Engineering Recombinant Protein Sensors for Quantifying Histone Acetylation.

Area of Science:

• Epigenetics and Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Histone H3 lysine 14 acetylation (H3K14ac) is a critical epigenetic modification implicated in diseases like cancer and neurological disorders.
• Existing methods for detecting H3K14ac are limited by complex sample preparation, inaccurate quantification, and antibody dependency.
• Accurate tools are needed to study H3K14ac's role in biological processes and disease.

Purpose of the Study:

• To engineer and validate a novel recombinant protein sensor for detecting and quantifying H3K14ac.
• To overcome the limitations of current H3K14ac detection techniques.
• To provide a tool for studying H3K14ac in both in vitro and in situ applications.

Main Methods:

• Engineered recombinant protein sensors utilizing bromodomain sequences from human polybromo-1 (PB1) as H3K14ac recognition domains.
• Tested various sensor designs using nuclear extracts and live cells.
• Validated sensor performance through in vitro assays, live-cell imaging in HEK293T cells, and correlation with antibody-based methods.

Main Results:

• A sensor with dimeric bromodomain repeats demonstrated high efficacy in quantifying H3K14ac.
• The sensor exhibited a linear detection range of 0.5-50 nM with nuclear extracts.
• In situ assays showed sensor colocalization with H3K14ac antibodies and correlated well with in vitro measurements.

Conclusions:

• Developed a novel recombinant sensor for accurate H3K14ac detection and quantification.
• The sensor is effective for both in vitro analysis of nuclear extracts and in situ analysis of live cells.
• This tool offers a significant advancement for studying epigenetic modifications in biological research and disease contexts.