Applications of CUT&Tag Sequencing in Epigenetics and Gene Regulation

CUT&Tag, with its unique design of targeted cleavage and in-situ library construction, has rapidly become a new paradigm in epigenetics research. Compared to ChIP-seq, it eliminates the need for cross-linking and sonication, requiring only a single-cell sample for experiments, and boasts a signal-to-noise ratio improvement of over 50%. This provides a novel tool for elucidating disease mechanisms (such as cancer metabolic reprogramming) and developmental biology (such as organoid differentiation).

This article aims to systematically review the technical principles, core advantages, and cutting-edge applications of CUT&Tag, and, through typical cases in histone modification, transcription factor regulation, and disease models, elucidate how it propels epigenetics from "population average" to "single-cell precision."

Technical Principles and Core Advantages

CUT&Tag (Cleavage Under Targets and Tagmentation) is an antibody-guided DNA-protein interaction research technology. It uses an engineered Tn5 transposase to cleave DNA near the target protein binding site and simultaneously add sequencing adapters to directly construct sequencing libraries.

• Its core steps include:
  • Antibody Targeting Binding: The primary antibody recognizes the target protein (such as histone modifiers or transcription factors), and the secondary antibody enhances the signal before introducing the Protein A/G-Tn5 fusion enzyme complex.
  • Targeted Cleavage and Labeling: The Tn5 enzyme cleaves DNA at the target protein binding region and inserts sequencing adapters, releasing fragmented DNA into the extracellular space, significantly reducing background noise.
  • Library Construction and Sequencing: No cross-linking or sonication steps are required in traditional ChIP-seq; library preparation can be completed solely through PCR amplification, with the entire process completed within one day.
• Technical Advantages:
  • Low Sample Requirements: Starting cell quantity can be as low as a single cell (some optimized schemes).
  • High signal-to-noise ratio: Targeted cleavage reduces non-specific binding, resulting in over 80% effective data.
  • High resolution: Capable of detecting fine binding sites ranging from 200-500 bp.
  • Wide compatibility: Applicable to various research scenarios including histone modification, transcription factors, and chromatin accessibility.

For a detailed introductory guide to CUT&Tag sequencing, please refer to "What is CUT&Tag Sequencing? A Complete Beginner’s Guide".

Reproducibility and efficiency of CUT&Tag (Kaya-Okur HS et al., 2019)

Precise Localization of Histone Modifications

Histone modifications (such as H3K4me3, H3K27ac, and H3K27me3) are a core mechanism of epigenetic regulation, regulating gene expression by altering chromatin structure. CUT&Tag technology, through antibody-targeted binding and in-situ cleavage, enables high-resolution detection of histone modification distribution across the entire genome, providing crucial "molecular markers" for elucidating gene expression regulation.

• Organoid Development: Duong P et al. developed an improved CUT&Tag protocol to verify its effectiveness in analyzing chromatin modifications (using H3K4me3 as an example) during fin regeneration in adult zebrafish, revealing the dynamic changes in regeneration-related chromatin. In uninjured fin tissue, CUT&Tag detected nearly 48,000 H3K4me3 enrichment sites. During the critical regeneration period (2 days post-regeneration), the regeneration-specific peak (FOS motif) gained H3K4me3, while the uninjured-specific peak (FOX/KLF motif) lost H3K4me3, reflecting the reactivation of the embryonic developmental program. Chromatin accessibility increased in regions gaining H3K4me3, while accessibility stabilized in those losing it, synergistically regulating regeneration. This protocol confirms the high efficiency of CUT&Tag in regeneration epigenetic studies, revealing the consistency and molecular characteristics of H3K4me3 reprogramming and developmental program reactivation in fin regeneration.

• Mapping Modifications: Zhong Z et al. used CUT&Tag technology to map the genome-wide epigenetic regulation of H3K4me3 in dermal cells (DPCs) of Shaanbei white cashmere sheep. They found that H3K4me3 modification was mainly confined to the vicinity of the transcription start site (TSS); the chromosomal peak distribution showed widespread and relatively uniform modification, with the signal intensity ±3 kb at the peak center exhibiting an approximately normal distribution. This clarified the chromosomal characteristics of the H3K4me3 peak (such as peak width, enrichment, significance, and proximity to the TSS).

• Identifying specific targets: Janssens, D.H. used CUT&Tag to map the fusion-specific targets of various KMT2A fusion proteins (such as KMT2A–AF9, KMT2A–ENL, etc.), identifying common and tumor subtype-specific sites of abnormal chromatin regulation; the fusion binding sites were enriched with active promoter markers (H3K4me3, RNAP2S5p, H3K27ac) and genomic markers (H3K4me1, H3K36me3), lacked silencing markers (H3K27me3/H3K9me3), and some sites had bivalent (H3K4me3+H3K27me3) characteristics. Single-cell CUT&Tag showed their intercellular heterogeneity (suggesting lineage plasticity).

Adaptation of CUT&Tag for full automation (Janssens DH et al., 2021)

A Detailed Analysis of Transcription Factor Regulation

Transcription factors (TFs) regulate gene transcription by binding to gene promoter or enhancer regions, serving as key nodes in gene expression regulation. CUT&Tag technology can pinpoint TF binding sites at high resolution, and combined with ATAC-seq (chromatin accessibility) or RNA-seq (gene expression), it reveals the TF-mediated gene regulatory network.

• Follicular Maturation Mechanism: Zhang H, using CUT&Tag, discovered that biomarkers significantly elevated during ovulation (such as Cyp11a1 and Star) exhibited strong H3K4me3 signaling at their promoters, but this signal was absent in enhancer regions anchored by H3K27ac, indicating that promoter occupancy plays a central role in chromatin accessibility during follicular maturation. FoSl2 CUT&Tag revealed that FoSl2 showed significantly enhanced whole-genome signaling in pGCs at different maturation stages; the average motif score of the differentially occupied peak was lower than that of the constitutive peak, suggesting that increased FoSl2 expression can bind to low-affinity sites. FoSl2 has an increased genome-wide occupancy rate, and its unique GC-activated access region (GAA) overlaps with the FoSl2 gain peak in 75% of ovulations. It regulates GAA opening and downstream gene expression, maintains chromatin state, and is a key factor in follicle maturation.

• AtSPL9 genome-wide target distribution in Arabidopsis: Tao XY et al. used the nuclei of 21-day-old shoots for B-CUT&Tag analysis and identified 3183 peaks, of which 60%-70% were shared with the peaks of inflorescence samples. Compared with ChIP-seq (14-day-old branches): ChIP-seq produced 5841 peaks (corresponding to 4744 genes), of which 36.1% (1713 genes) and 41.1% (1951 genes) overlapped with the target genes of inflorescence and shoots in B-CUT&Tag, respectively, indicating that the regulation of AtSPL9 has spatial (inflorescence vs. shoot) and temporal (21 days vs. 14 days) variations.

• Dynamics of GAF recovery on newly synthesized chromatin: Wooten M et al. used the newborn CUT&Tag technique to analyze the binding of GAF (a key transcription factor for Drosophila development) to newly synthesized DNA. Overall recovery showed that 40% of the GAF signal on newly synthesized DNA recovered rapidly, while the remaining signal gradually recovered within 4 hours. Site heterogeneity analysis revealed two different recovery patterns: early recovery sites (265 sites) showed lower signal, shorter GAF motifs, and narrower peak widths (median width 942 bp), enriched with cell cycle-related functions, and were immediately occupied after the replication fork passed through; late recovery sites (157 sites) showed significant signal, longer and degenerated motifs, and wider peak widths (median width 1603 bp), enriched with development-related functions, and their signal reached its maximum after 4 hours.

• Downstream target gene screening: Gao K et al. analyzed the genomic binding sites of transcription factor XBP1s using CUT&Tag technology, identifying a total of 4723 significant binding peaks. They found that the binding characteristics were predominantly in the promoter region (nearly 70% of the peaks were located in the promoter region, and 66.48% of the peaks were less than 1 kb away from the promoter region), suggesting that XBP1s mainly exerts its regulatory role by directly binding to the promoters of target genes. Further research revealed that MAP3K2, a key gene in the MAPK/ERK pathway, is a direct downstream target gene of XBP1s. XBP1s overexpression significantly increased the mRNA and protein levels of MAP3K2, while XBP1 knockout (KO) significantly inhibited its expression, clarifying the epigenetic regulatory mechanism of the XBP1s-MAP3K2-MAPK/ERK axis in endometritis-induced EMT(Epithelial-Mesenchymal Transition).

CUT&Tag assay reveals downstream gene targets of XBP1s (Gao K et al., 2025)

Single-cell epigenetic heterogeneity

Single-cell CUT&Tag technology, with its low starting quantity (100-1000 cells) and single-cell resolution, solves the problem that traditional bulk sequencing cannot resolve cellular heterogeneity, revealing the epigenetic differences among different cell subpopulations in complex tissues.

• Multimodal chromatin profiling: Bartosovic M et al. developed the nano-CUT&Tag (nano-CT) technology, which uses a nanobody-Tn5 fusion protein to simultaneously localize three epigenomic modalities (chromatin accessibility ATAC, active marker H3K27ac, and repressive marker H3K27me3) at single-cell resolution. Its advantages include low starting material requirements (25,000-200,000 cells), 16-fold higher fragment count per cell compared to single-cell CUT&Tag, and significantly improved sensitivity. Applied to analysis of young mouse brains, it was found that simultaneous multimodal detection can distinguish more cell types/states, resolve oligodendrocyte lineage chromatin velocity and H3K27me3 repressive waves, revealing the crucial role of multimodal collaboration in cellular heterogeneity and differentiation dynamics.

nano-CUT&Tag (nano-CT) (Bartosovic M et al., 2023)

Technological Optimization and Cutting-Edge Applications

With technological advancements, CUT&Tag has been continuously optimized, giving rise to novel technologies such as single-cell CUT&Tag, spatial CUT&Tag, and long-read CUT&Tag, further expanding its application boundaries.

• Single-cell CUT&Tag: By combining with the 10x Genomics single-cell platform, it enables analysis of histone modifications or TF binding sites at the single-cell level, solving the problem that traditional bulk sequencing cannot resolve cellular heterogeneity.

• Spatial CUT&Tag: By combining with spatial transcriptomics technology, it enables in situ epigenetic analysis of tissues.

• Long-read CUT&Tag: By combining with Nanopore sequencing technology, it enables long-read epigenetic analysis, solving the problem that short-read sequencing cannot resolve complex genomic regions (such as repetitive sequences and structural variations).

Conclusion

CUT&Tag sequencing technology, as a core tool in epigenetics and gene regulation research, demonstrates irreplaceable value in multiple fields, including histone modification localization, transcription factor regulation, single-cell heterogeneity analysis, and disease mechanism exploration, due to its advantages of low starting amount, high signal-to-noise ratio, and rapid efficiency. With continuous optimization of the technology (such as single-cell, spatial, and long-read CUT&Tag), its application boundaries will be further expanded, providing a more powerful tool for precision medicine (such as tumor immunotherapy and metabolic disease treatment). In the future, CUT&Tag is expected to be combined with more omics technologies (such as RNA-seq, ATAC-seq, and proteomics) to achieve full-chain analysis of "epigenetics-gene expression-cell function," providing a more comprehensive perspective for revealing the essence of life phenomena.

References

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