Quantifying genome-wide transcription factor binding affinities for chromatin using BANC-seq.

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Transcription factors (TFs) regulate gene expression by binding specific DNA sequences.
• TF-DNA binding affinity is influenced by local chromatin structure and DNA accessibility, complicating accurate measurement.
• Existing methods often fail to capture these crucial in vivo factors.

Purpose of the Study:

• To present a detailed protocol for BANC-seq (binding affinities in native chromatin by sequencing).
• To enable accurate genome-wide measurement of TF-DNA binding affinities under native chromatin conditions.
• To provide a method reproducible by molecular biologists with basic bioinformatics skills.

Main Methods:

• BANC-seq involves incubating intact mammalian nuclei with epitope-tagged TFs across a concentration range.
• Chromatin immunoprecipitation or nuclease cleavage with spike-in DNA is used to quantify TF binding.
• Apparent dissociation constants (K_d^App) are determined across the genome.

Main Results:

• A detailed, stepwise protocol for BANC-seq is provided.
• The method allows for the determination of TF-DNA binding affinities in their native chromatin context.
• BANC-seq experiments can be performed in 1.5 days, excluding data analysis.

Conclusions:

• BANC-seq offers a robust method for measuring TF-DNA binding affinities, considering chromatin accessibility.
• The protocol is accessible to molecular biologists, though data analysis requires bioinformatics expertise.
• This technique advances the understanding of transcription regulation by incorporating crucial genomic context.