Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging

Area of Science:

• Molecular Biology
• Epigenetics
• Structural Biology

Background:

• Nucleosomes are fundamental to eukaryotic genome organization and gene regulation.
• Post-translational modifications of histones are key epigenetic marks influencing chromatin accessibility.
• The single-molecule structural basis of nucleosome function and epigenetic regulation remains incompletely understood.

Purpose of the Study:

• To investigate the conformational landscape of specific histone-modified nucleosomes using high-throughput atomic force microscopy.
• To elucidate the single-molecule structural implications of H3K36me3, H3S10phos, and H4K5/8/12/16ac modifications on nucleosome unwrapping dynamics.

Main Methods:

• High-throughput atomic force microscopy (AFM) imaging and analysis pipeline.
• Quantitative analysis of over 25,000 nucleosomes, including wild-type and modified variants.
• Characterization of nucleosomal DNA unwrapping steps and cooperativity.

Main Results:

• Identified nucleosomal unwrapping steps of approximately 5 base pairs of DNA.
• H3K36me3 modified nucleosomes exhibit significantly increased and stochastic unwrapping from both sides, unlike wild-type nucleosomes.
• H3S10phos and H4K5/8/12/16ac modifications did not alter nucleosome unwrapping populations or anticooperativity compared to wild-type.

Conclusions:

• H3K36me3 directly influences nucleosome structure at the single-molecule level, affecting DNA accessibility.
• H3S10phos and H4K5/8/12/16ac likely regulate gene expression through higher-order chromatin processes rather than direct structural changes.
• The developed AFM pipeline is a valuable tool for high-resolution studies of nucleoprotein complex conformations.