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Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis
Published on: December 12, 2017
Single-epitope recognition imaging of native chromatin.
Hongda Wang1,2, Yamini Dalal3,4, Steven Henikoff3,5
1Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA.
Epigenetics & Chromatin
|December 19, 2008
Summary
Atomic force microscopy (AFM) reveals that centromeric nucleosomes, containing centromere-specific histone 3 (CenH3), are predominantly tetramers. This technique allows direct visualization of native nucleosomes under physiological conditions.
Area of Science:
- Chromatin biology
- Epigenetics
- Atomic Force Microscopy (AFM)
Background:
- Direct visualization of chromatin offers insights into epigenetic processes.
- Atomic force microscopy (AFM) visualizes single nucleosomes under physiological conditions.
- Previous AFM studies focused on in vitro reconstituted chromatin, not native nucleosomes.
Purpose of the Study:
- To explore the potential of AFM for dissecting native nucleosomes.
- To directly identify centromere-specific histone 3 (CenH3) within native centromeric chromatin particles.
Main Methods:
- Applied a recognition mode of AFM imaging to native Drosophila chromatin.
- Isolated histone core particles containing CenH3.
- Confirmed specificity using CenH3 peptide blocking and quantified interaction strength via force measurements.
Main Results:
- Over 90% of identified particles were tetrameric in height.
- AFM imaging successfully identified CenH3 within histone core particles from native centromeric chromatin.
- Results indicate these particles are mature CenH3-containing hemisomes.
Conclusions:
- Tetramers are the predominant form of centromeric nucleosomes in mature native chromatin.
- AFM enables efficient, highly specific recognition of CenH3 in native nucleosomes.
- This approach provides a direct, rapid method for studying native nucleosomes in physiological salt concentrations.
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