Model for recruiting and concentrating NuRD compound in cell nucleus by using transcription factor SALL4 and application

Abstract

The invention relates to a model for recruiting and concentrating a NuRD compound in a cell nucleus by using a transcription factor SALL4 and application. The construction method of the model for recruiting and concentrating the NuRD compound in the cell nucleus by using the transcription factor SALL4 comprises the following steps: 1) constructing an SALL4-fluorescent protein fusion protein coding fragment; 2) constructing an SALL4-fluorescent protein fusion protein virus expression vector; 3) packaging the virus for expressing the SALL4-fluorescent protein fusion protein; 4) performing primary isolation / in-vitro culture on target cells; and infecting the target cells in the step 4) by utilizing the virus vector constructed in the step 3), namely expressing SALL4-fluorescent protein fusion protein in the target cells to obtain the cell model for recruiting and concentrating the NuRD compound in the cell nucleus by utilizing the transcription factor SALL4. By utilizing the scheme provided by the invention, the model for recruiting and concentrating the NuRD compound in the cell nucleus can be established. The model provided by the invention can be applied to intracellular NuRD in-situ electron microscope structure analysis.

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 is composed of pericentromeric 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 localization 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, which provides a new method for in-situ high-resolution structure model analysis of NuRD.

[0013] The object of the application can be achieved by the following technical scheme: 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: 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 the EGFP sequence; * is a stop codon.

[0014] The present application further provides an application of the transcription factor SALL4 in a cell model for constructing a nuclear recruited and concentrated NuRD complex, in which the transcription factor SALL4 is combined with a chromocenter and a 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:

[0015] 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.

[0016] 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: 1) Constructing a SALL4-fluorescent protein fusion protein coding fragment: wherein the amino acid sequence of the Sall4-fluorescent protein fusion protein is shown in SEQ ID NO. 1; 2) Constructing a SALL4-fluorescent protein fusion protein virus expression vector 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; 3) Packaging a virus expressing SALL4-fluorescent protein fusion protein Packaging a virus expressing SALL4-fluorescent protein fusion protein using a tool cell, i.e. obtaining a virus vector; 4) Primary isolation / in vitro culture of target cells: According to the type of target cells to be identified, primary target cells are isolated or target cells are expanded in vitro; 5) Expressing SALL4-fluorescent protein fusion protein in target cells Infecting the target cells in 4) with the virus vector constructed in 3) to express SALL4-fluorescent protein fusion protein in the target cells, and obtaining a cell model for recruiting and concentrating NuRD complex in the nucleus by transcription factor SALL4.

[0017] In one embodiment of the present application, in step 1), the 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 a cDNA library is synthesized using reverse transcriptase, and according to the DNA sequence on the NCBI website, the upstream and downstream PCR primers are designed, and then the Sall4 gene fragment can be cloned and amplified from the cDNA library, and based on a similar method, the upstream and downstream PCR primers are designed at the same time, and the coding gene fragment of EGFP, i.e. egfp gene fragment, can be amplified; and then based on the principle of bridge PCR, the upstream and downstream primers of the SALL4-fluorescent protein fusion protein coding gene fragment are designed at the same time, and the SALL4-fluorescent protein fusion protein coding gene fragment is amplified.Sall4 gene fragment and egfp The gene fragment is used as a template, and Sall4 an upstream primer of the gene fragment and egfp a downstream primer of the gene fragment are used to connect the two fragments, so as to obtain a SALL4-fluorescent protein fusion protein coding fragment (i.e. Sall4-egfp a gene fragment).

[0018] In an embodiment of the present application, in step 2), after the SALL4-fluorescent protein fusion protein coding fragment is recombined with the virus carrier, the E. coli is transformed for monoclone purification, the monoclone is picked and expanded, and sequencing is performed; the carrier with correct sequencing is the SALL4-fluorescent protein fusion protein virus expression carrier, which can be used as a main tool of the present application. In step 2), based on the type of cells to be infected, the virus expression carrier used is selected.

[0019] In an embodiment of the present application, in step 3), the virus expression carrier is determined to be used for determining a packaging tool cell, and the virus expression carrier is transferred into the packaging cell. The fusion protein virus carrier is transfected into the virus packaging cell by using a transfection reagent such as PEI, and then the virus supernatant is collected.

[0020] For example, when the cells to be infected are mouse embryonic fibroblasts, the virus expression carrier used is determined to be PMXs, and the packaging tool cell determined according to the virus expression carrier is Plat-E. Then, the virus expression carrier can be transferred into the packaging cell by using a transfection reagent such as PEI. Finally, the virus supernatant is collected and stored at room temperature.

[0021] 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, 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 after trypsin digestion, the pieces can be cultured, and when the healing degree reaches about 80%, the pieces can be frozen, and after thawing, the pieces can be directly expanded for subsequent use.

[0022] In an embodiment of the present application, after step 5), the recruitment and binding effect are identified, and the specific method is as follows: 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 time when the expression is the strongest is determined to be 48 h after the serum-free culture medium is replaced. At the time when the expression is the strongest, the cells are fixed, and the endogenous NuRD subunit is dyed by using an immunofluorescence technology. At this time, the NuRD specific subunits GATAD2A / 2B, RBBP4 / 7, etc. can be selected, and finally the recruitment and binding effect of SALL4 on NuRD are determined by microscopic examination.

[0023] According to the inventors' previous research, the binding of SALL4 and NuRD is disrupted by BMP4. Since a certain amount of BMP4 exists in serum, a serum-free culture medium is needed to obtain a stronger recruitment effect.

[0024] In one embodiment of the present invention, a method for processing and studying a cell model after recruiting and concentrating the NuRD complex in the cell nucleus is also provided, comprising the following steps: S1. Preparation of frozen samples: Before freezing, the grid was subjected to glow discharge using a plasma cleaner. The grid used was a stainless steel grid (Xinxing Bairui, 200 mesh). The cell model samples, in which the NuRD complex was recruited and concentrated within the cell nucleus, were then frozen using an injection-type cryostat EMGP (Leica).

[0025] S2, Cryo-Focused Ion Beam Preparation of Cryo-Slices Frozen sections were prepared using a three-beam microscope (ELI-Tri-Scope, Institute of Biological Sciences, Chinese Academy of Sciences) employing fluorescence beam / focused iron bean (FIB) / scanning electron beam.

[0026] The entire sample thinning process consists of the following steps: Platinum is sputtered onto a grid to reduce the charge effect on the sample. Then, a platinum organometallic compound is injected onto the sample using a gas injection system, causing the compound to deposit on the sample surface to form a protective layer. Subsequently, the cell samples are subjected to fluorescence observation to locate the staining centers—SALL4-NuRD particles—within the cell nuclei, followed by subsequent cutting and thinning according to the indicated locations.

[0027] S3, cryo-electron computed tomography (CETCT) image data collection Tomographic imaging was performed using a TitanKrios G3 transmission electron microscope (Thermo Fisher Scientific), with Serial EM software assisting in image acquisition. Fluorescence localization indicators were used to locate the target protein on the frozen section, and data collection points were selected based on these locations.

[0028] S4, Cryo-electron computed tomography image data processing The raw images of the frozen electron tomography are drift-corrected and tilt series are generated using Warp software. Data alignment is performed using Aretomo. Subsequently, the alignment parameters are input into Warp, and tomograms are denoised using software such as deconv, cryoCARE in the Warp software package. All NurD particles are manually picked up in Dynamo software respectively. Subtomos are generated from the picked particles using Warp software, and subsequent calculations are performed in Relion and M. Model building, correction and subsequent structure analysis are completed by software such as AlphaFold2, SWISS-MODEL, Coot, Chimera.

[0029] The application also provides a model for recruiting and concentrating NuRD complexes in the nucleus by using transcription factor SALL4, which is prepared by the above method.

[0030] The application further provides an application of the model for recruiting and concentrating NuRD complexes in the nucleus by using transcription factor SALL4, which is used for in-situ electron microscopy structural analysis of NuRD.

[0031] Further, the model for recruiting and concentrating NuRD complexes in the nucleus by using transcription factor SALL4 is used for drug screening of targeted drugs for NuRD complexes.

[0032] The application first discovers that overexpression of SALL4 molecules in mouse fibroblasts can form a shell structure around the chromocenter, and this shell structure can bind NuRD complexes uniformly expressed in the nucleus, thereby playing a role of 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.

[0033] By using the scheme provided by the application, NuRD complexes can be recruited and bound in the nucleus, and the cell model for recruiting and concentrating NuRD complexes in the nucleus can be directly analyzed by electron microscopy, and then in-situ structural analysis of NuRD in cells is performed. Compared with the prior art, the application has the following advantages and beneficial effects: (1) The model is novel, and the chromocenter positioning of SALL4 and the double binding characteristics of NuRD are used to concentrate NuRD complexes near the chromocenter, thereby improving the particle picking efficiency, significantly reducing the amount of in-situ electron microscopy data collection, and reducing costs and increasing efficiency.

[0034] (2) The operation is simple, and the technology only needs to add a SALL4-fluorescent protein overexpression step before in-situ electron microscopy structural analysis of NuRD, without affecting the subsequent entire process, and can be nested with any in-situ electron microscopy analysis technology.

[0035] (3) The amount of cells used is low. The diameter of the staining center-SALL4-NuRD particle 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, which provides the possibility of operating some cells that are not easy to obtain or cultivate.

[0036] (4) The application can be widely used. The present technology can be applied to many different types of mammalian cultured cells.

[0037] (5) The present application recruits the target complex molecule NuRD by finding the docking site in the nucleus-staining center and the intermediate bridging 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 the 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

[0038] Figure 1 Technical process for recruiting and concentrating NuRD complex in the nucleus by using transcription factor SALL4; Figure 2 : A, flow chart of SALL4 virus infection of mouse embryonic fibroblasts; B, immunofluorescence and 3D simulation show the particle structure of SALL4-staining center with a shell wrapped around it; 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; Figure 4 : A, transmission electron microscopy results after ultrathin sectioning show the location after SALL4 overexpression; B, transmission electron microscopy results after ultrathin sectioning show the location of the staining center-SALL4-NuRD before SALL4 overexpression; Figure 5 : A, fluorescence localization of SALL4 under a cryo-optical microscope; B, results after focused ion beam cutting of the staining center-SALL4-NuRD; C, fluorescence localization of SALL4 on the thin section after focused ion beam thinning under a cryo-optical microscope; D, suspected staining center-SALL4-NuRD region in the cryo-section tomography data. DETAILED DESCRIPTION

[0039] The present application will be described in detail below in conjunction with the drawings and specific examples.

[0040] In the following examples, cDNA: complementary deoxyribonucleotide PCR: polymerase chain reaction EGFP: enhanced green fluorescent protein PEI: polyethylenimine, commonly used for cell transfection NCBI is the name of the website Warp, Aretomo, deconv, cryoCARE, Dynamo, Relion, M, AlphaFold2, SWISS-MODEL, Coot, Chimera, etc. are all software names.

[0041] Example 1 Reference Figure 1 The present embodiment provides a method for recruiting and concentrating NuRD complex in the nucleus by transcription factor SALL4, and the flow is as follows Figure 1 , specifically comprising the following steps: A method for constructing a model for recruiting and concentrating NuRD complex in the nucleus by transcription factor SALL4, comprising the following steps: 1) Constructing SALL4-fluorescent protein fusion protein coding fragment: Wherein, the amino acid sequence of the Sall4-fluorescent protein fusion protein is shown as SEQ ID NO. 1; 2) Constructing SALL4-fluorescent protein fusion protein virus expression vector Select the enzyme cutting site on the target virus expression vector, and design the recombination arm according to the sequence near the enzyme cutting site, recombine the SALL4-fluorescent protein fusion protein coding fragment into the expression vector, and then transform the competent bacteria to obtain the SALL4-fluorescent protein fusion protein virus expression vector; 3) Packaging virus expressing SALL4-fluorescent protein fusion protein Use tool cells to package virus expressing SALL4-fluorescent protein fusion protein, i.e. obtain virus vector; 4) Primary separation / in vitro culture of target cells: According to the type of target cells to be identified, primary target cells are isolated or target cells are expanded in vitro; 5) Expressing SALL4-fluorescent protein fusion protein in target cells Use the virus vector constructed in 3) to infect the target cells in 4), i.e. express 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.

[0042] In step 1), the 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 is extracted, and a cDNA library is synthesized by using reverse transcriptase, and according to the DNA sequence on the NCBI website, upstream and downstream PCR primers are designed, and then the coding gene fragment of SALL4 can be cloned and amplified from the cDNA library. Sall4 Based on similar methods, the coding gene fragment of EGFP, i.e. egfp can be amplified by designing upstream and downstream PCR primers. Sall4 Based on the principle of bridge PCR, the upstream primer of egfp and the downstream primer of Sall4 are used to connect the two fragments to obtain the coding fragment of SALL4-fluorescent protein fusion protein (i.e. egfp ). Sall4-egfp

[0043] In step 2), after the coding fragment of SALL4-fluorescent protein fusion protein is recombined with the viral vector, E. coli is transformed for single colony purification, and then the single colony is picked and amplified, and sequencing is performed. The vector with correct sequencing is the SALL4-fluorescent protein fusion protein viral expression vector, which can be used as the main tool of the application.

[0044] In this embodiment, when the cells to be infected are mouse embryonic fibroblasts, the viral expression vector used is PMXs, and the packaging tool cell is Plat-E according to the viral expression vector. Then the viral expression vector is transfected into the packaging cell by using PEI and other transfection reagents. Finally, the virus supernatant is collected and stored at room temperature.

[0045] 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 trypsin digestion is performed. The culture is performed, and when the healing degree reaches about 80%, it can be frozen for storage, and then it can be directly recovered and expanded for subsequent use.

[0046] In addition, in this embodiment, the recruitment and restraint of the mouse embryonic fibroblasts are identified, and the specific method is as follows: ​After successful infection, the target cell culture medium is changed and serum-free medium is used for culture. The expression of fluorescent protein is tracked under a fluorescence microscope to determine the strongest expression time, 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 technology. At this time, NuRD-specific subunits such as GATAD2A / 2B, RBBP4 / 7, etc. can be selected. Finally, the recruitment and binding of SALL4 to NuRD are determined by microscopy.

[0047] According to the results of the inventors' previous studies, the combination of SALL4 and NuRD is disrupted by BMP4, and there is a certain amount of BMP4 in serum. To obtain a stronger recruitment effect, serum-free medium needs to be used.

[0048] In this embodiment, the cell model with recruited and concentrated NuRD complex in the nucleus is treated and the method of research includes the following steps: S1, frozen sample preparation: 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 Bairui, 200 mesh). Then, the cell model sample with recruited and concentrated NuRD complex in the nucleus is frozen by an injection-type freezing instrument EMGP (Leica).

[0049] S2, frozen focused ion beam preparation of frozen sections 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 Institute of Biology, Chinese Academy of Sciences).

[0050] 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, so that the platinum organometallic compounds are deposited on the sample surface to form a protective layer. Subsequently, the cell sample is observed under fluorescence, the staining center-SALL4-NuRD particle in the nucleus is located, and subsequent cutting and subsequent thinning are performed according to the indicated position.

[0051] S3, frozen electron tomography image data collection Tomographic imaging is performed using a transmission electron microscope Titan Krios G3 (Thermo Fisher Scientific), and the Serial EM software is used to assist image acquisition. Through fluorescence positioning indication, the position of the target protein on the frozen section is found, and the data collection point is selected based on this.

[0052] S4, cryo-electron tomography image data processing The raw images of cryo-electron tomography were drift-corrected using Warp software and tilt series were 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, and all NurD particles were manually picked up in Dynamo software, respectively. Subtomos were generated from the picked particles 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, Chimera, etc.

[0053] The SALL4-EGFP fusion protein was overexpressed in the nucleus of mouse embryonic fibroblasts to verify its ability to bind to the chromocenter. The results of Z-axis projection overlay and 3D simulation using a Zeiss 900 confocal microscope showed that it formed a shell-like structure around the chromocenter, rather than overlapping with the chromocenter, as shown in Figure 2 -A, Figure 2 -B. In mouse fibroblasts, immunofluorescence and high-resolution microscopy showed that the NuRD subunit GATAD2B was almost uniformly expressed in the entire nucleus, as shown in Figure 3 -A. To verify its recruitment ability, on the basis of Figure 2 , immunofluorescence was used to locate and identify the specific components of NuRD subunit GATAD2B, as shown in Figure 3 -B, the results proved that NuRD subunits could be recruited and concentrated at the SALL4-chromocenter position, indicating that the PBP model was established.

[0054] Ultra-thin (~70 nm) sections and transmission electron microscopy were used to verify the recruitment and concentration phenomenon. Compared with the control group, as shown in Figure 4 -B, after overexpression of SALL4, SALL4-NuRD concentrated and aggregated particles (red arrow position) were observed near the chromocenter (gray line segment marked area), as shown in Figure 4 -A.

[0055] Finally, cryo-optical microscopy and cryo-electron microscopy were used for correlated imaging. First, the fluorescence of the cell sample after vitrification was observed, and the chromocenter-SALL4-NuRD particles were located in the nucleus, as shown in Figure 5-A. The results show that the SALL4 fluorescent protein still retains the staining center localization characteristics under frozen conditions. Then the correlation of the fluorescent image and the electron microscopy image is achieved by aligning the position of the marker in the focused ion beam device, and then the ion beam is cut according to the fluorescent position of the target molecule. For example Figure 5 -B, Figure 5 -C. Finally, the electron microscopy carrier net with multiple thin slices is transferred to a 300kV cryo transmission electron microscope to collect the sequence tilt electron tomography data, and then the existing software is used for image alignment, reconstruction of the body, particle selection, structure analysis, etc. For example Figure 5 -D. The above experimental results show that the NURD complex can be recruited and concentrated by using the method of constructing a PBP model of SALL4, so as to be applied to the in situ structure analysis of NuRD in different cells.

[0056] The above description of the embodiments is for the purpose of facilitating the understanding and use of the invention by those skilled in the art. Those skilled in the art can obviously make various modifications to these embodiments, and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present application is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art within the scope of the present application without departing from the scope of the present application should be within the protection scope of the present application.

Claims

1. A SALL4-fluorescent protein fusion protein, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1. It can bind to the staining center, recruit the NuRD complex, and indicate the nuclear localization of protein SAAL4.

2. The application of transcription factor SALL4 in constructing a cell model that recruits and concentrates the NuRD complex within the cell nucleus, characterized in that... In the cell model, the transcription factor SALL4 binds to the staining center and NuRD respectively, forming a staining center-SALL4-NuRD complex structure. The amino acid sequence of the transcription factor SALL4 is shown in SEQ ID NO.

2.

3. 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.

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 3, 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.

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 3, 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.

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 3, characterized in that, In step 4), the target cells are mouse fibroblasts.

7. The method for constructing a model using transcription factor SALL4 to recruit and concentrate the NuRD complex in the cell nucleus according to claim 3, 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.

8. A model that utilizes the transcription factor SALL4 to recruit and concentrate the NuRD complex in the cell nucleus, characterized in that, It is prepared by any one of the methods described in claims 3-6.

9. An application of the model described in claim 8, which utilizes the transcription factor SALL4 to recruit and concentrate the NuRD complex in the cell nucleus, characterized in that, Used for in-situ electron microscopy structure analysis of NuRD.

10. An application of the model described in claim 8, which utilizes the transcription factor SALL4 to recruit and concentrate the NuRD complex in the cell nucleus, characterized in that... Drug screening for targeted therapies against NuRD complexes.

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