Cell Lysate Microarray for Mapping the Network of Genetic Regulators for Histone Marks

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Proteins execute cellular functions, and their abundance and post-translational modifications are tightly regulated.
• Understanding gene-protein function relationships is crucial for cell biology.
• Genetic perturbations offer a powerful tool to investigate these regulatory networks.

Purpose of the Study:

• To develop a high-throughput method for exploring genome-wide genetic perturbations.
• To establish a workflow for identifying regulators of protein abundance and post-translational modifications.
• To investigate the regulators of histone modifications in yeast.

Main Methods:

• Development of a cell lysate microarray on kilo-conditions (CLICK) platform.
• Utilizing 4837 yeast knockout (YKO) and 322 temperature-sensitive (ts) mutant strains.
• Application of the CLICK array for global identification of upstream regulators, exemplified by histone marks.

Main Results:

• A general workflow for global identification of upstream regulators was established using the CLICK array.
• The CLICK array successfully identified known regulators of H3K4me3 in yeast, demonstrating its efficacy.
• Several protein groups were found to negatively regulate H3K4me3, and Cab4p and Cab5p were identified as key enzymes in histone acylation.

Conclusions:

• The CLICK array is a versatile and applicable tool for rapid, global identification of protein/enzyme regulators.
• This method can be used to study the regulation of protein levels and post-translational modifications for both histone and non-histone proteins.
• The study provides insights into the regulation of histone modifications and identifies novel enzymes involved in histone acylation.