Single-Molecule Investigations on Histone H2A-H2B Dynamics in the Nucleosome

Lee, Jaehyoun; Lee, Tae Hee

doi:10.1021/acs.biochem.6b01252

Cited by 28 publications

(37 citation statements)

References 50 publications

(108 reference statements)

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“…Single-molecule fluorescence measurements are widely utilized to investigate protein dynamics including the detection of conformational changes, stoichiometry, and protein mobility (17,19,20). Experiments with this level of detail are only feasible after purification of the protein from its physiological cellular environment (21,22). This type of purification is not achievable for many types of membrane proteins, which lose their structural and functional integrity when removed from their native cellular environment.…”

mentioning

confidence: 99%

Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking

Fox-Loe¹,

Moonschi²,

Richards

2017

Journal of Biological Chemistry

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Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and β subunits. Many nAChR subtypes can assemble with varying subunit ratios, giving rise to multiple stoichiometries exhibiting different subcellular localization and functional properties. In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds and activates nAChRs and influences their trafficking and expression on the cell surface. Currently, no available technique can specifically elucidate the stoichiometry of nAChRs in the ER those in the plasma membrane. Here, we report a method involving single-molecule fluorescence measurements to determine the structural properties of these membrane proteins after isolation in nanoscale vesicles derived from specific organelles. These cell-derived nanovesicles allowed us to separate single membrane receptors while maintaining them in their physiological environment. Sorting the vesicles according to the organelle of origin enabled us to determine localized differences in receptor structural properties, structural influence on transport between organelles, and changes in receptor assembly within intracellular organelles. These organelle-specific nanovesicles revealed that one structural isoform of the α4β2 nAChR was preferentially trafficked to the cell surface. Moreover, nicotine altered nAChR assembly in the ER, resulting in increased production of the receptor isoform that traffics more efficiently to the cell surface. We conclude that the combined effects of the increased assembly of one nAChR stoichiometry and its preferential trafficking likely drive the up-regulation of nAChRs on the cell surface upon nicotine exposure.

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mentioning

confidence: 99%

Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking

Fox-Loe¹,

Moonschi²,

Richards

2017

Journal of Biological Chemistry

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“…Between pauses, gradually changing fluorescence intensities are observed, suggesting that the DNA is unwrapping as the elongation complex translocates. Gradual dissociation of H2A-H2B with no change in DNA is another possibility, which is extremely unlikely considering the structure of the nucleosome and according to our published data even at an elevated salt concentration [49]. If it is still desired to rule out this possibility, a control experiment can be carried out with a FRET pair labeled at both gyres of DNA near the dimer-DNA contact region.…”

Section: Data Interpretationmentioning

confidence: 97%

“…Single-molecule methods provide a powerful means to interrogate such a complex process in real-time in a timeresolved manner. This review is to focus on our single-molecule experimental systems and methods that we reported to investigate nucleosome dynamics [44][45][46][47][48][49][50][51][52][53][54][55][56][57]. In particular, we recently reported an experimental system and method with which we can investigate the real-time dynamics of RNAPII translocation along nucleosomal DNA [56].…”

Section: Introductionmentioning

confidence: 99%

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Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II

Lee

Crickard

Reese

et al. 2019

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Transcription elongation through the nucleosome is a precisely coordinated activity to ensure timely production of RNA and accurate regulation of co-transcriptional histone modifications. Nucleosomes actively participate in transcription regulation at various levels and impose physical barriers to RNA polymerase II (RNAPII) during transcription elongation. Despite its high significance, the detailed dynamics of how RNAPII translocates along nucleosomal DNA during transcription elongation and how the nucleosome structure dynamically conforms to the changes necessary for RNAPII progression remain poorly understood. Transcription elongation through the nucleosome is a complex process and investigating the changes of the nucleosome structure during this process by ensemble measurements is daunting. This is because it is nearly impossible to synchronize elongation complexes within a nucleosome or a sub-nucleosome to a designated location at a high enough efficiency for desired sample homogeneity. Here we review our recently developed single-molecule FRET experimental system and method that has fulfilled this deficiency. With our method, one can follow the changes in the structure of individual nucleosomes during transcription elongation. We demonstrated that this method enables the detailed measurements of the kinetics of transcription elongation through the nucleosome and its regulation by a transcription factor, which can be easily extended to investigations of the roles of environmental variables and histone post-translational modifications in regulating transcription elongation.

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“…In theory, two global pathways for nucleosome disassembly may be envisaged, either dissociation of the octamer as a single entity from the DNA or sequential release of histones or groups of histones; the second hypothesis is now generally accepted. A series of studies of salt-induced dissociation based on FRET (Forster Resonance Energy Transfer) approaches [1], 2, 11, 12, [11-14] [2) or TR-SAXS (Time-Resolved Small Angle X-ray Scattering) {Chen, 2014 #3253, 15,16] [17] provided coherent arguments in favour of a pathway with two major successive phases: a first step leading to the release of H2A-H2B dimers and a second, distinct step corresponding to (H3-H4)2-DNA dissociation.…”

Section: Introductionmentioning

confidence: 99%

Nucleosome assembly and disassembly pathways

Hatakeyama

Retureau

Pasi

et al. 2020

Preprint

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Nucleosome assembly and disassembly play a central role in the regulation of gene expression. Here we use PhAST (Photochemical Analysis of Structural Transitions) to monitor at the base pair level, structural alterations induced all along DNA upon histone binding or release. By offering the first consistent, detailed comparison of nucleosome assembly and disassembly in vitro, we are able to reveal similarities and differences between the two processes. We identify multiple intermediate states characterised by specific PhAST signatures; revealing a complexity that goes beyond the known sequential events involving (H3-H4)2 tetramer and H2A-H2B heterodimers. Such signatures localise and quantify the extent of the asymmetry of DNA/histone interactions with respect to the nucleosome dyad. This asymmetry is therefore defined by the localisation and amplitude of the signals. The localisation of the signal is consistent between assembly and disassembly and dictated by the DNA sequence. However, the amplitude component of this asymmetry not only evolves during the assembly and disassembly but does so differently between the two processes.Understanding the regulation of gene expression requires a complete knowledge of nucleosome dynamics. Our unexpected observation of differences between assembly and disassembly opens up new avenues to define the role of the DNA sequence in these processes. Overall, we provide new insights into how the intrinsic properties of DNA are integrated into a holistic mechanism that controls chromatin structure. 2 Keywords: Nucleosome. PhAST . Nucleosome assembly. Nucleosome disassembly. DNA sequence effect. Statement of SignificanceThis manuscript addresses the question of nucleosome dissociation compares with association. We used PhAST which is a non-intrusive photochemical technique to follow nucleosome dynamics at base pair resolution. We observed structural asymmetry during nucleosome turnover. We also showed for the first time that the process of nucleosome dissociation is not a reversal of association. This asymmetry favours intermediate states involved in chromatin organisation suggesting novel models for the role of nucleosome turnover in the epigenetic regulation of gene expression.

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Single-Molecule Investigations on Histone H2A-H2B Dynamics in the Nucleosome

Cited by 28 publications

References 50 publications

Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking

Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking

Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II

Nucleosome assembly and disassembly pathways

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