Single-molecule states link transcription factor binding to gene expression.

Area of Science:

• Molecular Biology
• Genetics
• Biophysics

Background:

• Gene expression in mammalian cells is driven by transcription factors (TFs) binding to genomic enhancers.
• The precise molecular mechanisms linking enhancer sequences to TF binding, promoter status, and transcription levels are not fully understood.

Purpose of the Study:

• To investigate the quantitative relationships between TF binding, nucleosome occupancy, and gene expression.
• To dissect the contributions of TF binding affinity and activation domains to gene regulation.

Main Methods:

• Single-molecule footprinting was employed to simultaneously measure TF, nucleosome, and regulatory protein occupancy.
• Engineered enhancer-promoter constructs with varying TF binding sites were utilized for both synthetic and endogenous TFs.

Main Results:

• TF binding to nucleosome-free DNA is independent, but activation domains recruit cofactors that destabilize nucleosomes, promoting TF binding cooperativity.
• Average TF occupancy shows a linear correlation with promoter activity.
• TF binding strength was successfully decomposed into distinct binding and activation components.

Conclusions:

• The study provides a quantitative framework for dissecting gene expression regulation.
• Models were developed to predict enhancer binding microstates and gene expression dynamics.
• Key contributors to gene expression, including TF properties and chromatin regulator recruitment, were identified.