Single Gene Analysis using Chromatin DNA Rehybridization to Identify Transcription Factors

Identification of transcription factors (TF) assembled at the promoter region of a gene is vital to understand molecular mechanisms of gene regulation. Most TF identification methods are based on DNA/protein interactions in vitro, which may not reflect DNA/protein interactions in vivo and may overlook protein complexes. Moreover, existing methods for chromatin pull‐down target pre‐identified proteins rather than DNA and are unlikely to discover new proteins that may bind to specific genomic sequences. We are pursuing a new approach, chromatin DNA rehybridization (CDR), which preserves and detects DNA/protein and protein/protein interactions that occur in live cells and targets specific DNA sequences rather than associated proteins. We are using ribosomal RNA gene (rDNA), the most actively transcribed gene, and its associated TFs as the model system to establish the CDR technique. Biotinylated DNA probes that target the core and upstream control elements are utilized to pull down rDNA promoter fragments and associated TF complexes. TFs are then released from the DNA for analysis. This work is supported by National Institutes of Health through grant R21 CA134419‐01A1.