Abstract Epigenetic drugs (or epidrugs) can reshape transcriptional programs by altering the chromatin landscape, yet their global impact on transcription factor (TF) binding remains poorly understood1-3. While we know that epidrugs affect chromatin association and transcription factor (TF) function, we lack detailed information on specific TF changes when chromatin is disrupted by epidrugs. This issue is more complex for TFs with similar motifs or those not directly binding DNA. However, current methods are inadequate to test individual TFs at the necessary scale. In this project, we aim to systematically evaluate how a selected epidrug influences TF-DNA interactions across the genome. To achieve this, we employ a cell library produced by High-throughput Insertion of Tags Across the Genome (HITAG) comprising more than 500 HEK293T derived clones4. Each clone expresses a unique FLAG-tagged transcription factor (TF), facilitating the parallel analysis of binding profiles for multiple individual TFs within a single experiment. Our experimental approach will combine in situ reverse transcription with nanoCUT&Tag5 on the 10x Genomics platform. This strategy will provide single-cell-level data for FLAG-TF identification and TF occupancy in a high-throughput manner. Multiplexed profiling of TF binding at this scale has not been previously achieved and holds the potential to reveal the effects of chromatin remodelers on TF occupancy across a broad range of specific targets. References 1. Tomaselli D, Lucidi A, Rotili D, Mai A. Epigenetic polypharmacology: A new frontier for epi‐drug discovery. Medicinal Research Reviews 2020; 40: 190–244. 2. Navakauskienė R, Navakauskas D, Borutinskaitė V, Matuzevičius D. Chromatin Remodeling During Leukemia Cell Proliferation and Differentiation. In: Navakauskien˙e R, Navakauskas D, Borutinskait˙e V, Matuzevi˘cius D (eds). Epigenetics and Proteomics of Leukemia: A Synergy of Experimental Biology and Computational Informatics. Springer International Publishing: Cham, 2021, pp 67–122. 3. Suraweera A, O’Byrne KJ, Richard DJ. Epigenetic drugs in cancer therapy. Cancer Metastasis Rev 2025; 44: 37. 4. Kim J, Kratz AF, Chen S, Sheng J, Kim HK, Zhang L et al. High-throughput tagging of endogenous loci for rapid characterization of protein function. Sci Adv 2024; 10:eadg8771. 5. Bárcenas-Walls JR, Ansaloni F, Hervé B, Strandback E, Nyman T, Castelo-Branco G et al. Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution. Nat Protoc 2024; 19: 791–830.