Rehybridization of Promoter DNA Targets in Chromatin to Discover Transcription Factors of Oncogenes

Abstract

Identification of transcription factors (TF) assembled at promoter regions of oncogenes is vital to understand how gene expression is altered in cancer and in tumor microenvironments. Most TF identification methods are based on one‐on‐one DNA/protein interactions in vitro, which may not reflect DNA/protein interactions in vivo and may overlook protein complex information. Moreover, existing methods for chromatin pull down target pre‐identified proteins rather than DNA, which limits the ability to discover new proteins that may bind to specific genomic sequences. We are pursuing a new approach, chromatin rehybridization (CR), which preserves and detects DNA/protein and protein/protein interactions that occur in live cells and targets specific DNA sequences rather than associated proteins.The G‐quadruplex forming Pu27‐mer located upstream of the c‐MYC oncogene and its associated TFs including cellular nucleic acid binding protein (CNBP) serve as the model system for development of the CR technique. Preliminary results demonstrate that CNBP is pulled down with the targeted Pu27‐mer in MCF‐7 cells using CR. At least one unreported TF associated with c‐MYC was also detected and will be identified using mass spectrometry.The CR method addresses the need for full proteomic analysis of particular genomic regions. This work is supported by NIH through grant R21 CA134419‐01A1.