Understanding how immune cells develop and function requires insight into the epigenomic mechanisms that regulate gene expression. While many genomic studies focus on transcriptional outputs, changes in the chromatin landscape play a central role in shaping lineage commitment. The mammalian immune system is composed of highly diverse and dynamic cell types, but detailed epigenomic studies have been severely limited by technical challenges in profiling rare cell populations.
We developed and validated a low-input, automated CUT&RUN workflow that incorporates standardized sample preparation to ensure reliable generation of data at the consortium scale. This method minimizes sample handling and applies internal controls to monitor assay performance during experimental and sequencing stages.
Extensive optimization of assay conditions and antibody reagents enabled robust mapping of histone post-translational modifications (PTMs) from as few as 10,000 cells per reaction. Applying this approach, we profiled >170 immune subpopulations collected from 11 ImmGen consortium labs over two years.
These innovations establish a scalable, high-resolution platform for profiling chromatin landscapes from minimal cell inputs. Our automated CUT&RUN pipeline enables standardized, reproducible analysis across diverse immune cell types and can distinguish technical issues from true biological insights. Together, these advances lay the foundation for a companion study presenting the first comprehensive epigenomic atlas of immune lineages and provide a framework for studying chromatin regulation in rare or limited samples across the life sciences.
NIH R44 AI167215
Technological Innovations in Immunology (TECH)
Aaron J. Alcala, M. Marunde, C. L. Windham et al.· Journal of Immunology· 0 citations
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.
Keith E. Maier, James T. Anderson, Connor P. Frasier et al.· Clinical Cancer Research· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
