In a groundbreaking study set to transform our understanding of gene regulation, researchers have introduced a novel sequencing technology called Deaminase-Assisted single-molecule chromatin Fiber sequencing (DAF-seq). This pioneering approach enables unparalleled resolution in mapping the organization and protein occupancy along chromatin fibers within single cells, illuminating a layer of genetic regulation previously obscured in diploid organisms. Given the complexity of the human genome — composed of two homologous chromosome sets, each potentially exhibiting divergent regulatory states — DAF-seq’s capacity to resolve interactions at the single-molecule, single-nucleotide, and single-haplotype level represents a formidable leap forward in genomics.
Gene regulation fundamentally depends on the orchestrated co-binding of proteins across chromosomes. These proteins, including transcription factors, chromatin remodelers, and structural components, interact dynamically along chromatin fibers, determining how genes are expressed in specific cells. Yet, until now, current methodologies have fallen short in deciphering the heterogeneity in regulatory protein binding between homologous chromosomes or even between individual cells. What emerges is a blurred picture, averaging signals over populations and masking critical functional nuances necessary for understanding disease processes and cellular differentiation. DAF-seq surmounts these challenges by combining DNA sequence information with precise protein occupancy fingerprints on individual chromatin fibers.
At its core, the DAF-seq technique harnesses deaminase enzymes to conduct single-molecule footprinting, effectively tagging exact nucleotide positions occupied by proteins on the DNA strand. This approach not only pinpoints the precise loci of protein-DNA interactions with near-nucleotide resolution but also preserves the integrity of the DNA sequence, allowing simultaneous genotypic and epigenetic profiling. This dual profiling capability enables researchers to detect how somatic mutations or rare epiallelic variations influence chromatin state and protein occupancy in ways previously elusive to bulk assays.
The implications of being able to study co-binding of proteins along lengthy chromosomal regions in individual cells cannot be overstated. The DAF-seq platform also unlocked the first high-resolution maps revealing cooperative protein occupancy at individual regulatory elements, regions such as promoters, enhancers, and insulators that critically influence transcriptional activity. Indeed, the method demonstrated how proteins cluster or cooperate in situ along the fiber, painting a dynamic picture that informs not just static binding but functional complexes that fine-tune gene expression.
An especially remarkable advancement is the extension of DAF-seq into the single-cell domain, termed single-cell DAF-seq (scDAF-seq). This innovation makes it possible to generate comprehensive chromatin fiber maps spanning 99% of each single cell’s mappable genome. This breadth is unparalleled, offering insights into chromatin states across entire chromosomes rather than limited loci, fundamentally changing the scale at which chromatin architecture can be studied in cellular contexts.
The application of scDAF-seq has exposed a profound level of chromatin plasticity. Research findings indicate that chromatin actuation patterns — the functional occupancy states of proteins along chromatin — diverge by an astonishing 61% between the two haplotypes within a single cell. Even more strikingly, intercellular comparisons reveal a 63% divergence in chromatin actuation among different cells. These revelations underscore the remarkable epigenomic variability that has been suspected but difficult to quantify until the advent of this technology.
Such heterogeneity at the single-fiber level highlights biological processes that could underlie phenomena like allelic imbalance, imprinting, and differential gene expression, which are crucial in development, immune responses, and disease susceptibility. For example, somatic variants that had previously been discounted as irrelevant background noise can now be directly connected to alterations in protein occupancy and regulatory outcomes, deepening our understanding of genotype-phenotype relationships.
One of the most fascinating observations facilitated by scDAF-seq is the preferential co-actuation of regulatory elements along the same chromatin fiber, exhibiting a distance-dependent pattern reminiscent of cohesin-mediated chromatin loops. These loops, long thought to organize chromatin topology and enhance regulatory interactions by bringing distant DNA elements into close proximity, are now revealed to function in concord with protein occupancy states, corroborating models of 3D genome architecture and its impact on transcriptional regulation.
From a methodological perspective, the power of DAF-seq lies in its ability to integrate multiple layers of genetic and epigenetic information on a single DNA molecule. This integration is vital for dissecting complex regulatory networks because chromatin states are often heterogenous and context-dependent. By paralleling nucleotide-level sequence data with chromatin occupancy maps, researchers can now objectively evaluate how variants, both inherited and somatic, impact chromatin dynamics at the molecular scale.
The expansive coverage achieved by DAF-seq—mapping the chromatin fiber architecture over entire chromosomes—provides a broader context for understanding gene regulatory landscapes. Traditional assays have either fallen short in resolution or lacked single-cell granularity, but this technology bridges both gaps. It enables future exploration into how chromatin fiber architecture shifts during cellular differentiation, oncogenesis, or in response to environmental stimuli, offering a platform for novel diagnostic and therapeutic strategies.
Moreover, the potential applications in clinical genomics are substantial. As precision medicine increasingly seeks to understand patient-specific regulatory features influencing disease phenotypes, tools like DAF-seq could provide unmatched insights into somatic mutation impacts and rare epigenetic modifications. This precise molecular detail will be instrumental in deciphering the functional consequences of genetic alterations and how they manifest in cell behavior and pathology.
This research shines a light on the complexity and dynamism of chromatin biology, challenging previous assumptions of uniform chromatin states within diploid cells. By revealing extensive compartmentalization and divergence in regulatory protein binding patterns even within a single cell, DAF-seq invites a re-evaluation of regulatory paradigms and models of chromatin function.
DAF-seq thus marks not merely an incremental improvement but a paradigm shift—a technology capable of revealing the intimate choreography of protein-DNA interactions with unprecedented resolution, scale, and single-cell specificity. This capacity heralds a new era in chromatin biology and genomics, opening pathways to understanding the fundamental mechanics of gene regulation in health and disease with exquisite detail.
In conclusion, the advent of DAF-seq and scDAF-seq introduces a cutting-edge toolkit for scientists probing the epigenomic underpinnings of cellular identity and variability. Moving forward, the integration of such single-molecule and haplotype-aware regulatory maps promises to unravel the intricacies of the genome’s functional organization, heralding transformative insights into cell biology, development, and pathogenesis.
Subject of Research: Chromatin fiber architecture, single-molecule protein occupancy, and single-cell haplotype-resolved gene regulation.
Article Title: Mapping single-cell diploid chromatin fiber architectures using DAF-seq.
Article References:
Swanson, E.G., Mao, Y., Mallory, B.J. et al. Mapping single-cell diploid chromatin fiber architectures using DAF-seq. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02914-3
