CUT&RUN Profiling of the Budding Yeast Epigenome

Area of Science:

• Epigenetics and Genomics
• Molecular Biology
• Cellular Processes

Background:

• Epigenome mapping is crucial for understanding chromatin regulation of DNA processes like transcription.
• Chromatin Immunoprecipitation (ChIP) is widely used but has limitations like cross-linking issues.
• Accurate profiling of chromatin landscapes is essential for deciphering gene regulation.

Purpose of the Study:

• To introduce and validate Cleavage under targets and release using nuclease (CUT&RUN) as a superior chromatin profiling technique.
• To demonstrate the application of CUT&RUN and CUT&RUN.ChIP for high-resolution genome-wide mapping.
• To highlight the efficiency and advantages of CUT&RUN over traditional ChIP methods.

Main Methods:

• Utilized Cleavage under targets and release using nuclease (CUT&RUN) for in situ chromatin profiling.
• Employed antibody-targeted Micrococcal Nuclease for controlled cleavage and solubilization of protein-DNA complexes.
• Developed CUT&RUN.ChIP for simultaneous genome-wide mapping of two epitopes on single molecules.

Main Results:

• Achieved precise genome-wide maps of histone modifications, variants, transcription factors, and chromatin remodelers in budding yeast.
• Demonstrated CUT&RUN's ability to avoid cross-linking and solubilization issues inherent in ChIP.
• Identified transient genomic features, including dynamic nucleosome-remodeling intermediates, due to low background and high resolution.

Conclusions:

• CUT&RUN and CUT&RUN.ChIP are powerful, high-resolution techniques for epigenome mapping.
• These methods offer significant advantages over ChIP, enabling detailed analysis of chromatin dynamics.
• The 2-day protocol from cells to sequencing library preparation enhances experimental efficiency.