FENGC: Targeted, Multiplexed, and Integrated Single-Molecule Profiling of Chromatin Accessibility and DNA Methylation.

Unraveling genomic regulatory complexity by adding chromatin accessibility to the long-read sequencing toolkit 2247245

Fiber-seq simultaneously profiles chromatin accessibility, DNA methylation, protein footprints, and genetic variation on single molecules at near—base-pair resolution, revealing how genetic and epigenetic features interact to regulate gene expression and provides a powerful new framework for dissecting immune cell function and disease mechanisms.

Unpacking Chromatin Accessibility with Fiber-seq

Chromatin accessibility has long been used as a marker for regions of DNA with regulatory potential. Fiber-seq detects chromatin accessibility on individual DNA fibers, enabling analyses beyond the identification of the accessible chromatin regions (ACRs). By providing single molecule level high resolution, Fiber-seq provides unprecedented qualitative descriptions, including potential categorizations of ACRs, identification of internal transcription factor footprints and nucleosome positioning within individual DNA fibers. As with all tools, the power of this technique depends on careful experimental design and data analysis -- incorrect usage will result in incorrect conclusions. Here we offer guidelines and flag potential pitfalls when generating and analyzing Fiber-seq data, such as (1) the optimum levels of adenosine methylation per-fiber, (2) the power of per-fiber state inference, (3) the importance of controlling for read depth and methylation rates when comparing across samples, (4) the limitations of long-read sequence mapping, and (5) suggestions for identification of differentially accessible peaks across samples.

Decoding the regulatory genome: single-cell four-omics integration

In a recent study published in Nature, Chen et al. introduced CHARM (single-cell assay for Chromatin conformation, Histone modi fi cation, chromatin Accessibility, and RNA expression Multi-omics pro fi ling), a platform that simultaneously captures four regulatory modalities within the same nucleus. 1 This integrated strategy provides a comprehensive framework for dissecting how multiple layers of epigenetic regulation converge to control gene expression at single-cell resolution. Gene regulation in eukaryotic cells is governed by a complex interplay of molecular and spatial mechanisms. Chromatin accessibility determines whether regulatory elements such as promoters and enhancers are available for transcription factor binding. Histone modi fi cations de fi ne chromatin states that either promote or repress transcription. In parallel, the three-dimensional organization of the genome establishes spatial proximity between distal regulatory elements and their target genes. Although each of these regulatory layers has been extensively studied, understanding how they operate together within the same cell has remained a major challenge. 2 Previous technologies have provided valuable insights into individual modalities. ATAC-seq pro fi les chromatin accessibility, CUT&Tag captures histone modi fi cations, and Hi-C reveals three-dimensional genome architecture. Recent single-cell platforms such as ChAIR and scHiCAR jointly pro fi le chromatin accessibility, RNA, and 3D contacts, but their 3D contact capture is anchored at accessible chromatin or candidate cis-regulatory elements, introducing structural bias into chromatin architecture reconstruction. 3,4 Moreover, regulatory modalities not captured by these platforms, such as histone modi fi cations, require separate pro fi ling and computational integration for broader cross-modality analysis. CHARM is distinguished from previous platforms by adding histone modi fi cation as a fourth same-cell modality and by using restriction-enzyme-based Hi-C

Assay for Transposase-Accessible Chromatin Sequencing (ATAC-seq) of Cancer Cells in Culture.

High-Efficiency Genomic Mapping of Chromatin-Associated Targets with CUT&RUN.

The precise regulation of chromatin composition is critical to gene expression and cellular identity, and thus a key component in development and disease. Robust assays to study chromatin features, including histone post-translational modifications (PTMs) and chromatin-associated proteins (e.g., transcription factors or PTM readers), are crucial for understanding their function and identifying novel therapeutic strategies. To this end, Cleavage Under Targets and Release Using Nuclease (CUT&RUN) has emerged as a powerful tool for high-resolution epigenomic profiling. The approach has been successfully applied to numerous cell and tissue types, providing insights into target genomic distribution with unprecedented sensitivity and throughput. Here, we provide a detailed CUT&RUN protocol from sample collection through data analysis, including best practices and defined controls to ensure specific, efficient, and robust target profiling.

Targeting DNA Methylation: New Paradigms and the Advent of Gene-Selective Tools.

The mechanisms and roles of DNA methylation in epigenetic regulation are examined, the current landscape of DNA methylation modulators are evaluated, from traditional DNMT inhibitors to cutting-edge CRISPR-dCas9 fusion systems and protein-protein interaction disruptors, and their clinical relevance are evaluated.