AUTHOR=Ning Duo , Deng Yuqing , Gao Tong , Yang Yang , Chen Gengzhan , Tian Simon Zhongyuan , Zheng Meizhen 

TITLE=TF-chRDP: a method for simultaneously capturing transcription factor binding chromatin-associated RNA, DNA and protein

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=Volume 13 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2025.1561540

DOI=10.3389/fcell.2025.1561540

ISSN=2296-634X

ABSTRACT=Transcription factors (TFs) play a crucial role in the regulation of gene expression and the structural organization of chromatin. They interact with proteins, RNA, and chromatin DNA to exert their functions. Therefore, an efficient and straightforward experimental approach that simultaneously captures the interactions of transcription factors with DNA, RNA, and proteins is essential for studying these regulatory proteins. In this study, we developed a novel method, TF-chRDP (Transcription Factor binding Chromatin-associated RNA, DNA, and Protein), which allows for the concurrent capture of these biomolecules in a single experiment. We enriched chromatin complexes using specific antibodies and divided the chromatin into three fractions: one for DNA library preparation to analyze the genomic binding sites of transcription factors, another for RNA library preparation to investigate the RNA associated with transcription factor binding, and the third for proteomic analysis to identify protein cofactors interacting with transcription factors. We applied this method to study the transcription factor p53 and its associated chromatin complexes. The results demonstrated high specificity in the enrichment of DNA, RNA and proteins. This method provides an efficient tool for simultaneously capturing chromatin-associated RNA, DNA and protein bound to specific TF, making it particularly useful for analyzing the role of protein-DNA-RNA complexes in transcriptional regulation.