Single-molecule chromatin configurations link transcription factor binding to expression in human cells

Area of Science:

• Molecular Biology
• Genetics
• Biophysics

Background:

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

Approach:

• Single-molecule footprinting (SMF) was employed to simultaneously measure TF, nucleosome, and transcription machinery occupancy.
• Engineered enhancer/promoter constructs with varying TF binding sites for synthetic and endogenous TFs were utilized.

Key Points:

• TF activation domains enhance competition with nucleosomes for DNA binding in a BAF-dependent manner.
• TF binding on nucleosome-free DNA suggests independent TF interactions.
• Average TF occupancy linearly correlates with promoter activation rates.

Conclusions:

• TF binding strength can be separated into independent binding and activation components.
• Developed thermodynamic and kinetic models quantitatively predict enhancer binding and gene expression dynamics.
• This provides a framework for dissecting gene activation contributors like chromatin remodelers, TF domains, and TF binding site configurations.