Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted...

Area of Science:

• Molecular Biology
• Proteomics
• Systems Biology

Background:

• Cellular abundance of transcription factors (TFs) critically influences their regulatory roles in gene expression.
• Accurate determination of TF copy numbers is essential for understanding gene regulation mechanisms.

Purpose of the Study:

• To develop and apply a sensitive mass spectrometry assay for quantifying absolute TF copy numbers.
• To profile key TF levels, including PPARγ and RXRα, during 3T3-L1 pre-adipocyte differentiation.
• To build a DNA binding model for PPARγ integrating copy number, binding energetics, and chromatin state.

Main Methods:

• Selected reaction monitoring-based mass spectrometry assay for simultaneous quantification of up to ten proteins.
• Application of the assay to profile absolute TF levels in differentiating mouse 3T3-L1 pre-adipocytes.
• Formulation of a biophysical DNA binding model for PPARγ.

Main Results:

• Demonstrated a method to determine absolute TF copy numbers per nucleus, ranging from ~250 to >300,000.
• Observed significant dynamic range (up to fivefold) in TF abundance during adipocyte differentiation.
• Developed a model explaining PPARγ binding site dynamics, correlating with copy number and chromatin state.

Conclusions:

• The developed mass spectrometry assay provides a powerful tool for quantitative proteomics of TFs.
• TF abundance varies dramatically and dynamically, impacting gene regulation during differentiation.
• The PPARγ DNA binding model elucidates the interplay between TF levels, binding affinity, and chromatin accessibility.