A Cytosine Deaminase-Based Genomic Footprinting Assay (cFOOT-seq) for Detecting Transcription Factor Occupancy

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Transcription factors (TFs) are crucial regulators of gene expression.
• Simultaneously assessing multiple TF bindings genome-wide is technically challenging.
• Current methods often require antibodies or lack base-resolution profiling.

Purpose of the Study:

• To develop a novel, antibody-independent method for genome-wide profiling of TF occupancy, chromatin accessibility, and nucleosome organization.
• To provide a reproducible protocol for base-resolution analysis of transcriptional regulation.
• To enable quantitative and comparative analysis of TF footprint patterns across the genome.

Main Methods:

• cFOOT-seq utilizes a cytosine deaminase (SsdAtox) to convert cytosine to uracil in accessible DNA regions.
• TF binding and nucleosomes protect DNA from deamination, creating footprints.
• The FootTrack analysis framework quantifies deamination patterns for base-resolution profiling.
• Compatibility with ATAC-seq enrichment enhances sensitivity and scalability.

Main Results:

• cFOOT-seq provides antibody-independent, base-resolution, genome-wide TF occupancy, chromatin accessibility, and nucleosome organization profiling in a single assay.
• The FootTrack framework enables quantitative and comparative analysis of TF footprint patterns.
• Genomic DNA integrity is preserved, allowing compatibility with ATAC-seq.
• ATAC-combined cFOOT-seq offers sensitive TF footprint analysis at low sequencing cost, supporting single-cell applications.

Conclusions:

• cFOOT-seq is a versatile and powerful tool for dissecting gene regulatory mechanisms.
• The protocol facilitates genome-wide TF footprint profiling across various biological contexts, including development, immunity, and cancer.
• This method advances the understanding of transcriptional regulation by enabling detailed analysis of TF binding and chromatin structure.