Abstract B011: Single-molecule protein footprinting with Fiber-seq resolves coordinated chromatin states across regulatory domain

Abstract

Gene regulation emerges from coordinated interactions among DNA sequence, chromatin accessibility, DNA methylation, nucleosome positioning, and transcription factor occupancy. These features are typically measured using separate short-read assays, fragmenting regulatory information across experiments and obscuring how regulatory states co-occur along individual DNA molecules. This limits mechanistic interpretation of cis-regulatory architecture, particularly within repetitive or structurally complex genomic regions that are poorly resolved by short-read approaches. Fiber-seq is a long-read, single-assay multiomic method that preserves regulatory context across individual DNA molecules by integrating chromatin accessibility footprinting with native long-read sequencing. Accessible adenines are enzymatically methylated using the N6-adenine methyltransferase Hia5 and sequenced alongside endogenous 5mC using PacBio or Oxford Nanopore Technologies platforms. Each long read therefore links chromatin accessibility, DNA methylation, nucleosome positioning, and transcription factor occupancy across extended regulatory domains with haplotype resolution. Fiber-seq recapitulates accessibility patterns observed with conventional assays while revealing chromatin architectures that are collapsed in short-read data. Single-molecule profiles resolve heterogeneous protein occupancy across individual DNA molecules, enabling direct detection of nucleosome positioning, transcription factor binding, and polymerase II recruitment at active regulatory elements. By distinguishing protected from accessible motifs within motif-dense regions, Fiber-seq supports composite motif analysis and prioritization of candidate regulatory elements and transcription factors for functional validation. These capabilities could support mechanistic studies of therapeutic response by enabling direct observation of regulatory state transitions following pharmacologic perturbation. For example, Fiber-seq could resolve loss of occupancy following transcription factor degradation together with local rearrangement of neighboring protein occupancy within the same cis-regulatory domain and on the same DNA molecule. This integrated single-molecule view of regulatory remodeling may support identification of adaptive resistance mechanisms, compensatory regulatory programs, pharmacodynamic biomarkers, and candidate synthetic lethal interactions relevant to epigenetic drug development. NOTE: Generative AI was used to assist in drafting the abstract text; all authors reviewed and approved the final content. Keith E. Maier, James T. Anderson, Connor P. Frasier, Allison R. Hickman, Sabrina R. Hunt, Zu-Wen Sun, Martis W. Cowles, Andrew Stergachis, Bryan J. Venters, Michael-Christopher Keogh. Single-molecule protein footprinting with Fiber-seq resolves coordinated chromatin states across regulatory domain [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B011.