Chromatin architectures underlying plasmid-based assays for regulatory variant effects

Area of Science:

• Molecular Biology
• Epigenetics
• Genomics

Background:

• Plasmids are crucial tools in molecular biology, but their chromatin structure in mammalian cells is poorly understood.
• Investigating plasmid chromatinization is essential for understanding gene regulation and developing gene therapies.

Purpose of the Study:

• To develop and apply a novel method for high-resolution mapping of chromatin architecture on transfected plasmids.
• To elucidate the principles of plasmid chromatinization and its impact on transcriptional activity.

Main Methods:

• Developed plasmid single-molecule chromatin fiber sequencing (plasmid Fiber-seq) for near-single-nucleotide resolution mapping.
• Applied plasmid Fiber-seq to diverse plasmids and cell lines.
• Integrated plasmid Fiber-seq with high-throughput reporter assays.

Main Results:

• Demonstrated that plasmids are chromatinized in an organized, sequence-dependent manner.
• Revealed heterogeneous and incomplete chromatin structures on plasmids compared to nuclear chromatin.
• Showed focal nucleosome and transcription factor occupancy is key to plasmid transcriptional activity.
• Identified molecular mechanisms of pathogenic non-coding variants by disentangling activator and repressor effects.

Conclusions:

• Plasmid chromatin architecture is organized and sequence-dependent, influencing transcriptional activity.
• Plasmid Fiber-seq provides a powerful tool for fine-scale mapping of chromatin-dependent regulatory grammar.
• Findings offer insights into gene regulation and pathogenic variants at near-single-nucleotide resolution.