Method for monitoring dynamic changes in histone methylation levels in live cells based on phase separation

Abstract

The application discloses a method for monitoring dynamic changes of histone methylation levels of living cells based on phase separation. First, a histone methylation probe of living cells is constructed, the methylation sensor in the probe comprises a short peptide containing a methylation site H3K9, a fluorescent protein and an oligomerization short peptide I, the phase separation reporter comprises a chromatin binding domain of Drosophila HP1 alpha and an oligomerization short peptide II, when the H3K9 site is modified by trimethylation, the chromatin binding domain of Drosophila HP1 alpha is recognized and combined, under the action of the oligomerization short peptides I and II, the methylation sensor and the phase separation reporter are aggregated into a multimer, and phase separation droplets are generated. The probe can be used for real-time and accurate quantitative detection of dynamic changes of histone methylation levels of living cells, has the characteristics of high signal-to-background ratio, strong specificity, good universality and good biocompatibility, and can be widely applied to quantitative detection of changes of cell methylation levels caused by diseases, aging, drugs or gene editing, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for detecting dynamic changes in histone methylation levels in living cells based on fluorescence microscopy and its application. Background Technology

[0002] Mammalian chromatin possesses a highly complex hierarchical structure and diverse dynamic processes to control complex life activities. The highly compressed chromatin makes it difficult for various regulatory factors to access DNA, hindering processes such as transcription and DNA replication. Therefore, rapid changes in chromatin structure are required for various cellular life activities, often accompanied by histone post-translational modifications. Histones exhibit various types of post-translational modifications, including methylation, phosphorylation, and acetylation. Among these, methylation is the most studied type; all histones can be methylated, and a single histone often has multiple methylation sites. Histones play an irreplaceable role in heterochromatin formation, DNA damage repair, and transcriptional regulation, and are closely related to various diseases such as cancer, heavy metal poisoning, and neurodegenerative diseases. Many histone methyltransferases and demethylases have become popular targets for anticancer drugs. Cellular histone methylation levels are constantly changing; therefore, developing a method to detect the dynamics of histone methylation in living cells is essential for studying the functional mechanisms of histone methylation in various life processes and for developing anticancer drugs.

[0003] Current techniques for detecting intracellular methylation levels include immunofluorescence, polyacrylamide gel electrophoresis, chromatin immunoprecipitation, and mass spectrometry. However, these methods are limited by the requirement for cell fixation or lysis, thus preventing real-time monitoring of histone post-translational modifications in live cells. Therefore, developing tools for detecting histone post-translational modifications in live cells is essential. Researchers have developed probes capable of detecting histone methylation, acetylation, and phosphorylation in live cells, which can be categorized into three main types based on their detection principles: those based on FRET principles, those based on fluorescent protein technology, and those based on antigen-antibody recognition.

[0004] Probes based on the FRET principle for detecting post-translational modifications of histones in live cells have been applied in various scenarios. One end of the probe is a histone-containing peptide responsible for sensing methyltransferase activity, while the other end's domain can bind to methylated histones. The middle portion of the probe contains two fluorescent proteins capable of FRET. When the corresponding methyltransferases are overexpressed in cells, an increase in the FRET signal can be observed. However, due to the limited range of changes in intracellular histone post-translational modifications, the amplitude of the FRET signal change is also insufficient, affecting the probe's sensitivity and accuracy. This has prevented FRET-based histone post-translational modification probes from being used in live animal imaging, as animal tissues scatter and absorb light, requiring a higher amplitude of signal change from the probe. This also indicates that current methods for detecting histone post-translational modifications still have room for improvement.

[0005] Besides probes designed based on the FRET principle, some fluorescent protein techniques can also be used to detect histone modifications, such as bimolecular fluorescence complementation (BiFC). These methods typically involve cleaving a fluorescent protein into two parts, each fused to one of two proteins. If these proteins interact and bind, the two segments of the fluorescent protein will reassemble into a single, complete fluorescent protein. However, since most fluorescent proteins do not separate after assembly, the BiFC system is irreversible and cannot accurately reflect the dynamics of histone post-translational modifications in real time.

[0006] The last method is based on antigen-binding fragments (Fab) for detecting histone modifications. This approach involves obtaining purified antibodies, preparing Fab via protease digestion, labeling with a fluorescent dye, and finally injecting it into live cells using microinjection. Compared to whole IgG molecules, Fab has the advantages of smaller size, weaker binding force, and less interference with normal cellular function. However, the Fab technique also has significant drawbacks, such as a very complex preparation process, difficult microinjection, and considerable cell damage. Furthermore, exogenous proteins have a short survival time within cells, which is unfavorable for long-term imaging. While using genetically encoded Fab can address this issue relatively well, another drawback is that this strategy cannot perform quantitative analysis because the total fluorescence intensity in the cell nucleus remains unchanged regardless of whether the Fab binds to endogenous histone methylation sites.

[0007] Therefore, in order to improve the sensitivity and accuracy of detecting histone methylation levels in living cells and to advance the development of related drugs, there is an urgent need in this field to develop a method that can detect the dynamic changes in histone methylation in living cells. Summary of the Invention

[0008] To overcome the shortcomings of existing technologies, this invention provides a method for detecting dynamic changes in intracellular methylation levels with high signal-to-background ratio, strong specificity, universality, and good biocompatibility. Based on the principle of phase separation, this method accurately and quantitatively detects the dynamic changes in histone methylation levels in live cells in real time, offering numerous advantages: First, the phase-separating droplet significantly increases the probe concentration within the droplet due to the phase separation principle, resulting in a high signal-to-background ratio, facilitating identification and statistical analysis, and making it suitable for in vivo animal imaging. Second, the probe exhibits high specificity; when methylation sites are mutated or intracellular histone methylation levels decrease, the number of phase-separating droplets decreases or even disappears, and the number of phase-separating droplets is positively correlated with the intracellular histone methylation level, while the methylation level at other sites does not affect the number of phase-separating droplets. Third, the probe has good universality, exhibiting consistent performance across different species and cell lines. Fourth, the probe has good biocompatibility, is non-toxic to cells, and does not affect cell proliferation rate.

[0009] The method for monitoring the dynamic changes in histone methylation levels in living cells based on phase separation provided in this invention is referred to as 5K9 in this paper, and the probe used is called the 5K9 probe.

[0010] In a first aspect of the invention, a gene-encoded fluorescent probe is provided for real-time monitoring of dynamic changes in histone methylation levels in living cells based on the principle of phase separation. This probe mainly consists of two parts: a methylation sensor and a phase separation reporter. The methylation sensor of the probe (see...) Figure 1 The methylation reaction substrate on the N-fragment of histone H3 is a short peptide (SEQ ID No: 1) truncated from the N-terminus of histone H3, containing the methylation site H3K9 of histone H3. The phase separation reporter of the probe (see...) Figure 1 The C fragment in the probe utilizes the chromatin-binding domain (SEQ ID No: 2) of Drosophila HP1α, with an equilibrium dissociation constant of 10 μM for the H3K9me3 peptide. To enhance probe flexibility and facilitate phase separation, the full-length HP1 sequence is not used. Furthermore, artificially designed oligomeric short peptides I and II (e.g., Hotag3 and Hotag6) are linked to the methylation sensor and phase separation reporter, respectively. These two short peptides can aggregate into multimers. When the intracellular methyltransferase content increases, the methylation sensor of the probe is methylated and specifically binds to the phase separation reporter, thereby generating phase-separated droplets. Conversely, when the intracellular demethylase content increases, the methylation sensor of the probe is demethylated, the phase separation reporter sequence no longer binds to the methylation sensor, and the phase separation droplets gradually disappear. For details on the principle, see [link to documentation]. Figure 1 .

[0011] To verify the effectiveness of the method for dynamically changing histone methylation levels in live cells according to the present invention, intracellular histone methylation levels were first altered using chemical reagents that affect methylation and demethylation. In the embodiments of the present invention, H3K9me3 levels were increased by inhibiting the activity of demethylases using the drugs IOX1 and ciclopirox, respectively, and H3K9me3 levels were decreased by inhibiting the activity of methyltransferases using the drug chaetocin. Changes in phase separation levels were then dynamically observed. However, the present invention does not limit any method of influencing H3K9me3 levels.

[0012] The live-cell histone methylation probe consists of two parts: a methylation sensor and a phase-separation reporter. The two fragments are expressed on the same plasmid and linked by a flexible amino acid linker P2A sequence. The P2A sequence can also be replaced with any flexible amino acid linker such as scFv or Myc. After protein expression, the protein self-cleaves into two segments, with the total length of the entire probe gene not exceeding 1800 bp. To improve P2A cleavage efficiency and prevent self-aggregation, GSG tripeptide sequences are added to both ends of P2A. In one embodiment of the invention, the N and C fragments contain c-Myc and SV40 nuclear localization sequences, respectively, AAKRVKLD (SEQ ID No: 9) and PKKKRKV (SEQ ID No: 10). These two nuclear localization sequences can be interchanged, or the same nuclear localization sequence can be used, or they can be replaced with any other peptide with nuclear localization function to ensure probe enrichment in the cell nucleus. Hotag3 and Hotag6 are artificially designed short peptides with sequences GEIAKSLKEIAKSLKEIAWSLKEIAKSLKG (SEQ ID No: 3) and TLREIEELLRKIIEDSVRSVAELEDIEKWLKKI (SEQ ID No: 4), respectively, with lengths of 30 and 33 amino acids. Hotag3 spontaneously forms a hexamer in cells, while Hotag6 spontaneously forms a tetramer. Expression of Hotag3 or Hotag6 alone is insufficient to induce phase separation, but phase separation can occur if the two peptides bind together due to multivalent interactions. The positions of these two peptides can be interchanged, or they can be replaced by other self-polymerizing amino acid sequences.

[0013] The biocompatibility and phase-separation ability of the two peptides have been verified. The methylation substrate in the probe is a short peptide cleaved from the N-terminus of histone H3, containing the H3K9 site, and its peptide sequence is ARTAQTAR. K STGG (SEQ ID No: 1) has its H3K4 lysine site mutated to alanine compared to the endogenous H3 peptide to eliminate interference from H3K4 methylation.

[0014] The methylation sensor of the probe is specifically composed of the following components: First, to improve the sensitivity of the probe, 3 to 8 identical peptide sequences containing the H3K9 site are tandemly linked at the N-terminus of the probe. The higher the number of tandem peptides, the more obvious the phase separation phenomenon. More than eight repetitive peptides will affect the normal function of the cell itself. These sequences are linked sequentially by peptide sequences composed of three to four flexible amino acids (proline and serine), and then linked to EGFP (enhanced green fluorescent protein) by a peptide sequence (e.g., GGLESGLRSGSGGGDI, SEQ ID No: 6). EGFP can be replaced by any other fluorescent protein, such as mcherry, DsRED, mStrawberry, mOrange, and mCitrine. Then, Hotag3 and the nuclear localization sequence SV40 are linked by an adapter sequence with the sequence GSGSAGGSAGGSAGGSAGGSAGGSAGGSR (SEQ ID No: 7). This adapter sequence can also be replaced by other sequences composed of flexible amino acids. The above components constitute all the elements of the methylation sensor of the live cell histone methylation probe. The sequence of the methylation sensor is connected to the sequence of the phase separation reporter via the P2A sequence, which is ATNFSLKQAGDVEENPGP (SEQ ID No: 5). The P2A sequence can be replaced with any other flexible linker.

[0015] In the phase separation reporter element of the probe of the present invention, the chromatin binding domain of Drosophila HP1α is used to identify and bind the trimethylated H3 peptide. The equilibrium dissociation constant KD for the trimethylated peptide H3K9me3 is 10 μM, but it has no affinity for the monomethylated peptide H3K9me1 and the dimethylated peptide H3K9me2. To simplify the probe structure, increase its flexibility to facilitate phase separation, and reduce interference from exogenously expressed HP1 on normal cellular function, the full-length HP1 protein is not used here. The peptide sequence employed is EEEYAVEKIIDRRVRKGKVEYYLKWKGYPETENTWEPENNLDCQDLIQQYEASRKD (SEQ ID No: 2). Finally, a flexible peptide linker connects Hotag6 and the nuclear localization sequence SV40. The amino acid sequence of this flexible peptide linker is GSGSAGGSAGGSAGGSAGGSAGGSAGGSAGGSR (SEQ ID No: 8). This linker sequence can also be replaced by other flexible amino acid sequences. These are all the components of the phase separation reporter of the live-cell histone methylation probe.

[0016] The expression cassette of the above-mentioned live cell histone methylation probe, as well as the vector, host bacteria or cell line containing the encoding gene of the live cell histone methylation probe, are all within the protection scope of this invention.

[0017] Therefore, this invention provides a method for detecting dynamic changes in intracellular histone methylation levels with high signal-to-backflow ratio, strong specificity, good universality and biocompatibility. See the flowchart for details. Figure 1 For any changes in H3K9me3 levels caused by any factor, quantitative detection can be performed using the following steps:

[0018] (1) Construct a plasmid expressing the live cell histone methylation probe, and transfect the cell with the live cell histone methylation probe plasmid by lentivirus method to express the probe in the cell.

[0019] (2) Cells expressing fluorescent proteins were sorted by flow cytometry and cultured. Cell lines with different expression levels were sorted according to fluorescence intensity to avoid affecting the expression level of the probe in the cell due to different amounts of transfected plasmids in the cell, which in turn affects the degree of phase separation.

[0020] (3) For the selected cell line, change the methylation level in the cell and observe and photograph the changes in the number and size of phase separation droplets in the cell under different conditions.

[0021] (4) Count the number of phase-separated droplets in the cell, among which the number of small droplets can reflect the changes in the methylation level in the cell.

[0022] Preferably, in step (4), ImageJ is used to count the number of phase-separated droplets in the cell. First, the microscopic image is converted into an 8-bit format, then a threshold is set to distinguish the droplets from the fluorescent background in the cell nucleus, and finally, large droplets are distinguished from small droplets according to their diameter, and the number of small droplets is counted. Droplets with a diameter greater than 1.5 micrometers are classified as large droplets. The reason why the number of large droplets is not counted is that large droplets in the cell nucleus are often located around the nucleolus. Since the nucleolus is rich in heterochromatin, endogenous HP1 forms aggregates here, recruiting a large number of methylation sensors of probes, which easily form large droplets. The generation of these large droplets cannot reflect the dynamic changes in the methylation level in the cell.

[0023] The beneficial effects of this invention include:

[0024] (1) This invention develops a live cell histone methylation probe based on the principle of phase separation. This probe reflects the histone methylation state in live cells through the formation and depolymerization of phase-separated droplets. The probe has strong identifiability, a high signal-to-background ratio of phase-separated droplets, and the number of droplets is easy to count.

[0025] (2) The probe of the present invention has good specificity. The phase separation of the probe depends on the trimethylation of the H3K9 site of the methylation sensor and is not affected by the methylation or acetylation level of other sites.

[0026] (3) The probe of this invention has good universality. The live-cell histone methylation probe is highly universal, exhibiting good performance in cell lines of other species besides HeLa cells. Phase separation can occur within a wide concentration range. Finally, the probe has good biocompatibility. It is non-toxic to cells, does not cause apoptosis or necrosis in the short term, and does not affect cell viability. Long-term culture does not affect cell proliferation rate. Therefore, compared to probes based on the FRET principle for detecting live-cell histone post-translational modifications, it has higher sensitivity; compared to probes designed based on the BiFC principle, it can reflect the kinetic state of histone post-translational modifications in real time; and compared to Fab-based detection methods, it can perform long-term detection and quantitative analysis of cells. This probe is widely applicable for quantitatively detecting changes in cell methylation levels caused by various factors such as disease, aging, drugs, or gene editing, and has a very broad application prospect in molecular biology and clinical diagnosis. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the principle of the histone methylation probe for living cells of the present invention, wherein: A is a schematic diagram of the structure of each part of the methylation probe; B is a schematic diagram of the formation and depolymerization principle of phase separation droplets.

[0028] Figure 2 The results of the verification experiment on the specificity of the 5K9 probe in Example 2 of this invention are as follows: A shows the distribution of H3K9me3 in HeLa cells expressing the 5K9 probe before and after chaetocin treatment, detected by immunofluorescence; B shows the quantitative results of intracellular H3K9me3 levels in A, N=15; C shows the response of 5K9 to different histone methylation and acetylation agonists. Scale bar: 10 micrometers.

[0029] Figure 3 This is the result of a verification experiment on the effect of the 5K9 probe on normal cell function in Example 2 of the present invention. A shows a scatter plot of flow cytometry analysis of cells in each experimental group after apoptosis staining. 10,000 cells were analyzed in each group. The positive control group had a puromycin concentration of 1 μg / mL (below the median lethal concentration). B compares the apoptosis and necrosis rates of cells in each group. C shows the cell viability of each group analyzed by the CCK-8 assay. D shows the effect of the 5K9 probe on cell proliferation rate. Scale bar: 10 micrometers.

[0030] Figure 4This is an example of observing the effect of phase-separated droplets using different magnification objectives in Example 2 of the present invention. A shows the imaging effect of the same cell under different magnification objectives using a laser confocal microscope; B shows the fluorescence distribution of the corresponding microscopic images under different objectives in A. Scale bar: 10 micrometers. Detailed Implementation

[0031] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0032] Example 1.5 Construction of K9 probe plasmid.

[0033] (I) Materials

[0034] 1. Trans5α competent cells

[0035] 2. High-fidelity DNA polymerase

[0036] (II) Steps

[0037] The methylation sensor and phase separation reporter components of the 5K9 probe of this invention are mainly constructed using the Gibson ligation method to build the plasmid. Specific steps include:

[0038] 1. DNA Fragment Amplification. Design suitable primers for amplifying the target gene. The Tm value of the primer binding to the template region is generally between 60-66℃. Add a 15-25 bp homologous fragment to the template binding region for subsequent homologous recombination. Based on experience, for amplifying DNA fragments shorter than 4kb, use the Phanta Max Super-Fidelity DNA Polymerase enzyme system. The amplification program is as follows: pre-denaturation at 95℃ for 30 seconds, followed by 35 cycles of denaturation at 95℃ for 15 seconds, annealing at 55-65℃ for 15 seconds, and extension at 72℃ for a few seconds. Perform a final extension at 72℃ for 5 minutes, and then cool to room temperature.

[0039] In some cases, it is necessary to amplify long DNA fragments (>4kb). For example, if the vector lacks specific restriction enzyme sites, only the entire vector can be amplified. To ensure efficiency, 2×Phanta Flash Master Mix can be used for amplification. The reaction procedure is as follows: 98℃ pre-denaturation for 30 seconds, cycling stage: 98℃ denaturation for 10 seconds, annealing for 5 seconds, extension at 72℃ for several seconds, for 35 cycles, followed by a 72℃ extension for 1 minute, and finally cooling to room temperature.

[0040] For ultrashort gene fragments (less than 100 bp), a pair of complementary DNA single strands are directly synthesized, followed by denaturation and annealing to form double strands. The annealing program is as follows: heat at 95°C for 1 minute, then slowly cool at a rate of 5°C per minute until reaching 25°C. The reaction product can be used directly for subsequent homologous recombination reactions, but it needs to be diluted to an appropriate concentration.

[0041] In the DNA fragment digestion step, digestion at 37°C for 1 hour is sufficient to ensure the digestion effect.

[0042] 2. Agarose gel electrophoresis is used to examine the amplification effect of the target fragment, including fragment length and specificity. DNA is then recovered using the appropriate kit. Care should be taken to avoid ethanol residue in this step, as it may reduce the efficiency of subsequent DNA ligation.

[0043] 3. Recombinase ligation. The amount of each DNA fragment used should be roughly calculated using this formula: Optimal amount = [0.02 × number of base pairs per fragment] ng. To ensure accurate loading, if the concentration of the recovered DNA product is too high, it can be diluted in advance to ensure that the loading volume of each component is not less than 0.5 μL. The homologous recombination reaction system should be prepared on ice. Incubate at 50°C for 15 minutes. The product is used for subsequent transformation of competent cells. For homologous recombination reactions involving more than four fragments, to improve the ligation success rate, a non-ligase-dependent multi-fragment one-step cloning kit should be used. After preparing the reaction system, incubate at 37°C for 30 minutes to complete the reaction.

[0044] 4. The product from the homologous recombination reaction was added to Escherichia coli Trans5α competent cells for heat shock transformation to obtain a monoclonal strain.

[0045] 5. Select single-clone colonies for colony PCR verification.

[0046] 6. Culture bacteria, extract plasmids, and perform sequencing verification.

[0047] Example 2.5: Relative quantitative characterization of H3K9me3 levels in HeLa cells using K9 probes.

[0048] (I) Materials

[0049] 1. Cell line: HeLa cells

[0050] 2. Opti-MEM is a serum-free culture medium used as a reagent for cell transfection.

[0051] 3. Chaetocin is an inhibitor of SUV39H1 / 2 and is the most commonly used drug in the field of epigenetics to reduce H3K9me3 levels. In this experiment, the working concentration of chaetocin was 50 nM, which is higher than its half-maximal inhibitory concentration (IC50).

[0052] 4. IOX1 is an inhibitor of H3K9me3 demethylases KDM3 and KDM4.

[0053] 5. Ciclopirox is an inhibitor of various H3K9me3 demethylases, including KDM4B.

[0054] 6. The CCK-8 assay kit contains WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt). In the presence of an electron carrier, WST-8 is oxidized and reduced by intracellular dehydrogenases to produce a water-soluble orange-yellow formazan dye that dissolves in tissue culture medium. The amount of formazan produced is directly proportional to the number of viable cells. The CCK-8 method is a highly sensitive, non-radioactive assay used to determine the number of viable cells in cell proliferation or toxicity experiments.

[0055] Color detection method

[0056] 7. Annexin V-Alexa Fluor 647, used to detect apoptosis.

[0057] (II) Steps

[0058] 1. Construction of cell lines using lentivirus method

[0059] First, seed healthy HeLa cells onto six-well plates and culture them at a density of 60%-80% for transfection. Replace the medium with fresh medium before transfection and continue culturing for 5 hours. Add PsPAX2, pMD2.G, and the target plasmid to 200 μL of opti-MEM medium. Add Neofect transfection reagent at a 1:1 ratio and mix thoroughly. Incubate at room temperature for 15 minutes. Add the transfection solution to the HeLa cells and gently shake to mix. Continue culturing for 18 hours and check for successful transfection under a fluorescence microscope. Replace with 3 mL of fresh DMEM medium. After culturing for 48 hours, collect the supernatant and centrifuge at 500×g for 5 minutes. Filter through a 0.45 μm filter to obtain the viral solution. Generally, the viral solution does not need to be concentrated. Mix the viral solution with fresh medium at a 1:2 volume ratio and add it to the cells to be infected, culturing for at least 12 hours. Replace with fresh medium and continue culturing for at least 72 hours. Cells expressing fluorescent proteins were sorted by flow cytometry and cultured further. Cell lines with different expression levels were also sorted based on fluorescence.

[0060] 2. Cellular drug stimulation

[0061] To further verify the probe's specificity, intracellular H3K9me3 levels were altered by drug stimulation, and changes in probe distribution were observed. L-poly-L-lysine solution (0.1 mg / mL) was prepared in advance. The poly-L-lysine solution was spread on a 35 mm glass dish and incubated at 37°C for 20 minutes. The solution was discarded, and any remaining solution was aspirated from the dish. The dish was then placed on a cell culture bench for 10 minutes to allow it to dry completely. Cells were seeded at a density below 40%. Cells were cultured for at least 12 hours until they adhered firmly. Different concentrations of drug-containing culture media, such as chaetocin, ciclopirox, DMSO, etc., were prepared in EP tubes and thoroughly mixed. The culture medium in the dish was discarded, and the drug-containing medium was immediately added. Cells were continued to culture in an incubator for the specified time according to experimental requirements. Cell samples were directly imaged using a microscope.

[0062] from Figure 2 As shown in Figure A, after treatment with 25 nM chaetocin, the small droplets in the nucleus of HeLa cells expressing the 5K9 probe completely disappeared. Maintaining consistent experimental conditions, immunofluorescence was used to compare the H3K9me3 levels in cells before and after chaetocin treatment. The fluorescence intensity per unit area of ​​each cell was used to reflect the H3K9me3 level. Figure 2 As can be seen from Figure B, compared with the DMSO-treated group, the overall H3K9me3 level of cells treated with chaetocin decreased by approximately 40%. Figure 2 As can be seen from the data in Figure C, for the 5K9 probe, treatment with UNC1999 to reduce the level of intracellular H3K27me3, or TSA to increase the level of intracellular histone acetylation, has no effect on the phase separation of the 5K9 probe.

[0063] 3. Apoptosis rate detection

[0064] As gene-encoded live-cell probes, one of the fundamental principles is that they should not affect normal cellular function; otherwise, their use would be severely limited. Because the H3 peptide of the 5K9 probe is almost identical to the endogenous histone sequence and expressed at high levels, it interacts with endogenous proteins such as HP1, potentially interfering with normal intracellular signaling pathways. Therefore, it is necessary to investigate the effect of the 5K9 probe on normal cellular function. Several detectable indicators exist, including the proportion of apoptosis and necrosis, cell viability, and cell growth rate. First, a comparison of apoptosis rates will be conducted.

[0065] The proportion of apoptotic cells is an important indicator for evaluating cell health. Many harmful factors, such as ultraviolet radiation, hypoxia, and microbial contamination, can lead to apoptosis. The widely used Annexin V staining technique is used to detect apoptosis. In early apoptotic cells, the phosphatidylserine residue on the inner membrane flips outward, allowing Annexin V to bind. In late apoptotic and necrotic cells, the staining is permeable to the cellular dye PI. Double staining with Annexin V and PI can analyze the proportion of apoptotic and necrotic cells.

[0066] First, seed the cells in a culture dish, ensuring they adhere firmly. Digest the cells with EDTA-free trypsin. The digestion time should not be too long to avoid false positives, nor too short to prevent cell damage during pipetting before the cells detach from the culture dish. For HeLa cells, digestion time is 3 minutes. Centrifuge the cell suspension at 300×g for 5 minutes and collect the cells. Wash the cells once with PBS and centrifuge at 300×g, 4°C for 5 minutes. Add 250 μL of...

[0067] Resuspend cells in 1× Binding Buffer and dilute to a cell density of approximately 1×10⁻⁶. 6 Cells / mL. Take 100 μL of cell suspension in an EP tube, add 5 μL of Annexin V-Alexa Fluor 647, and mix gently. Incubate at room temperature for 15 minutes in the dark. Wash again with 1× Binding buffer, and centrifuge at 300×g for 5 minutes at 4°C. Add 400 μL of Binding Buffer and mix well. Analyze using flow cytometry within one hour. Add PI staining solution before flow cytometry analysis, mix well, and then perform flow cytometry analysis. Throughout the experiment, be sure to keep the cells gently pipetting to prevent damage. Analyze using software such as Flowjo, and plot a scatter plot with Alexa Fluor 647 on the x-axis and PI on the y-axis. Data from 10,000 cells were collected for each sample.

[0068] from Figure 3 As shown in Figure A, to verify the reliability of the experiment, the positive control group was treated with a low dose of puromycin. Flow cytometry analysis revealed that after 24 hours of puromycin treatment, the cell death and apoptosis rate in the positive control group reached 16%, while the apoptosis and necrosis rates in the 5K9-expressing cell line and the wild-type HeLa cell line were both less than 3%. Figure 3 As can be seen from B, there is no statistically significant difference. Therefore, it can be concluded that the expression...

[0069] 5K9 does not increase the proportion of apoptosis or necrosis.

[0070] 4. Comparison of cell viability

[0071] Cell viability was assessed using the CCK-8 assay, a common method for evaluating cell viability. In this assay, colorless WST-8 is reduced by mitochondrial dehydrogenases to a highly water-soluble orange-yellow formazan product, with a maximum absorption wavelength of 450 nm, which can be read using a microplate reader. Higher cell viability indicates stronger mitochondrial function, enabling the conversion of more WST-8 to formazan, resulting in a higher absorbance value. For a given cell number, the absorbance of the solution after the reaction is directly proportional to cell viability.

[0072] The specific experimental procedure begins by seeding the cell lines to be compared in culture dishes, ensuring they are in good condition and that the cell numbers are roughly similar. Cells are then digested and collected, and resuspended in culture medium. Cell density is calculated using a cell counting chamber. The cell suspension is diluted to the same density. Cells are then seeded into 96-well plates, with approximately 20,000 cells added to each well, maintaining a consistent cell count across wells. Fresh culture medium is added to bring the total volume to 100 μL per well. Cells are incubated in a cell culture incubator for 12-18 hours to allow for full cell adhesion. 10 μL of CCK-8 solution is gently added to each well along the well wall, gently agitating to avoid air bubbles. Incubation continues for 1-4 hours. Finally, the absorbance at 450 nm is measured using a microplate reader. The absorbance value represents cell viability.

[0073] from Figure 3 As shown in Figure C, HeLa cells treated with puromycin were still used as the positive control group in the CCK-8 experiment. According to the statistical results, the viability of HeLa cells in the positive control group decreased significantly by about 20%, while there was no statistically significant difference in viability between the cell line expressing 5K9 and wild-type HeLa cells. Therefore, it can be concluded that expressing the 5K9 probe does not affect cell viability.

[0074] 5. Comparison of cell proliferation rates

[0075] Cell proliferation rate can be used to assess the impact of probe expression on long-term cell function.

[0076] Cell lines expressing the 5K9 probe were cultured with wild-type HeLa cells under the same conditions for one week. After resuspending the cells, 80 μL of cells were placed in the grooves of a cell counting chamber and observed and counted under a microscope at 10x magnification. The cell counting chamber has a square grid area divided into 9 square cells. The number of cells in the four corner square cells was counted. During counting, some cells may be located on the boundary lines of the squares. It was uniformly stipulated that cells on the top and left boundaries were included in the count, while cells on the bottom and right boundaries were excluded. The average number N was obtained from the four values, and the cell density was N × 10⁻⁶. 4 10 cells / mL. 4The cells were reseeded into 12-well plates and cultured, and the count was repeated the next day. To calculate the cumulative cell doubling number, the total number of cells on the second day was divided by the number of cells seeded the previous day, and then the logarithm of 2 was taken to obtain the doubling number. The cumulative doubling numbers for each day were accumulated to obtain the cumulative doubling number (CPD), from which a cell doubling curve could be plotted.

[0077] The cell doubling curve shows that after expressing 5K9, within one week, the cell proliferation rate increased significantly. Figure 3 As can be seen from Figure D, there was no statistically significant difference in the growth rate of the cell line compared to wild-type HeLa cells. Therefore, it can be determined that, in the long term, expression of the 5K9 probe has no effect on cell proliferation.

[0078] 6. Observe the effect of phase separation droplets using objective lenses of different magnifications.

[0079] In microscopy, high-magnification objectives allow for the observation of finer structures, but offer a smaller field of view. Low-magnification objectives provide a larger field of view, but have limited resolution. When using laser confocal microscopy for live animal imaging, the highest magnification objectives are often unavailable due to limitations in objective working distance. Even for some live animals, such as nematodes, where high-magnification objectives are possible, researchers often prefer to observe multiple cells with a larger field of view using low-magnification objectives to obtain more information. Therefore, it is necessary to test the imaging performance of phase-separation probes under different magnification objectives. For the same cell, 20x, 40x, 63x, and 100x objectives were used for observation, and the fluorescence signal-to-background ratio of the droplets was analyzed. When using 63× and 100× oil immersion lenses (numerical aperture is 100%)...

[0080] 1.4) During imaging, from Figure 4 As can be seen in image A, small droplets are clearly visible, and the signal-to-background ratio (STR) reaches approximately 4. When using a 40× water microscope (numerical aperture 1.2), the numerical aperture of the objective lens decreases, reducing the microscope resolution, but the STR for small droplets remains around 2-3. Figure 4 As can be seen from B, it remains clearly distinguishable. However, if the objective magnification is reduced to 20x, the fluorescence signal-to-background ratio and resolution decrease significantly, making it difficult to distinguish small droplets. In conclusion, when imaging droplets on a 5K9 probe using a laser confocal microscope, from... Figure 4 As can be seen from point A, the objective lens magnification is at least 40x. This provides guidance for the subsequent application of the probe.

Claims

1. A live-cell histone methylation probe, comprising a methylation sensor and a phase separation reporter, wherein the methylation sensor includes a short peptide containing a methylation site H3K9 cleaved from the N-terminus of histone H3, as well as a fluorescent protein and an oligomeric short peptide I; the phase separation reporter includes a chromatin-binding domain of Drosophila HP1α and an oligomeric short peptide II; wherein, The sequence of the short peptide containing the methylation site H3K9 is ARTAQTARKSTGG, and the sequence of the chromatin-binding domain of the Drosophila HP1α is EEEYAVEKIIDRRVRKGKVEYYLKWKGYPETENTWEPENNLDCQDLIQQYEASRKD. When the methylation site H3K9 in the methylation sensor is trimethylated, it is recognized and bound by the chromatin-binding domain of the Drosophila HP1α in the phase separation reporter. Under the action of oligomerized short peptides I and II, the methylation sensor and the phase separation reporter aggregate into a polymer, generating phase-separated droplets.

2. The live-cell histone methylation probe as described in claim 1, characterized in that, The oligopeptide I is Hotag3, and the oligopeptide II is Hotag6; or, the oligopeptide I is Hotag6, and the oligopeptide II is Hotag3; wherein: the sequence of Hotag3 is GEIAKSLKEIAKSLKEIAWSLKEIAKSLKG, and the sequence of Hotag6 is TLREIEELLRKIIEDSVRSVAELEDIEKWLKKI.

3. The live-cell histone methylation probe as described in claim 1, characterized in that, The fluorescent protein is selected from one of EGFP, mcherry, DsRED, mStrawberry, mOrange, and mCitrine.

4. The live-cell histone methylation probe as described in claim 1, characterized in that, The methylation sensor, from N-terminus to C-terminus, consists of 3 to 8 tandem segments of a short peptide containing the methylation site H3K9, a flexible peptide, a fluorescent protein, a flexible linker sequence, an oligomerized short peptide I, and a nuclear localization sequence; the phase separation reporter, from N-terminus to C-terminus, consists of a chromatin-binding domain of Drosophila HP1α, a flexible linker sequence, an oligomerized short peptide II, and a nuclear localization sequence.

5. The live-cell histone methylation probe as described in claim 4, characterized in that, The methylation sensor and phase separation reporter are expressed on the same plasmid and are connected by a flexible amino acid linker sequence.

6. The live-cell histone methylation probe as described in claim 5, characterized in that, The amino acid linker sequences connecting the methylation sensor and the phase separation reporter were selected from P2A, scFv, and Myc, with the P2A sequence being ATNFSLKQAGDVEENPGP.

7. The live-cell histone methylation probe as described in claim 4, characterized in that, The short peptides containing the methylation site H3K9 are linked by three to four flexible amino acids; the sequence of the flexible peptide linking the short peptide containing the methylation site H3K9 to the fluorescent protein is GGLESGLRSGSGGGDI; the flexible linker sequence between the fluorescent protein and the oligomeric short peptide I is GSGSAGGSAGGSAGGSAGGSAGGSAGGSR; the flexible linker sequence between the chromatin-binding domain of Drosophila HP1α and the oligomeric short peptide II is GSGSAGGSAGGSAGGSAGGSAGGSAGGSAGGSR; the nuclear localization sequence is selected from c-Myc and SV40, wherein the sequence of c-Myc is AAKRVKLD and the sequence of SV40 is PKKKRKV.

8. The expression cassette of the live cell histone methylation probe according to any one of claims 1 to 7, comprising a vector, host bacterium, or cell line containing the encoding gene of the live cell histone methylation probe.

9. A method for monitoring dynamic changes in histone methylation levels in living cells, comprising the following steps: 1) Construct a plasmid expressing any of the live cell histone methylation probes according to claims 1 to 7, and transfect cells with the live cell histone methylation probe plasmids by lentivirus method to express the probes in cells; 2) Select cells expressing fluorescent proteins for further culture, and sort cell lines with different expression levels according to fluorescence intensity; 3) For the selected cell line, observe and photograph images of the changes in the number and size of phase-separated droplets in the cells under different conditions; 4) Count the number of phase-separated droplets in the cell, where the number of small droplets reflects the change in the methylation level in the cell.

10. The method as described in claim 9, characterized in that, Step 4) Use ImageJ to count the number of phase-separated droplets in the cell. First, convert the microscopic image into 8-bit format, then set a threshold to distinguish the droplets from the fluorescent background in the cell nucleus, and count the number of small droplets with a diameter ≤ 1.5 micrometers.

Images