: a Python framework for single-molecule dynamics and localization analysis

Chromatin behavior in living cells: Lessons from single‐nucleosome imaging and tracking

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