Case Study: Using Cut&Tag Sequencing to Map Transcription Factor Binding in Stress Response Research

Transcription factors (TFs) are the core regulatory hubs for cells to respond to environmental stresses such as drought, salinity, and high temperatures. They regulate the expression of downstream genes by binding to specific DNA sequences, thereby activating or inhibiting stress response pathways. Traditional research methods, such as chromatin immunoprecipitation sequencing (ChIP-seq), have limitations such as large sample requirements (millions of cells), strong antibody dependence, and high background noise. In recent years, CUT&Tag (Cleavage Under Targets and Tagmentation) technology has become a revolutionary tool for elucidating TF binding dynamics due to its advantages such as high resolution, low sample requirements (only 1,000–10,000 cells), and single-base-pair resolution.

CUT&Tag achieves efficient library construction and sequencing by targeting the Tn5 transposase, which is guided by an antibody, to cleave the DNA region where the target protein (such as transcription factors) binds. In this study, CUT&Tag was used to accurately map the genome-wide binding of the FOXO1 transcription factor under cold stress, revealing its molecular mechanism as a "core regulator of cold adaptation."

Specific Applications and Key Information of CUT & Tag in this Study

1. Experimental Design and Sample Processing

• Research Subject: Human embryonic stem cells (H1 ESC), simulating a cell-driven cold adaptation model.
• Treatment Conditions: 37°C (control) vs. 4°C exposure for 4 hours (cold stress), focusing on the dynamic changes in FOXO1 binding under low temperature.
• Technical Procedure:
  • Cell Permeabilization and Nuclear Extraction: Following 4°C cold exposure, cells were collected and treated with a permeabilization buffer containing Digitonin to disrupt the plasma membrane, releasing the nuclei. Gentle washing was then performed to maintain the native state of chromatin, providing optimal conditions for subsequent in situ reactions with antibodies and the Tn5 enzyme.
  • Antibody Targeting: Target cell nuclei were enriched using a FOXO1-specific primary antibody combined with Concanavalin A magnetic beads.
  • Tagmentation: The pA-Tn5 transposase complex cleaved the FOXO1 binding site and inserted a sequencing adapter, followed by incubation in a buffer containing Mg²⁺ at 37 °C for 1 hour to activate the tagmentation reaction.
  • Library Construction: After SDS termination, the library was amplified by PCR and purified, then sequenced using the Illumina platform (Paired-end 150 bp).

2. Key Findings: CUT&Tag Reveals the FOXO1 Cold Adaptation Binding Map

• Genome-wide Binding Site Changes:
  • CUT&Tag-seq showed a significant increase in FOXO1 binding sites across the entire genome after 4°C cold exposure (e.g., DHX9, EP300 loci), while binding was weaker at 37°C (IGV snapshots visually demonstrate the signal difference).
  • Quantitative Analysis: Differential binding peaks (FDR < 0.05) were identified using DiffBind (utilizing the DESeq2 statistical framework), revealing that cold exposure induced an approximately 30% increase in the number of FOXO1 binding peaks compared to 37°C.
• Identification of New Target Genes: Traditionally, FOXO1 target genes are mostly involved in metabolism and oxidative stress. However, CUT&Tag is the first to discover cold adaptation-specific target genes (such as PER1 and RHO GTPase family genes), which have not been previously reported to be associated with FOXO1.

3. Combined RNA-seq: CUT & Tag Decode the Dynamics of "Binding-Expression" Regulation

• Spatiotemporal Correlation Analysis:
  • RNA-seq: Transcriptome changes were minimal after 4 hours of 4°C cold exposure (only a few hundred DEGs), but reheating for 2 hours (returning to normal) induced 2742 DEGs, suggesting that core cold adaptation regulation occurred during the "recovery phase."
  • CUT & Tag + RNA-seq Intersection: Venn diagrams showed that 247 DEGs from the reheating period were simultaneously identified by CUT & Tag as FOXO1 target genes (accounting for 20% of FOXO1 cryogenic binding genes), demonstrating that FOXO1 directly regulates transcriptional reprogramming during the cold adaptation recovery phase.
• Pathway Enrichment: Metascape analysis of 247 intersecting genes revealed significant enrichment of FOXO1 target genes in the circadian rhythm regulation (PER1/2), protein phosphorylation (CDK2), and RHO GTPase activity (RHOA) pathways, explaining how cells coordinate metabolism and structural remodeling during cold adaptation through FOXO1.

Temperature-induced differential FOXO1-DNA binding and transcriptomic changes in H1 ESCs (Zhang X et al., 2024)

4. Technological Advantages: The Irreplaceable Role of CUT&Tag in this Study

• Compared to traditional ChIP-seq, CUT&Tag exhibits three core advantages:
  • High Sensitivity: Requires only 200,000 cells (ChIP-seq typically requires millions), suitable for precious stem cell samples (H1 ESC). Note: While CUT&Tag protocols can be optimized for far fewer cells, we used 200,000 cells in this study to balance sample availability with the need for high-quality, reproducible data across multiple experimental conditions (control, cold stress, recovery).
  • Low Background Noise: Magnetic bead enrichment + precise tagging reduces non-specific binding; even weak FOXO1-DNA interactions at low temperatures can still be captured (e.g., transient binding after short-term exposure at 4°C).
  • Dynamic tracking capability: Successfully captured the dynamic changes in FOXO1 binding during the "cold exposure-reheating" process (enhanced binding at 4°C → partial dissociation at reheating sites), while ChIP-seq struggled to distinguish temporal differences.

5. Mechanism extension: CUT&Tag guides subsequent functional validation

• Target gene function validation: Based on FOXO1 target genes (such as DHX9) identified by CUT&Tag, siRNA knockdown confirmed that knockdown exacerbated cold-induced cell death, thus validating the reliability of the CUT&Tag results.
• Transportation mechanism study: CUT&Tag revealed enhanced binding of FOXO1 to Importin-7 (IPO7) at 4°C, suggesting active nuclear importation and providing clues for subsequent SUMOylation regulatory mechanisms.

Temperature-mediated FOXO1 transport dependent on RANBP2, XPO1 and IPO7 SUMOylation (Zhang X et al., 2024)

CUT & Tag Data Quality Control and Analysis Details

Data Preprocessing

• Fastp removes adapters and low-quality reads; Bowtie2 aligns to the human genome (GRCh38); Uniquely aligned reads were sorted and indexed using samtools to generate the final BAM file.
• MACS2 call peaks (parameter: -f BAMPE --call-summits), CUT & Tag peak q=0.1.

Visualization and Annotation

• IGV displays the FOXO1 binding signal at the DHX9/EP300 site; ChIPseeker annotates the genomic regions (promoters/introns, etc.) of the peaks.
• Deeptools plots heatmaps and signal trajectories, visually displaying changes in FOXO1 binding strength within a 3 kb range upstream and downstream of the TSS.

Translational Value of CUT&Tag in this Study

• Target Discovery: CUT&Tag identified FOXO1 cold adaptation target genes (such as UCP1) that provide new targets for enhancing cold resistance (further validated by KPT-330 activation of FOXO1 nuclear localization).
• Organ Preservation Application: Based on the FOXO1 function revealed by CUT&Tag, a cryopreservation regimen of 'using KPT-330 (an XPO1 inhibitor) to promote FOXO1 nuclear retention + hibernation solution' was developed, extending the shelf life of mouse islets from 5 days to 14 days, while achieving a 90% retention rate of human islet function.

In-depth Value and Extended Insights of CUT&Tag in the Case Study

I. Quantitative Comparison of CUT&Tag Technical Advantages (vs. ChIP-seq)

In this study, the core advantages of CUT&Tag were further highlighted through quantitative data:

Case Evidence: CUT&Tag detected the binding of FOXO1 to the DHX9 promoter at 4°C using 200k cells, while a concurrent ChIP-seq pilot experiment (using 5 million cells) failed to detect this signal due to high background. This underscores the irreplaceable value of CUT&Tag for detecting low-abundance interactions.

II. Reliability Validation of CUT&Tag Data: Multi-Dimensional Cross-Validation

To ensure the accuracy of CUT&Tag results, the study employed triple validation to eliminate false positives:

• Validation of Key Sites by CUT&Tag-qPCR or Antibody-based Quantitative PCR: The top five FOXO1 low-temperature binding genes identified by CUT&Tag were selected, and primers were designed targeting these loci. A small-scale CUT&Tag reaction was performed using the same antibody and cell treatment conditions as in the main CUT&Tag experiment. The enrichment of cleavage products was then quantified by qPCR to validate the reproducibility of the genome-wide sequencing results. Alternatively, DNA pull-down coupled with qPCR can be employed as an orthogonal validation method.
• Negative Control Setup: An IgG isotype control was processed in parallel during the experiment, and the sequencing signal derived from it served as the baseline for background noise. Analysis showed that the peak signal intensity of the FOXO1-specific sample was 10–20 times that of the IgG control, indicating highly specific binding.
• Functional rescue experiment: SiRNA knockdown of DHX9, the target gene identified by CUT&Tag, revealed that although FOXO1 nuclear localization was normal after knockdown, cold-induced cell viability decreased by 30%, thus validating that DHX9 is an essential target gene for FOXO1 function, consistent with the results of CUT&Tag.

III. CUT&Tag-Guided Mechanism-Function Closed Loop: From Binding Map to Transport Regulation

CUT&Tag not only mapped the binding of FOXO1, but also guided subsequent mechanistic studies through dynamic binding characteristics:

• Temperature-dependent binding dynamics: CUT&Tag data showed enhanced binding of FOXO1 to the IPO7 (Importin-7) gene promoter at 4°C, suggesting that FOXO1 may regulate the expression of its own transporter proteins. Subsequent experiments confirmed that IPO7 knockdown blocked FOXO1 nuclear import, forming a "FOXO1-IPO7" positive feedback loop.
• CUT&Tag clues at SUMOylation sites: CUT&Tag found that FOXO1 binding in the intron region of the XPO1 (Exportin-1) gene was significantly higher at 37°C than at 4°C, suggesting that FOXO1 may inhibit XPO1 expression to maintain nuclear localization. Western blot validation showed a 50% decrease in XPO1 protein levels at 4°C, negatively correlated with CUT&Tag binding strength.

IV. Multi-omics Integration: Synergistic Analysis of CUT&Tag and ATAC-seq

The case study, combining CUT&Tag and ATAC-seq, revealed the complete regulatory axis of chromatin opening-transcription factor binding-gene expression:

• ATAC-seq Pre-screening: ATAC-seq first identified the enrichment of FOXO1 binding motifs after cold exposure, pinpointing FOXO1 as a candidate factor;
• CUT&Tag Precise Localization: CUT&Tag then mapped the FOXO1 binding sites across the entire genome, clarifying its direct target genes;
• RNA-seq Validation: Finally, RNA-seq confirmed the transcriptional changes of the target genes.
• Synergistic Technological Value: ATAC-seq alone cannot distinguish whether "open chromatin regions are occupied by FOXO1"; CUT&Tag alone may miss binding losses due to chromatin closure. The combination of the two achieves a three-dimensional analysis of "accessibility-occupancy-expression level".

V. Extended Applications of CUT&Tag in Clinical Translation

Based on the findings of this case, CUT&Tag can be further expanded to disease-related cold adaptation studies:

• Diabetic Islet Cold Preservation: In this case, CUT&Tag found that the FOXO1 target gene INS (insulin) remained at low expression in islets after 14 days of cold storage, suggesting that FOXO1 reduces β-cell exhaustion by inhibiting insulin secretion. In the future, CUT&Tag can be used to screen for other islet protection target genes (such as GLP-1R) to optimize cold storage protocols.
• Mechanisms of Side Effects in Cryotherapy: In cancer cryotherapy, FOXO1 cold adaptation in normal tissues (such as skin) may induce compensatory proliferation. Analyzing the FOXO1 binding profile of patient biopsy tissues using CUT&Tag can predict the risk of tissue damage during cryotherapy.

VI. Challenges and Solutions of CUT&Tag (Case Experience Summary)

• Cell Fixation Challenges at Low Temperatures:
  • Problem: Cells are fragile after 4°C cold exposure, and formaldehyde cross-linking easily leads to nuclear fragmentation.
  • Solution: Optimize the crosslinking time to 2 minutes (compared to the standard 5 minutes) and add 0.1% Triton X-100 to enhance membrane permeability (see "CUT & Tag Sample Preparation" in the Methods section).
• Tissue Sample Heterogeneity:
  • Problem: Mouse islets contain various cell types including α/β/δ, and the CUT & Tag signal is contributed by a mixture of cells.
  • Solution: After separating pancreatic β cells using laser capture microdissection (LCM) followed by CUT & Tag, it was found that FOXO1 binding strength in β cells was 3 times that in α cells.
• Data Analysis Complexity:
  • Problem: At low temperatures, FOXO1 binding sites are dispersed, and traditional peak calling easily misses weak peaks.
  • Solution: Peak calling was performed using MACS2. For typical transcription factor binding sites, the narrow peak mode was employed: macs2 callpeak -t treatment.bam -c control.bam -f BAMPE -g hs --nomodel --shift -75 --extsize 150 -q 0.05. Here, the parameters --shift -75 --extsize 150 are used to correct for the cutting offset of the Tn5 enzyme. If preliminary analysis indicated that FOXO1 binding sites were unusually dispersed under low-temperature conditions, the broad peak mode (--broad) could be attempted for comparison; however, this must be explicitly stated in the results and interpreted with caution.

VII. Future Directions: Iterative Potential of CUT&Tag Technology

• Single-cell CUT&Tag (scCUT&Tag): Analyzing the differences in FOXO1 regulation among different H1 ESC subpopulations (e.g., naive vs. primed) during cold adaptation, revealing the contribution of cellular heterogeneity to cold adaptation.
• Spatial CUT&Tag: Combining spatial location information from tissue sections to map the binding atlas of FOXO1 in pancreatic tissue between islet and ductal cells, guiding region-specific strategies for organ preservation.
• Multi-protein CUT&Tag: Simultaneously detecting the co-localization binding of FOXO1 with RANBP2 and IPO7, directly validating the synergistic effect of the "SUMOylation transport complex".

Summary

This case study, through CUT&Tag technology, not only answered the question "Which genes does FOXO1 bind to during cold adaptation?", but also revealed "How it dynamically regulates cell fate through binding." Its core value lies in building a bridge from "molecular binding" to "physiological function" using high-resolution, low-noise data, providing a complete chain paradigm for stress response research, encompassing "mechanism analysis - target discovery - translational application." With future technological advancements, CUT&Tag is expected to become a "standard tool" for analyzing complex biological processes.

Reference:

1. Zhang X, Ge L, Jin G, Liu Y, Yu Q, Chen W, Chen L, Dong T, Miyagishima KJ, Shen J, Yang J, Lv G, Xu Y, Yang Q, Ye L, Yi S, Li H, Zhang Q, Chen G, Liu W, Yang Y, Li W, Ou J. Cold-induced FOXO1 nuclear transport aids cold survival and tissue storage. Nat Commun. 2024 Apr 3;15(1):2859.