Comprehensive profiling of histone modifications using a label-free approach and its applications in determining...

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Histone post-translational modifications (PTMs) are crucial for regulating gene expression and DNA repair.
• Histone H3 lysine 56 acetylation (H3K56ac) is important for responding to DNA replication stress.
• Understanding the dynamics of histone PTMs requires robust analytical methods.

Purpose of the Study:

• To develop and validate a novel, two-pronged approach for comprehensive profiling of histone PTMs.
• To investigate the role of Rtt109 and Asf1 in regulating H3K56ac in Saccharomyces cerevisiae.
• To establish a generic strategy for assessing global changes in histone PTMs.

Main Methods:

• Utilized nano LC-MS to analyze intact core histones and infer PTMs from mass profiles.
• Employed fractionation, derivatization with propionic anhydride, and trypsin digestion for detailed analysis.
• Developed specialized peptide detection and clustering tools for data mining and PTM profiling.

Main Results:

• The new methodology successfully profiled histone PTMs in wild type and mutant yeast strains.
• Most common histone PTMs were unaffected in yeast lacking Rtt109 and Asf1.
• A significant reduction (17% of total H3) in H3K56ac was observed in both mutant strains compared to wild type.

Conclusions:

• The developed analytical strategy provides a rapid and comprehensive method for histone PTM profiling.
• Rtt109 and Asf1 are essential for maintaining normal levels of H3K56ac.
• This generic strategy can be applied to diverse sample sets for structure-function relationship studies.