Chromatin Stability at Low Concentration Depends on Histone Octamer Saturation Levels

Hagerman, Thomas A.; Fu, Qiang; Molinié, Benoit; Denvir, James; Lindsay, Stuart; Georgel, Philippe

doi:10.1016/j.bpj.2008.10.070

Cited by 11 publications

(16 citation statements)

References 40 publications

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“…3b ). The reconstituted nucleosome is first cross-linked with formaldehyde as nucleosomes become unstable upon dilution 30 , 31 . Cross-linked nucleosomes are incubated with target TFO (tTFO: can form triplex), with scrambled TFO (sTFO: cannot form triplex), or with buffer (no TFO), followed by incubation with streptavidin-conjugated beads.…”

Section: Resultsmentioning

confidence: 99%

Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation

Isogawa

Fuchs²,

Fujii³

2018

Sci Rep

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The goal of present paper is to develop a reliable DNA-based method for isolation of protein complexes bound to DNA (Isolation of DNA Associated Proteins: IDAP). We describe a robust and versatile procedure to pull-down chromatinized DNA sequences-of-interest by formation of a triple helix between a sequence tag present in the DNA and a complementary triple helix forming oligonucleotide (TFO) coupled to a desthiobiotin residue. Following optimization to insure efficient recovery of native plasmids via TFO probe in vitro, the procedure is shown to work under various experimental situations. For instance, it allows capture proteins associated to plasmids hosted in E. coli, and is also successfully applied to recovering nucleosomes in vitro opening many possibilities to study post translational modifications of histones in a genuine nucleosome context. Incubation in human nuclear extracts of a plasmid carrying a NF-κB model promoter is shown to pull-down a specific transcription factor. Finally, isolation of a specific locus from human genomic chromatin has been successfully achieved (Chromatin-of-Interest Fragment Isolation: CoIFI). In conclusion, the methodology can be implemented for capturing proteins that specifically bind to any sequence-of-interest, DNA adduct or secondary structure provided a short sequence tag for triple helix formation is located nearby.

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Section: Resultsmentioning

confidence: 99%

Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation

Isogawa

Fuchs²,

Fujii³

2018

Sci Rep

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show abstract

“…Many reports with optical and magnetic tweezers showed experiments performed at low salt and in absence of Mg 2+ , i.e. conditions where higher-order folding can be expected to be severely affected yielding variable results [45][46][47][48][49][50][51]. Sub-physiological salt solutions are also often used for fluid AFM operation to get stable imaging of nucleosomes [52,53].…”

Section: Afm Nanoindentation Spectroscopy Of Ex Vivo Purified Chromatinmentioning

confidence: 99%

Mechanical properties of nucleoprotein complexes determined by nanoindentation spectroscopy

Rakshit

Melters

Dimitriadis

et al. 2020

Nucleus

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The interplay between transcription factors, chromatin remodelers, 3-D organization, and mechanical properties of the chromatin fiber controls genome function in eukaryotes. Besides the canonical histones which fold the bulk of the chromatin into nucleosomes, histone variants create distinctive chromatin domains that are thought to regulate transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether histone variants translate distinctive biochemical or biophysical properties to their associated chromatin structures, and whether these properties impact chromatin dynamics as the genome undergoes a multitude of transactions, is an important question in biology. Here, we describe single-molecule nanoindentation tools that we developed specifically to determine the mechanical properties of histone variant nucleosomes and their complexes. These methods join an array of cutting-edge new methods that further our quantitative understanding of the response of chromatin to intrinsic and extrinsic forces which act upon it during biological transactions in the nucleus.

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“…Existing fluorescence-activated techniques for cell (19,20) and droplet (21) sorting lack the volume confinement necessary for sorting individual, single fluorophore-labeled molecules at picomolar concentrations and above that are favorable for protein or antibody binding and chromatin stability (22,23).…”

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confidence: 99%

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel

Cipriany

Murphy

Hagarman

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing. I n chromatin, chemical modifications to histone proteins and DNA alter the status of the epigenome and influence gene regulation and normal development. Their aberrant placement has been linked to the onset of cancer (1, 2) and other diseases. Bisulfite conversion and immunoprecipitation (IP) have been used extensively to examine these modifications on locus-specific and genome-wide scales. These approaches have limitations in terms of material handling or multiplexed detection. Conventional chromatin immunoprecipitation (ChIP) requires an abundance of input material, often 10 3 -10 6 cells for genome-wide studies, to compensate for >99% material loss during processing (3, 4). This problem compounds for sequential re-ChIP reactions, limiting the study of multivalent modifications (4), which could provide a clear view of epigenetic coordination. Whereas DNA methylation analysis using bisulfite conversion can operate on picogram quantities of DNA (5-7), the conversion causes degradation of >90% of the input DNA. Methods that combine ChIP and bisulfite sequencing in a sequential process (8) have demonstrated progress in multiplexed epigenetic analysis. There continues to be active research in reducing the input material requirements and in automation of the processes (9-11) for epigenetic analysis. Furthermore, there is interest in additional capability for simultaneous detection of multiple epigenetic modifications in the same material.Miniaturized fluidic devices offer a compelling toolset for multiplexed detection and efficient sample handling in analytical and preparatory systems. Microfluidics have performed complicated workflows that include nanoliter sample handling (12) and incorporate electrodes (13-16) or valves (12, 17) for sophisticated processing. Nanofluidics have achieved attoliter-scale fluid volume confinement to isolate and quantify the attributes of individual molecules that can ...

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Chromatin Stability at Low Concentration Depends on Histone Octamer Saturation Levels

Cited by 11 publications

References 40 publications

Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation

Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation

Mechanical properties of nucleoprotein complexes determined by nanoindentation spectroscopy

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel

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