Developing a robust method for analyzing transcription factor mediated chromatin interactions Transcription factors (TFs) mediate the interactions between distal regulatory elements and gene promoters to control specific gene expression; disruption of this process by mutations in the protein coding regions or the non-coding DNA binding sites of TFs could lead to dysregulation of transcription and human diseases. An increasing number of genomics tools (such as Hi-C) have been developed for mapping long range chromatin interactions. What is missing in these DNA-centric approaches are TFs and their cofactors, although bioinformatics approaches have been developed to infer the roles of TFs in chromatin interactions. Current technologies (such as HiChIP) for mapping protein-mediated chromatin interactions have worked well with high abundance proteins but not for most TFs. This is largely due to the high variability and low efficiency of antibodies used in the immunoprecipitation of chromatin complexes associated with the TF of interest. The introduction of in situ biotinylated TFs has greatly improved the sensitivity and reproducibility of ChIP-seq (a.k.a biochip-seq), but this strategy cannot be extended to the mapping of TF-mediated chromatin interactions. This is because all Hi-C based mapping techniques, including HiChIP, use biotinylated nucleotide to label the ligation junction to enrich contact signals. Consequently, streptavidin binding would pull down all chromatin contacts indiscriminately. To overcome this problem, the proposed studies aim to develop a new method for mapping TF-mediated chromatin interaction by adapting the HiChIP protocol to biotinylated TFs (referred to as bioHiChIP) (Aim 1) and test the application of bioHiChIP in mapping chromatin interactions mediated by biotinylated FOXP3 (Aim 2). The bioHiChIP is designed to be a simple and easy-to-operate protocol for robust mapping of protein-mediated chromatin interactions. It will serve as a powerful tool for the broad research community to take advantage of the increasing research resources of transgenic model organisms with biotinylated TFs to study the detailed mechanisms of transcription regulation in physiological functions and pathological diseases.