A model of transcription factor sall4 recruiting and concentrating nuRD complex in the nucleus and application thereof

Abstract

The present application relates to a model for recruiting and concentrating NuRD complex in the nucleus by transcription factor SALL4 and application. The method for constructing the model for recruiting and concentrating NuRD complex in the nucleus by transcription factor SALL4 comprises the following steps: 1) constructing a SALL4-fluorescent protein fusion protein coding fragment; 2) constructing a SALL4-fluorescent protein fusion protein virus expression vector; 3) packaging a virus for expressing SALL4-fluorescent protein fusion protein; 4) primary separation / in vitro culture of target cells; the target cells in 4) are infected by the virus vector constructed in 3), that is, SALL4-fluorescent protein fusion protein is expressed in the target cells, and a cell model for recruiting and concentrating NuRD complex in the nucleus by transcription factor SALL4 is obtained. According to the scheme provided in the present application, a model for recruiting and concentrating NuRD complex in the nucleus can be established. The model provided in the present application can be applied to in situ electron microscope structure analysis of NuRD in cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a model for recruiting and concentrating NuRD complex in a cell nucleus by using transcription factor SALL4 and application thereof. BACKGROUND

[0002] Proteins are the main bearers of life activities, which are compiled from various different amino acid residues, and the structure of the proteins determines their functions. The analysis of the three-dimensional mechanism of proteins and protein complexes not only helps to understand life activities and expand the theoretical boundaries, but also provides an accurate direction for drug research and development and disease targeted treatment.

[0003] NuRD (nucleosome remodeling and deacetylase, chromatin remodeling and deacetylase complex) is one of the main chromatin remodeling complexes in mammals, which is widely expressed in the cell nucleus and is composed of GATAD2A / B, CHD3 / 4, HDAC1 / 2 and other subunits, and can induce nucleosome sliding and histone deacetylation.

[0004] NuRD plays an indispensable role in the occurrence of diseases such as neural development, tumors and the balance of stem cell pluripotency, and the analysis of the in-situ electron microscope structure of NuRD in cells helps people to synthesize high-affinity targeted drugs, thereby protecting the health of human life.

[0005] However, the existing structural analysis of NuRD complex depends on in-vitro overexpression and reconstruction, and the artificially selected reconstructed subunits are quite different from the real subunits in the body, and the molecular environment in the body is lost. The drug efficacy cannot be guaranteed when designing targeted drugs based on the protein structure obtained by such methods. Moreover, due to the great flexibility of NuRD complex, the existing structural model has low resolution, and can only analyze the general spindle shape. The in-situ NuRD can be combined with various proteins and DNA in the body, and may have a more stable spatial structure, which can reduce the calculation difficulty to a certain extent.

[0006] In summary, the in-situ analysis of NuRD protein structure has great theoretical and application research value. However, there is currently a lack of in-situ NuRD structure model. SUMMARY

[0007] Based on the current situation that there is a lack of in-situ NuRD structure analysis model in the prior art, the application provides a model for recruiting and concentrating NuRD complex in a cell nucleus by using transcription factor SALL4 and application thereof.

[0008] The model provided by the application recruits and concentrates NuRD complex in the nucleus by transcription factor SALL4, can be applied to in-situ electron microscope structure analysis of NuRD, and can be further applied to drug screening of targeted drugs for NuRD complex.

[0009] In the process of obtaining the scheme of the application, the inventors found that NuRD is uniformly expressed in the nucleus, and the components in the nucleus are complex, and the contrast is low, so it is extremely difficult to directly select NuRD particles in situ. The centromere heterochromatin is composed of paracentromere heterochromatin, which has strong contrast under a transmission electron microscope and can be distinguished by naked eye, and can be used as a nuclear anchor point for binding NuRD complex. Moreover, the transcription factor SALL4 is not only a centromere positioning protein, but also can bind to NuRD complex through the N-terminal 12 amino acids.

[0010] Based on this, the application provides a cell model for recruiting and concentrating NuRD complex in the nucleus by transcription factor SALL4, and provides an application of the cell model in in-situ electron microscope structure analysis of NuRD.

[0011] The application utilizes the NuRD-centromere double binding ability of SALL4 and the characteristic that the centromere has a contrast that can be distinguished by naked eye under an electron microscope, and constructs a centromere-SALL4-NuRD model, which has a structure similar to a parking lot-bus-passenger (PBP) model.

[0012] Based on the scheme of the application, NuRD complex can be recruited and bound in situ in the nucleus, and a new method is provided for in-situ high-resolution structure model analysis of NuRD.

[0013] The object of the application can be achieved by the following technical scheme:

[0014] The application first provides a SALL4-fluorescent protein fusion protein (also referred to as Sall4-EGFP fusion protein), and the amino acid sequence is shown as SEQ ID NO. 1, and the specific amino acid sequence is as follows:

[0015]

[0016] Note: In the amino acid sequence of the above SALL4-fluorescent protein fusion protein, the amino acid sequence at position 1-1067 is the SALL4 amino acid sequence (i.e. SEQ ID NO. 2); TR at position 1068, 1069 is an inserted enzyme cutting site; GGSGG at position 1070-1074 is an inserted linker sequence; the amino acid sequence at position 1075-1313 is an EGFP sequence; * is a stop codon.

[0017] The present application further provides an application of the transcription factor SALL4 in constructing a cell model for recruiting and concentrating NuRD complex in the nucleus, in which the transcription factor SALL4 is combined with a chromocenter and NuRD respectively to form a chromocenter-SALL4-NuRD complex structure. The amino acid sequence of the transcription factor SALL4 is shown in SEQ ID NO. 2, and is specifically as follows:

[0018]

[0019] In one embodiment of the present application, the SALL4-fluorescent protein fusion protein is combined with the chromocenter and NuRD respectively to form a chromocenter-SALL4-fluorescent protein fusion protein-NuRD complex structure, which has a diameter of about 1-2 μm.

[0020] The present application also provides a method for constructing a model for recruiting and concentrating NuRD complex in the nucleus by transcription factor SALL4, comprising the following steps:

[0021] 1) Constructing a SALL4-fluorescent protein fusion protein coding fragment:

[0022] wherein the amino acid sequence of the Sall4-fluorescent protein fusion protein is shown in SEQ ID NO. 1;

[0023] 2) Constructing a SALL4-fluorescent protein fusion protein virus expression vector

[0024] Selecting a restriction site on the target virus expression vector, and designing a recombination arm according to the sequence near the restriction site, and recombining the SALL4-fluorescent protein fusion protein coding fragment into the expression vector, and then transforming the competent bacteria with the expression vector to obtain a SALL4-fluorescent protein fusion protein virus expression vector;

[0025] 3) Packaging a virus expressing SALL4-fluorescent protein fusion protein

[0026] Packaging a virus expressing SALL4-fluorescent protein fusion protein using a tool cell, i.e. obtaining a virus vector;

[0027] 4) Primary isolation / in vitro culture of target cells:

[0028] According to the type of target cells to be identified, primary target cells are isolated or target cells are expanded in vitro;

[0029] 5) Expressing SALL4-fluorescent protein fusion protein in target cells

[0030] Using the virus vector constructed in 3) to infect the target cells in 4), i.e. expressing SALL4-fluorescent protein fusion protein in target cells, to obtain a cell model for recruiting and concentrating NuRD complex in the nucleus by transcription factor SALL4.

[0031] In one embodiment of the present application, in step 1), the coding gene of transcription factor SALL4 is represented as Sall4 , Sall4SALL4 is a gene which is highly expressed in human and mouse pluripotent cells and tumor cells, so total RNA of such cells is extracted, and cDNA library is synthesized by using reverse transcriptase, and according to DNA sequence on NCBI website, upstream and downstream PCR primers are designed, and then the coding gene fragment of EGFP can be cloned and amplified from the cDNA library Sall4 based on similar method, the coding gene fragment of EGFP can be amplified, that is egfp based on similar method, the coding gene fragment of EGFP can be amplified, that is Sall4 based on similar method, the coding gene fragment of EGFP can be amplified, that is egfp based on similar method, the coding gene fragment of EGFP can be amplified, that is Sall4 based on similar method, the coding gene fragment of EGFP can be amplified, that is egfp based on similar method, the coding gene fragment of EGFP can be amplified, that is Sall4-egfp based on similar method, the coding gene fragment of EGFP can be amplified, that is

[0032] In an embodiment of the present application, in step 2), after the coding gene fragment of SALL4-fluorescent protein fusion protein is recombined with the virus expression vector, E. coli is transformed for single clone purification, and then the single clone is picked and amplified, and sequencing is performed, and the vector with correct sequencing is the SALL4-fluorescent protein fusion protein virus expression vector, which can be used as the main tool of the present application. In step 2), based on the type of cells to be infected, the virus expression vector to be used is selected.

[0033] In an embodiment of the present application, in step 3), the virus expression vector is transferred into the packaging cell according to the packaging tool cell determined by the virus expression vector. The fusion protein virus vector is transfected into the virus packaging cell by using PEI and other transfection reagents, and then the virus supernatant is collected.

[0034] For example, when the cells to be infected are mouse embryonic fibroblasts, the virus expression vector to be used is PMXs, and the packaging tool cell determined by the virus expression vector is Plat-E. Then the virus expression vector is transferred into the packaging cell by using PEI and other transfection reagents. Finally, the virus supernatant is collected and stored at room temperature.

[0035] In an embodiment of the present application, in step 4), the primary isolated mouse fibroblasts are used in the present application. The 13.5-day mouse embryo is collected, and the head, tail, limbs and internal organs of the mouse are removed in a biological safety cabinet. The remaining part is cut into pieces by using an ophthalmic forceps, and then trypsin digestion is performed. The pieces are cultured, and then frozen storage is performed when the healing degree reaches about 80%. The pieces can be directly recovered and expanded for subsequent use.

[0036] In an embodiment of the present application, after step 5), the recruitment and restraint effect is identified, and the specific method is as follows:

[0037] After successful infection, the target cell culture medium is changed and the cells are cultured using serum-free medium. The expression of fluorescent protein is tracked under a fluorescence microscope to determine the time of strongest expression, which is 48 hours after changing to serum-free medium. At the time of strongest expression, the cells are fixed and stained for endogenous NuRD subunits using immunofluorescence techniques. At this time, specific NuRD subunits such as GATAD2A / 2B, RBBP4 / 7, etc. can be selected. Finally, the recruitment and binding of SALL4 to NuRD are determined by microscopy.

[0038] According to the results of the inventors' previous studies, the binding of SALL4 to NuRD is disrupted by BMP4, and there is a certain amount of BMP4 in serum. To achieve strong recruitment, serum-free medium is required.

[0039] In one embodiment of the present application, a method for treating and studying a cell model in which NuRD complexes are recruited and concentrated in the nucleus is also provided, comprising the following steps:

[0040] S1, sample preparation:

[0041] Before freezing the sample, a plasma cleaning instrument is used to perform glow discharge on the grid. The grid used is a stainless steel grid (Xinxing Baishi, 200 mesh). Then, the sample of the cell model in which NuRD complexes are recruited and concentrated in the nucleus is frozen using an injection-type freezing instrument EMGP (Leica).

[0042] S2, preparation of frozen sections by focused ion beam

[0043] The preparation of frozen sections is performed using a fluorescence beam / focused ion beam (FIB) / scanning electron beam three-beam microscope ELI-Tri-Scope (Biological Imaging Center of the Institute of Biology, Chinese Academy of Sciences).

[0044] The entire sample thinning process is divided into the following steps: sputtering platinum on the grid to reduce the charge effect of the sample. Then, a gas injection system is used to inject platinum organometallic compounds above the sample, allowing the platinum organometallic compounds to deposit on the sample surface to form a protective layer. Subsequently, the cell sample is observed under fluorescence, the staining center-SALL4-NuRD particles are located in the nucleus, and subsequent cutting and subsequent thinning are performed according to the indicated position.

[0045] S3, collection of frozen electron tomography image data

[0046] Tomogram imaging was performed using a transmission electron microscope Titan Krios G3 (Thermo Fisher Scientific) with Serial EM software to assist image acquisition. The location of the target protein on the cryo-section was found by fluorescence localization indication, and the data collection point was selected based on this.

[0047] S4, processing of cryo-electron tomography image data

[0048] The original images of cryo-electron tomography were drift-corrected using Warp software, and a tilt series was generated. Data alignment was performed using Aretomo. Subsequently, the alignment parameters were input into Warp, and tomograms were denoised using software such as deconv, cryoCARE in the Warp software package. All NurD particles were manually selected in Dynamo software. Subtomos were generated from the selected particle information using Warp software, and subsequent calculations were performed in Relion and M. Model building, correction, and subsequent structural analysis were completed using software such as AlphaFold2, SWISS-MODEL, Coot, and Chimera.

[0049] The application also provides a model for recruiting and concentrating NuRD complexes in the nucleus using transcription factor SALL4, which is prepared by the above method.

[0050] The application further provides an application of the model for recruiting and concentrating NuRD complexes in the nucleus using transcription factor SALL4, which is used for in situ electron microscopy structural analysis of NuRD.

[0051] Further, the model for recruiting and concentrating NuRD complexes in the nucleus using transcription factor SALL4 is used for drug screening of targeted drugs for NuRD complexes.

[0052] The application first discovers that overexpression of SALL4 molecules in mouse fibroblasts can form a shell-like structure around the chromocenter, and this shell-like structure can bind NuRD complexes uniformly expressed in the nucleus, playing a role in recruiting and concentrating NuRD complexes. This method can be applied to in situ electron microscopy analysis of NuRD structures, and provides a theoretical basis for the development of targeted drugs for NuRD complexes.

[0053] Using the scheme provided by the application, NuRD complexes can be recruited and bound in the nucleus, and the cell model with recruited and concentrated NuRD complexes in the nucleus can be directly analyzed by electron microscopy, and then in situ structural analysis of intracellular NuRD is performed. Compared with the prior art, the application has the following advantages and beneficial effects:

[0054] (1) The model is novel, and it utilizes the chromocenter localization of SALL4 and the double-binding characteristics of NuRD to concentrate the NuRD complex near the chromocenter, thereby improving the particle selection efficiency, significantly reducing the amount of in-situ electron microscope data collection, and reducing costs and increasing efficiency.

[0055] (2) The operation is simple. This technology only needs to add a SALL4-fluorescent protein overexpression step before NuRD in-situ electron microscope structure analysis, without affecting the subsequent entire process, and can be nested with any in-situ electron microscope analysis technology.

[0056] (3) The cell input amount is low. The chromocenter-SALL4-NuRD particle diameter is about 1-2 μm, which is suitable for the data collection range of a 300 kV electron microscope. In theory, one cell can collect multiple sets of data, providing operation possibilities for some cells that are not easy to obtain or cultivate.

[0057] (4) The application scenarios are wide. This technology can be applied to many different types of mammalian cultured cells.

[0058] (5) This application recruits the target complex molecule NuRD by finding the docking site in the nucleus-chromocenter and the intermediate bridge molecule SALL4, thereby concentrating it in the nucleus. This scheme can be used to recruit and concentrate other protein complexes, for example, taking a certain organelle as a docking site in the cytoplasm, and then fusing the localization peptide segment of the organelle and the recruitment peptide segment of the target complex to be overexpressed in the cell, and finally playing the role of recruiting and concentrating the complex to the organelle. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 The technical process for recruiting and concentrating NuRD complex in the nucleus by using transcription factor SALL4;

[0060] Figure 2 : A, flow chart of SALL4 virus infection of mouse embryonic fibroblasts; B, immunofluorescence and 3D simulation show the formation of SALL4-chromocenter particles with a shell wrapped structure;

[0061] Figure 3 : A, immunofluorescence results show the nuclear distribution and localization of NuRD specific subunit GATAD2B before SALL4 overexpression; B, immunofluorescence results show the nuclear distribution and localization of SALL4 and NuRD specific subunit GATAD2B after SALL4 overexpression;

[0062] Figure 4: A, the result of transmission electron microscopy after ultrathin sectioning shows the location of SALL4 overexpression; B, the result of transmission electron microscopy after ultrathin sectioning shows the location of SALL4 before overexpression of the staining center-SALL4-NuRD;

[0063] Figure 5 : A, the fluorescence localization of SALL4 under the cryo-optical microscope; B, the result of cryo-focused ion beam cutting of the staining center-SALL4-NuRD; C, the fluorescence localization of SALL4 on the thin section after focused ion beam thinning under the cryo-optical microscope; D, the suspected staining center-SALL4-NuRD region in the cryo-section tomography data. DETAILED DESCRIPTION

[0064] The present application will be described in detail below in conjunction with the accompanying drawings and specific examples.

[0065] In the following examples,

[0066] cDNA: complementary deoxyribonucleotide

[0067] PCR: polymerase chain reaction

[0068] EGFP: enhanced green fluorescent protein

[0069] PEI: polyethylenimine, commonly used for cell transfection

[0070] NCBI is the website name

[0071] Warp, Aretomo, deconv, cryoCARE, Dynamo, Relion, M, AlphaFold2, SWISS-MODEL, Coot, Chimera, etc. are all software names.

[0072] Example 1

[0073] Reference Figure 1 The present embodiment provides a method for recruiting and concentrating NuRD complex in the nucleus by using transcription factor SALL4, and the flow is as follows Figure 1 , which specifically comprises the following steps:

[0074] A method for constructing a model for recruiting and concentrating NuRD complex in the nucleus by using transcription factor SALL4, comprising the following steps:

[0075] 1) Constructing SALL4-fluorescent protein fusion protein coding fragment:

[0076] The amino acid sequence of the Sall4-fluorescent protein fusion protein is shown in SEQ ID NO. 1.

[0077] 2) Constructing SALL4-fluorescent protein fusion protein virus expression vector

[0078] Selecting enzyme cutting site on target virus expression vector, and designing recombination arm according to sequence near enzyme cutting site, recombining SALL4-fluorescent protein fusion protein coding fragment into expression vector, and then transforming competent bacteria with expression vector to obtain SALL4-fluorescent protein fusion protein virus expression vector;

[0079] 3) Packaging virus expressing SALL4-fluorescent protein fusion protein

[0080] Packaging virus expressing SALL4-fluorescent protein fusion protein with tool cell, to obtain virus vector;

[0081] 4) Primary separation / in vitro culture of target cells

[0082] Separating primary target cells or expanding target cells in vitro according to target cell type to be identified;

[0083] 5) Expressing SALL4-fluorescent protein fusion protein in target cells

[0084] Infecting target cells in 4) with virus vector constructed in 3) to express SALL4-fluorescent protein fusion protein in target cells, to obtain cell model in which transcription factor SALL4 recruits and concentrates NuRD complex in cell nucleus.

[0085] In step 1), coding gene of transcription factor SALL4 is represented as Sall4 , Sall4 is a gene highly expressed in human and mouse pluripotent cells and tumor cells, so total RNA of such cells needs to be extracted, and then cDNA library is synthesized with reverse transcriptase, and then upstream and downstream PCR primers are designed according to DNA sequence on NCBI website, and then Sall4 gene fragment can be cloned and amplified from the cDNA library, and based on similar method, EGFP coding gene fragment, i.e. egfp gene fragment, can be amplified; then based on principle of bridge PCR, taking Sall4 gene fragment and egfp gene fragment as templates, and using upstream primer of Sall4 gene fragment and downstream primer of egfp gene fragment, the two fragments are connected to obtain SALL4-fluorescent protein fusion protein coding fragment (i.e. Sall4-egfp gene fragment).

[0086] In step 2), after the SALL4-fluorescent protein fusion protein coding fragment is recombined with the virus carrier, the E. coli is transformed for single colony purification, the single colony is picked and expanded, and the sequencing is performed. The virus expression carrier with correct sequencing is the SALL4-fluorescent protein fusion protein virus expression carrier, which can be used as the main tool of the application.

[0087] In the embodiment, when the cells to be infected are mouse embryonic fibroblasts, the virus expression carrier used is PMXs, and the packaging tool cell is Plat-E according to the virus expression carrier. Then, the virus expression carrier is transfected into the packaging cell by using a transfection reagent such as PEI. Finally, the virus supernatant is collected and stored at room temperature.

[0088] In step 4), the primary isolated mouse fibroblasts are used in the application. The 13.5-day mouse embryo is collected, and the head, tail, limbs and internal organs of the mouse are removed in a biological safety cabinet. The remaining part is cut with an ophthalmic forceps and then digested with trypsin. The cells can be cultured, and can be frozen when the healing degree reaches about 80%. The subsequent use can be directly recovered and expanded.

[0089] In addition, in the embodiment, the recruitment and binding effect are identified, and the specific method is as follows:

[0090] After successful infection, the target cell culture medium is replaced with a serum-free culture medium for culture. The expression amount of the fluorescent protein is tracked under a fluorescence microscope, and the strongest expression time is determined to be 48 h after the serum-free culture medium is replaced. At the strongest expression time, the cells are fixed, and the endogenous NuRD subunit is stained by using an immunofluorescence technology. At this time, the NuRD specific subunits GATAD2A / 2B, RBBP4 / 7, etc. can be selected. Finally, the recruitment and binding effect of SALL4 on NuRD are determined by microscopic examination.

[0091] According to the previous research results of the inventors, the combination of SALL4 and NuRD is destroyed by BMP4, and a certain amount of BMP4 exists in the serum. If a stronger recruitment effect is required, a serum-free culture medium needs to be used.

[0092] In the embodiment, after the cell model in which the NuRD complex is recruited and concentrated in the nucleus is prepared, the cell model is treated and the method for research includes the following steps:

[0093] S1, frozen sample preparation:

[0094] Before the sample is frozen, the grid is subjected to glow discharge by using a plasma cleaning instrument. The grid used is a stainless steel grid (Xinxing Bairui, 200 mesh). Then, the sample of the cell model in which the NuRD complex is recruited and concentrated in the nucleus is frozen by using an injection type freezing instrument EMGP (Leica).

[0095] S2. Cryo-focused ion beam preparation of cryo-sections

[0096] The preparation of cryo-sections was performed using a fluorescent beam / focused ion beam (FIB) / scanning electron beam triple microscope ELI-Tri-Scope (Center for Biological Imaging, Institute of Biology, Chinese Academy of Sciences).

[0097] The whole sample thinning process is divided into the following steps: sputtering metal platinum on the support grid to reduce the charge effect of the sample. Then, the gas injection system is used to inject platinum organometallic compounds above the sample, so that the platinum organometallic compounds are deposited on the sample surface to form a protective layer. Subsequently, the cell sample is observed by fluorescence, the staining center-SALL4-NuRD particles in the nucleus are located, and subsequent cutting and subsequent thinning are performed according to the indicated position.

[0098] S3. Cryo-electron tomography image data collection

[0099] Tomography imaging was performed using a transmission electron microscope Titan Krios G3 (Thermo Fisher Scientific), and the Serial EM software was used to assist image collection. Through fluorescence positioning indication, the position of the target protein on the cryo-section was found, and the data collection point was selected based on this.

[0100] S4. Cryo-electron tomography image data processing

[0101] The original images of cryo-electron tomography were drift-corrected using Warp software, and a tilt sequence was generated. Data alignment was performed using Aretomo. Subsequently, the alignment parameters were input into Warp, and the tomogram was denoised using software such as deconv, cryoCARE in the Warp software package. All NurD particles were manually selected in the Dynamo software. Subtomo was generated by Warp software according to the selected particle information, and then calculation was performed in Relion and M. Model building, correction, and subsequent structure analysis were completed using software such as AlphaFold2, SWISS-MODEL, Coot, and Chimera.

[0102] The SALL4-EGFP fusion protein was overexpressed in the nucleus of mouse embryonic fibroblasts to verify its staining center binding ability. The Z-axis projection superposition results and 3D simulation results of the Zeiss 900 confocal microscope showed that it was wrapped in the shell structure outside the staining center, rather than overlapping with the staining center, as shown in Figure 2 -A、 Figure 2-B. In mouse fibroblasts, NuRD subunit GATAD2B was observed to be almost uniformly expressed in the whole nucleus by immunofluorescence and high-resolution microscopy, as shown in Figure 3 -A. To verify its recruitment ability, on the basis of Figure 2 , the specific component GATAD2B of NuRD subunit was located and identified by immunofluorescence, as shown in Figure 3 -B. The results proved that NuRD subunit could be recruited and concentrated in the SALL4-staining center position, indicating that the PBP model was established.

[0103] The recruitment and concentration phenomenon was verified by ultrathin (~70 nm) section and transmission electron microscopy, and it was found that compared with the control group, such as Figure 4 -B, near the staining center (gray line segment marked area) after overexpression of SALL4, SALL4-NuRD concentrated aggregation particles (red arrow position) could be observed, as shown in Figure 4 -A.

[0104] Finally, the correlation imaging of cryo-optical microscopy and cryo-electron microscopy was carried out. First, the fluorescence observation was carried out on the cell sample after glass freezing, and the staining center-SALL4-NuRD particles were located in the nucleus, as shown in Figure 5 -A. The results showed that under the condition of freezing, the SALL4 fluorescent protein still maintained the staining center positioning characteristics. Then, the correlation of fluorescence image and electron microscopy image was realized by aligning the position of the marker in the focused ion beam equipment, and then the sample was cut by ion beam according to the fluorescence position of the target molecule, as shown in Figure 5 -B, Figure 5 -C. Finally, the electron microscopy carrier net with multiple thin sections was transferred to the 300kV cryo-transmission electron microscope to collect the sequence tilt electron tomography data, and then the existing software was used for image alignment, reconstruction body generation, particle selection, structure analysis, etc. As shown in Figure 5 -D. The above experimental results show that by using the method of constructing PBP model by SALL4, NURD complex can be recruited and concentrated, so as to be applied to in situ structure analysis of NuRD in different cells.

[0105] The above description of the embodiments is for the convenience of those skilled in the art to understand and use the invention. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. The application of SALL4-fluorescent protein fusion protein in constructing a cell model that recruits and concentrates NuRD complexes within the cell nucleus, characterized in that... In the cell model, the SALL4-fluorescent protein fusion protein binds to the staining center and NuRD, respectively, recruits the NuRD complex that is uniformly distributed in the cell nucleus and concentrates it at the staining center to form a staining center-SALL4-fluorescent protein fusion protein-NuRD complex structure with a diameter of 1-2 μm. The amino acid sequence of the transcription factor SALL4 is shown in SEQ ID NO.2; The amino acid sequence of the SALL4-fluorescent protein fusion protein is shown in SEQ ID NO.

1. The SALL4-fluorescent protein fusion protein can indicate the nuclear localization of the protein SALL4.

2. A method for constructing a model that utilizes the transcription factor SALL4 to recruit and concentrate the NuRD complex in the cell nucleus, characterized in that, Includes the following steps: 1) Construct the SALL4-fluorescent protein fusion protein encoding fragment: The amino acid sequence of the SALL4-fluorescent protein fusion protein is shown in SEQ ID NO.1; 2) Constructing a viral expression vector for the SALL4-fluorescent protein fusion protein Enzyme restriction sites were selected on the target viral expression vector, and recombinant arms were designed based on the sequences near the enzyme restriction sites. The coding fragment of the SALL4-fluorescent protein fusion protein was recombined into the expression vector, and the expression vector was then transformed into competent bacteria. After amplification, the SALL4-fluorescent protein fusion protein viral expression vector was obtained. 3) Packaging viruses expressing SALL4-fluorescent protein fusion protein Viruses expressing the SALL4-fluorescent protein fusion protein were packaged using tool cells to obtain a viral suspension. 4) Primary isolation / in vitro culture of target cells: Isolate primary target cells or expand target cells in vitro according to the type of target cells to be identified; 5) Expression of SALL4-fluorescent protein fusion protein in target cells The viral vector constructed in 3) was used to infect the target cells in 4), that is, to express the SALL4-fluorescent protein fusion protein in the target cells, thus obtaining a cell model that uses the transcription factor SALL4 to recruit and concentrate the NuRD complex in the cell nucleus.

3. The method for constructing a model using transcription factor SALL4 to recruit and concentrate the NuRD complex in the cell nucleus according to claim 2, characterized in that, In step 2), after the SALL4-fluorescent protein fusion protein coding fragment is recombined and ligated with the viral vector, it is transformed into E. coli for single-clone purification, then single clones are picked and amplified, the vector is extracted and sequenced, and the vector with correct sequencing is the SALL4-fluorescent protein fusion protein viral expression vector.

4. The method for constructing a model using transcription factor SALL4 to recruit and concentrate the NuRD complex in the cell nucleus according to claim 2, characterized in that, In step 3), the packaging tool cells are determined based on the viral expression vector, and the viral expression vector is transformed into the packaging cells using the PEI transformation method.

5. The method for constructing a model using transcription factor SALL4 to recruit and concentrate the NuRD complex in the cell nucleus according to claim 2, characterized in that, In step 4), the target cells are mouse fibroblasts.

6. The method for constructing a model using transcription factor SALL4 to recruit and concentrate the NuRD complex in the cell nucleus according to claim 2, characterized in that, After step 5), its recruitment and binding effects are assessed, specifically through the following methods: After successful infection, the target cell culture medium was changed to serum-free medium for culture. The expression level of fluorescent protein was tracked under a fluorescence microscope, and the time of strongest expression was determined to be 48 hours after changing to serum-free medium. At the time of strongest expression, the cells were fixed with 4% paraformaldehyde fixative, and the endogenous NuRD subunits were stained using immunofluorescence technology. At this time, NuRD specific subunits GATAD2A / 2B and RBBP4 / 7 were selected. Finally, microscopic examination was used to determine the recruitment and binding effect of SALL4 on NuRD.

Images