Current detection methods related to epigenetics primarily include Chromatin Immunoprecipitation (ChIP), Mass Spectrometry (MS)^[1], CUT&Tag technology^[2], and others. Chromatin Immunoprecipitation, often referred to as ChIP, is a technique used to study the interactions between proteins and specific regions of genomic DNA^[3]. ChIP can selectively detect histones, histone modifications, and transcription factors, providing insights into chromatin states and gene transcription.

The first step of this technique is to fix the DNA-protein complexes. Formaldehyde is the most commonly used crosslinking agent, with the advantage of enabling tight binding between DNA and proteins while being easily reversible. Add 1% formaldehyde to the culture medium in the cell culture flask or plate. After fixation is complete, add 0.125 M glycine to terminate the reaction.

Note: The optimal conditions for formaldehyde crosslinking need to be determined through preliminary experiments or based on literature references. Typically, nucleosomal proteins crosslink more quickly, while non-histone proteins require longer incubation times. Insufficient fixation time may lead to inadequate fixation and excessively small DNA fragments, which are unfavorable for subsequent immunoprecipitation analysis. Conversely, excessively long fixation times may result in chromatin that is difficult to fragment.

The sample is subjected to multiple cycles of sonication to fragment the DNA into 100-500 bp pieces. Factors influencing sonication efficiency include sample volume, depth of the sonication probe, sonication intensity, and duration.

Note: The sample volume should generally not exceed 1 mL. If the sample is placed in a microcentrifuge tube, the probe should be inserted to a depth of at least 1 cm. Since the chromatin solution contains sodium dodecyl sulfate (SDS), it may foam, reducing sonication efficiency. Additionally, the sample must be kept on ice to maintain a low-temperature environment.

Immunoprecipitation is the most critical step in ChIP. Specific antibodies targeting the protein of interest are immobilized on magnetic beads and thoroughly mixed with the protein-DNA complexes. The mixture is incubated overnight on a magnetic rack, followed by centrifugation to remove the supernatant, yielding the protein-DNA complexes.

Note: Rabbit-derived antibodies exhibit similar binding affinity for both Protein A and Protein G, while goat-derived antibodies are more suitable for Protein G.

The magnetic beads are placed in 250 μL of 1% SDS and 0.1 M NaHCO₃ and incubated at room temperature for 15 minutes to elute the immunoprecipitated DNA. The eluted protein-DNA complexes are incubated at 68°C for 6 hours to overnight to reverse the formaldehyde crosslinking. Subsequently, they are incubated at 55°C for 50 hours with 200 mM NaCl, 10 mM EDTA (pH 8.0), 40 mM Tris-HCl (pH 6.5), and 50 μg/mL proteinase K to digest the proteins. DNA is then extracted using a phenol-chloroform-isoamyl alcohol mixture (25:24:1 ratio). To remove any residual salts and ensure the ChIP DNA is as pure as possible, the DNA is washed with pre-cooled 70% ethanol, air-dried, and resuspended in double-distilled water or TE buffer (pH 8.0).

After isolating the target DNA, various detection and quantification methods can be employed to study the isolated gene fragments. The most commonly used methods include PCR, q-PCR, DNA microarray hybridization (ChIP-chip), and high-throughput sequencing (ChIP-seq).

This study focuses on the biological functions and molecular mechanisms of METTL14 (Methyltransferase 14, N6-Adenosine-Methyltransferase Subunit) in colorectal cancer (CRC). METTL14 is a key RNA N6-adenosine methyltransferase that regulates RNA function and participates in tumor progression. The authors' previous research found that METTL14 expression is significantly downregulated in CRC tissues and is associated with poor prognosis. Additionally, the enrichment of histone H3K4 trimethylation (H3K4me3) in the promoter region of METTL14 is significantly reduced in CRC cells, while KDM5C (Lysine Demethylase 5C)-mediated demethylation of H3K4me3 suppresses METTL14 transcription. Therefore, the research team hypothesized that METTL14 may regulate CRC progression by influencing the expression of specific genes. The authors used ChIP experiments to investigate whether KDM5C directly binds to the promoter region of METTL14 and causes changes in H3K4me3 levels, thereby inhibiting METTL14 transcription.

The authors treated human colorectal cancer (CRC) cell lines HCT116 and HCT8 with a KDM5C inhibitor (KDM5A-IN-1). After treatment, CRC cells were collected, and chromatin was fragmented using sonication to generate DNA fragments of 200–500 bp. Immunoprecipitation was performed using KDM5C antibody, H3K4me3 antibody, and non-specific IgG as a negative control. The cell lysates were incubated with antibodies and Protein A/G overnight to obtain chromatin fragments specifically bound to the antibodies. DNA was then eluted and purified using a kit. qPCR was used to analyze the binding of KDM5C and H3K4me3 to the METTL14 promoter region.

The experimental results indicate that KDM5C can directly bind to the METTL14 promoter region and suppress METTL14 transcription by demethylating H3K4me3, thereby influencing the progression of CRC.

Thiostrepton induces ferroptosis in pancreatic cancer cells through STAT3/GPX4 signalling^[7].

In this study, the authors focus on the role of Thiostrepton (TST) in pancreatic cancer treatment, particularly its ability to induce ferroptosis. Ferroptosis is a novel form of programmed cell death that depends on intracellular iron and lipoxygenase, as well as Glutathione Peroxidase 4 (GPX4). GPX4 inhibits ferroptosis by converting lipid peroxides into non-toxic lipid alcohols. The researchers found that TST reduces the viability of pancreatic cancer cells and is accompanied by intracellular iron overload, reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) overexpression, and depletion of glutathione peroxidase (GSH-PX). The STAT3/GPX4 signaling pathway plays a key role in regulating ferroptosis, as STAT3 can bind to the GPX4 promoter region and promote its transcription. These findings suggest that TST may inhibit GPX4 expression by regulating STAT3. To gain a deeper understanding of how TST modulates the STAT3/GPX4 signaling pathway at the molecular level and subsequently influences ferroptosis, the researchers employed chromatin immunoprecipitation (ChIP) experiments.

In the experiment, the authors first used the JASPAR website (http://jaspar.genereg.net/) to predict potential STAT3 binding sites on the GPX4 promoter. They then conducted ChIP-qPCR experiments to validate these sites. After HEK293T cells reached 90% confluency, they were fixed using a cross-linking reagent. The cells were lysed with an SDS buffer, and DNA was fragmented into 100–500 bp pieces using sonication. Specific antibodies against STAT3 and normal mouse IgG (as a control) were used to precipitate DNA fragments bound to STAT3. After washing, elution, and reverse cross-linking of the DNA, qPCR was performed to detect the enriched sequences.

The ChIP experiment results confirmed that STAT3 directly binds to a specific region (P2) of the GPX4 promoter, but not to other predicted binding sites (P1, P3, and P4). TST promotes ferroptosis by regulating the expression and activity of STAT3, thereby affecting GPX4 transcription. This provides a new strategy for the treatment of pancreatic cancer.

Epigenetics not only helps to understand the regulatory mechanisms of gene expression, but also provides a new perspective for the prevention, diagnosis and treatment of diseases. By screening compounds that can influence specific epigenetic modifications, researchers can identify potential therapeutic targets and thereby develop new drugs!