CN110885831A

CN110885831A - Modified Bach1 gene and application thereof

Info

Publication number: CN110885831A
Application number: CN201911275165.9A
Authority: CN
Inventors: 孟丹; 魏香香; 郭阶雨; 李沁涵
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2018-11-28
Filing date: 2018-11-28
Publication date: 2020-03-17
Anticipated expiration: 2038-11-28
Also published as: CN110885831B; CN109517826B; CN109517826A

Abstract

The invention relates to a modified Bach1 gene and application thereof in stem cell pluripotency, self-renewal, proliferation and/or reprogramming, wherein the activity or expression of BACH1 protein of a cell is inhibited or the activity or expression of BACH1 protein of the cell is increased by modifying a transcription factor Bach1 gene, and then the modified Bach1 gene interacts with a deubiquitinating protease Usp7, a pluripotency factor Nanog, Sox2 and/or Oct4, a PRC2 complex, a Wnt/β -catenin signaling pathway and/or a Nodal/Smad2/3 signaling pathway, so that the stem cell self-renewal and/or proliferation are regulated.

Description

Modified Bach1 gene and application thereof

The application is a divisional application, and the application date of the original application is as follows: 11 and 28 in 2018, with the application numbers: 201811432995.3, and the name: a modified Bach1 gene and application thereof.

One, the technical field

The invention belongs to the technical field of biology, and particularly relates to a modified Bach1 gene and application thereof in adjusting stem cell pluripotency, self-renewal, proliferation, reprogramming and/or differentiation.

Second, background Art

The most important characteristics of stem cells are their self-renewal ability and the potential for multipotential differentiation. At present, the molecular mechanisms regulating self-renewal and differentiation of Human Embryonic Stem Cells (Human Embryonic Stem Cells, hESCs) have not been fully elucidated. In the early stage of embryonic development, the epigenetic modification such as histone modification and chromatin remodeling and the coordination of various signal paths in cells play an important role in embryonic development and stem cell differentiation. The transcription factor Bach1(BTB and CNC homology 1, BTB-CNC homolog 1) belongs to a member of the alkaline leucine zipper protein family, which is widely present in various tissues of mammals and is involved in regulating transcription of genes. However, the role of Bach1 in stem cell self-renewal and differentiation is not clear.

In addition, the culture method of human embryonic stem cells mainly uses a culture medium with complex components and high price, such as mTesR1, and the added substances such as growth factors are not easy to obtain, and the preparation process is complex. Therefore, there is an urgent need for a simpler and less expensive method for culturing the same.

Third, the invention

The invention researches the action and molecular mechanism of Bach1on the pluripotency, self-renewal, proliferation, reprogramming or differentiation of stem cells, and discovers that the pluripotency, self-renewal, proliferation, reprogramming and/or differentiation of stem cells can be regulated and controlled by modifying Bach1 gene, therefore,

in a first aspect, the invention relates to a modified transcription factor Bach1 gene, wherein the transcription factor Bach1 gene is modified such that the activity or expression of a Bach1 protein of a cell is inhibited or the activity or expression of a Bach1 protein of a cell is increased.

In a second aspect, the invention relates to the use of a modified transcription factor Bach1 gene for modulating stem cell pluripotency, self-renewal, proliferation, reprogramming and/or differentiation.

In a third aspect, the invention relates to the use of a regulatory agent for the transcription factor Bach1 for modulating stem cell pluripotency, self-renewal, proliferation, reprogramming and/or differentiation.

Further, the regulating agent regulates the transcription factor Bach1on the level of genes, mRNA, protein and the like.

In a fourth aspect, the invention relates to a method of modulating pluripotency, self-renewal, proliferation, reprogramming and/or differentiation of a stem cell, wherein the Bach1 gene of the stem cell is modified such that the Bach1 protein activity or expression of the stem cell is inhibited or the Bach1 protein activity or expression of the stem cell is increased.

In a fifth aspect, the invention relates to a stem cell line, wherein the Bach1 gene of the stem cell line is modified, so that the activity or expression of Bach1 protein of stem cells is inhibited or the activity or expression of Bach1 protein of stem cells is increased.

In the above invention, preferably, the transcription factor BACH1 protein interacts with deubiquitinating protease USP7, pluripotency factor NANOG, SOX2 and/or OCT4, PRC2 complex, Wnt/β -catenin signaling pathway, and/or Nodal/Smad2/3 signaling pathway, thereby modulating stem cell pluripotency, self-renewal, proliferation, reprogramming and/or differentiation.

Further, the genetic modification results in inhibition of BACH1 protein activity or expression of the stem cell, thereby inhibiting stem cell pluripotency, self-renewal, proliferation, and/or reprogramming.

Further, the gene modification enables the mRNA level of the Bach1 gene of the stem cell to be reduced, and further the expression of the BACH1 protein is inhibited.

Further, the gene modification enables the activity or expression of BACH1 protein of the stem cell to be inhibited, thereby promoting the differentiation of the stem cell to mesendoderm and inhibiting the differentiation of neuroectoderm.

Further, the gene modification enables the activity or expression of BACH1 protein of the stem cell to be inhibited, so that the enrichment of EZH2 protein and H3K27me3 protein in the promoter region of mesendoderm differentiation genes is reduced, and the expression of the differentiation genes is promoted.

Further, the gene modification enables the activity or expression of BACH1 protein of the stem cell to be inhibited, so that Wnt/β -catenin and Nodal/Smad2/3 signal paths are activated, enrichment of β -catenin and Smad2/3 in a promoter region of a mesendoderm differentiation gene is increased, and the expression of the mesendoderm differentiation gene is promoted.

Further, the gene modification enables the activity or expression of BACH1 protein of the stem cell to be increased, so that the pluripotency of the stem cell is maintained, the self-renewal capacity of the stem cell is maintained, the proliferation of the stem cell is promoted, the reprogramming of the stem cell is promoted, and the differentiation of the stem cell is inhibited.

Further, the genetic modification increases the activity or expression of BACH1 protein of the stem cell, thereby increasing the expression of deubiquitinating protease USP7 protein, increasing the interaction of the pluripotency factors NANOG, SOX2 and/or OCT4 protein and USP7 protein, and further increasing the protein stability of the pluripotency factors.

Further, the genetic modification increases the activity or expression of BACH1 protein of the stem cell, thereby increasing the efficiency of somatic reprogramming.

Preferably, exon 1, exon 2, exon 3, exon 4 and/or exon 5 of Bach1 gene are genetically modified.

Preferably, the gene modification means which allows the activity or expression of the BACH1 protein to be inhibited comprises point mutation, deletion, insertion, antisense polynuceotides, siRNA, microRNA or gene editing technology; further preferably, the gene editing technology comprises embryonic stem cell-based DNA homologous recombination technology, CRISPR/Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease and other gene editing technologies.

More preferably, the genetic modification comprises knockout of exon 2 of Bach1 gene using CRISPR/Cas9 technology.

Preferably, the genetic modification means for increasing or overexpressing the activity of BACH1 protein comprises point mutation, linking a strong promoter, linking an enhancer, increasing copy number, viral vector, transposon gene insertion.

Further preferably, the gene modification mode is that the Piggy Bac transposon system is used for constructing tetracycline Dox inducible Bach1 overexpression.

Further, the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell; preferably, the embryonic stem cell is a murine or human embryonic stem cell.

In a sixth aspect, the invention relates to a sgRNA sequence that targets the transcription factor Bach1 gene of a cell, while the sgRNA is unique in the target sequence on the transcription factor Bach1 gene to be altered and complies with the sequence arrangement rule of 5 '-NNN (20) -NGG 3' or 5 '-CCN-N (20) -3'.

More preferably, the target site of the sgRNA in the cell of the transcription factor Bach1 gene is located on

exon

1, 2, 3, exon 4 and/or exon 5 of the transcription factor Bach1 gene, respectively. Further preferably, the transformation region to be changed is part or all of exon 2.

Still further preferably, the sequence of the 5' target site targeted by the sgRNA is as set forth in SEQ ID NO: 1, the sequence of the sgRNA-targeted 3' end target site is shown as SEQ ID NO: 2, respectively.

The seventh aspect of the invention relates to a method for culturing a stem cell line, wherein the method adds BACH1 protein to culture the stem cell line.

Further, the stem cell line is embryonic stem cells, adult stem cells, induced pluripotent stem cells; preferably, the embryonic stem cell is a murine or human embryonic stem cell.

In an eighth aspect, the present invention relates to a stem cell line obtained by the above culture method.

In a ninth aspect, the invention relates to a use of the above stem cell line in the preparation of a pharmaceutical composition.

In a tenth aspect, the present invention relates to a pharmaceutical composition, wherein the pharmaceutical composition comprises the above stem cell line.

Further, the pharmaceutical composition is used for treating cardiovascular diseases.

According to the technical scheme provided by the invention, 1, the inventor discovers for the first time that the pluripotency, self-renewal, proliferation, reprogramming and/or differentiation of stem cells can be regulated through a modified Bach1 gene, the Bach1 gene interacts with a deubiquitinating protease Usp7, a pluripotency factor Nanog, Sox2 and/or Oct4, a PRC2 complex, a Wnt/β -catenin signaling pathway and/or a Nodal/Smad2/3 signaling pathway, so as to regulate the pluripotency, self-renewal, proliferation, reprogramming and/or differentiation of stem cells, 2 and Bach1 inhibit the differentiation of mesendoderm cells through various mechanisms, while cells of a cardiovascular system are obtained by further downstream differentiation of mesendoderm cells, diseases of the cardiovascular system are head killers harmful to human health, while stem cells of the stem system based on stem cell source are an important method for treating the mesendoderm diseases, the discovery, the Bach1 can promote the stem cells to be more favorable for clinical application of stem cells regeneration, such as stem cells, stem cells can be cultured, and promote the clinical regeneration of stem cells, and promote the regeneration of stem cells, and promote regeneration of stem cells.

The foregoing is merely a summary of aspects of the invention and is not, and should not be taken as, limiting the invention in any way.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following documents. For example:

1、Molecular Cloning ALaboratory Manual，2ndEd.，ed.By Sambrook，Fritschand Maniatis(Cold Spring Harbor Laboratory Press：1989)；

2、L.Jiang et al.,Bach1 represses Wnt/beta-Catenin signaling andangiogenesis.Circ Res117,364-375(2015)；

3、L.Jiang et al.,The Transcription Factor Bach1 Suppresses theDevelopmental Angiogenesis of Zebrafish.Oxid Med Cell Longev 2017,2143875(2017)；

4、Oligonucleotide Synthesis(M.J.Gaited.，1984)；O.Shalem et al.,Genome-scale CRISPR-Cas9knockout screening in human cells.Science 343,84-87(2014)；

5、S.Tu et al.,Co-repressor CBFA2T2 regulates pluripotency andgermline development.Nature534,387-390(2016).

all patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention. The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

Drawings

In all the drawings, WT is a wild type, Bach1-KO or KO is Bach1 knock-out embryonic stem cells, Bach1-KO + Dox is Bach1-KO plus Dox treatment transfected by DoxBach1, Bach1-KO-Dox is Bach1-KO not plus Dox treatment transfected by DoxBach1, and M is Marker

FIG. 1:

a: protein expression of Bach1 and Oct4 in six embryonic phases of mice analyzed by quantitative published mass spectrometry (left); published RNA-SEQ data were analyzed for expression of Bach1, T, Mesp1 in mouse embryos of E5.5(cavity stage, Cav), E6.0 (prestage stage, PS), E6.5 (early-stream stage, ES), E7.0(late-middle stream stage, LMS), and E7.5 (early-but stage, EB) (right). P, posterior; epi, ectoderm.

B: the upper diagram: confocal immunofluorescence analysis of Oct4 (left 2) and Bach1 (left 1) expression in E4.5 blastocysts. Scale bar, 20 μm;

the following figures: t (left 2) and Bach1 (left 1) expression in E7.5 mouse embryos. DAPI (nuclear marker, left 3). Scale bar, 100 μm (top) and 50 μm (bottom).

C: WT hescs were immunofluorescently stained to express Bach1 (left 1) and nuclei counterstained with DAPI (left 2). Scale bar, 100 μm.

D: a Bach1 knockout hESC strategy map was generated by CRISPR-Cas9 genome editing.

E: schematic representation of Dox-inducible Bach1 hESC generated by PiggyBac transposon system.

F: nanog, Sox2, Oct4 and Bach2 mRNA levels in WT and Bach1-KO hESC were measured by qRT-PCR; the results were normalized to the measurement in WT hESC (n-3).

G-evaluation of WB detection of Nanog, Sox2, Oct4 and BACH1 protein levels (left panel) and quantification (right panel) in DoxBach1 transfected WT hESCs treated with or without Dox (Dox-), (β -actin) was used to evaluate equivalent loading (n-3), P < 0.05;. P <0.01 vs. Dox-; t test.

H: cell cycle distribution of WT and Bach1-KO hESC. Percentage of cells in the indicated cell cycle phase (n-3). P < 0.05; p <0.01 compared to WT; t test.

I: apoptosis was quantified by identifying annexin V-labeled cells by flow cytometry (n ═ 3).

Data were collected from three independent replicates and shown as mean ± SD.

FIG. 2:

a: cloned Bach1 protein levels (left) and Alkaline Phosphatase (AP) staining (right) by immunoblot analysis of Bach1-KO hescs transfected with or without DOX-treated WT hESC and DoxBach1 (right), scale bar, 500 μm.

B: the average percentage of differentiated (Diff), mixed (some cells AP positive, some negative) and undifferentiated (Undiff) clones in WT hESCs and Bach1-KO hESCs. (n-3). P < 0.05; p <0.01 compared to WT; t-test.

C: WT, Bach1-KO-Dox and Bach1-KO + Dox hESC were seeded into Matrigel-coated wells (3X 10)⁴Individual cell/well) and proliferation was assessed by monitoring cell counts during the subsequent 4-day culture period. (n-3). P<0.05；**P<0.01 vs. WT; # P<0.05，##P<0.01, one-way ANOVA compared to Bach 1-KO-Dox.

D Nanog, Sox2, Oct4 and Bach1 protein levels in WT, Bach1-KO-Dox and Bach1-KO + Dox hESC were assessed and quantified by Western blotting (left panel), β -actin levels indicated the same loading (n-3.) P <0.01, compared to WT, # P <0.01, compared to Bach1-KO-Dox, one-way anova.

E: WT and Bach1-KO hESC were immunofluorescent stained with Sox2 or Oct4, and nuclei were stained with DAPI. Scale bar, 100 μm.

F: average percentage of AP-stained and differentiated, mixed and undifferentiated cell clones for clones in hESCs treated with either a lentiviral control shRNA (Lv-Con) or a lentiviral Bach1-shRNA (Lv-shBach 1). Scale bar, 500 μm. P < 0.05; p <0.01, compared to Lv-Con; t test.

G-H: hESCs infected with Lv-Con or Lv-shBach1 were analyzed 4 days later for expression level of pluripotency gene (left) and quantification of cell number (right) (n. about.3) by Western Blot. P <0.05, P <0.01 compared to Lv-Con; t test.

I: overexpression of Bach1 enhanced the reprogramming efficiency of human dermal fibroblasts. Left panel: AP staining of reprogrammed clones. And (3) right: quantification and statistical analysis of AP positive clones (n-4). P <0.05 compared to AdGFP; t test.

Data were collected from three or four independent replicates and shown as mean ± SD.

FIG. 3:

a: WT hESCs transfected with DoxBach1 with or without Dox treatment were treated with 10. mu.g/ml Cycloheximide (CHX) and the levels of Nanog, Sox2 and Oct4 proteins were determined by immunoblotting at the indicated times. The intensity of immunoblots was quantified (n-3). P < 0.05; p <0.01 vs Dox-, ttest.

B: WT and Bach1-KO hESC were treated with (MG132) or without (DMSO) proteasome inhibitor MG132(10 μ M) for 6 hours and levels of Nanog, Sox2 and Oct4 proteins were determined by WB and quantitation (n-3). P <0.05 compared to WT, DMSO; # P <0.05# # P <0.01 vs Bach1-KO, DMSO; and (4) carrying out one-way analysis of variance.

C: WT hESCs transfected with DoxBach1 with or without Dox treatment were treated with MG132 (10. mu.M) for 6 hours. The ubiquitinated protein was pulled down using ub antibody, and ubiquitination of Nanog protein was detected using anti-Nanog antibody.

D: bach1 immunoprecipitated from WT hESC; WB then assayed for the amount of Usp7, Nanog, Sox2, Oct4 and c-Myc present in the precipitate.

E-F Usp7mRNA (E), and protein (F) levels in WT hESCs and Bach1-KO hESCs were assessed and quantified by qRT-PCR or WB, and β -actin levels indicated the same loading (n 3). P < 0.05;. P <0.01, ttest compared to WT.

G: bach1 signal transcription of the representative locus Usp7 in hescs of published ChIP-seq data and binding of Usp7 to the promoter sequence of Bach1 was assessed in WT hescs by ChIP-qPCR and IgG was used as negative control (n ═ 3). P <0.05 compared to IgG, ttest.

H: among WT hescs transfected with DoxBach1, treated with (Dox +) or without (Dox-) Dox and transfected with (Usp7si) or without (Consi) Usp7 siRNA (n ═ 3), WB analyses were performed for changes in pluripotency proteins. P < 0.05; p <0.01 compared to Dox-, Consi; # P <0.01 compared to Dox +, Consi; and (4) carrying out one-way analysis of variance.

Detection of Nanog ubiquitination in DoxBach1 transfected WT hESCs after transfection of non-specific or Usp7 specific siRNAs in the presence and absence of Dox. IP was performed using ub antibody and the level of Nanog was determined by immunoblotting.

Data were collected from three independent replicates and shown as mean ± SD.

FIG. 4

A: WT hESCs transfected with DoxBach1 with or without Dox treatment were treated with addition of MG132 (10. mu.M) for 6 hours. The ubiquinated proteins were pulled down using ub antibodies and detected by western blotting using anti-Sox 2 antibody for Sox2 ubiquitinated proteins. Oct4 was immunoprecipitated and examined for ubiquitinated protein of Oct4 by Western blotting using ub antibodies.

B: plasmid expressing Flag-Bach1 was transfected into HEK293T cells, MS was performed with Flag antibody purification, and the number of peptides knocked out was counted.

C-D WT hESCs transfected with DoxBach1, treated with (Dox +) or without Dox (Dox-) and assessed and quantified for Usp7mRNA (C) and protein (D) by qRT-PCR or WB, β -actin was used to assess equivalent loading (n 3). P < 0.05;. P <0.01 vs. Dox-; ttest.

E: ubiquitination of nanogs in WT hescs after transfection with non-specific or Usp 7-specific sirnas. Immunoprecipitation was performed using ub antibody and detected by anti-Nanog antibody.

F: expression of Usp7 and pluripotency genes was detected on WT hescs, WB treated with different concentrations of (0,5,10 μ M) Usp7 inhibitor P5091.

G: WT hescs, WB transfected with DoxBach1 with or without treatment with Usp7 inhibitor P5091(10 μ M) were tested for expression of pluripotency genes.

H: WT and Bach1-KO hESC were transfected with or without Usp7 plasmid, and the levels of Nanog, Sox2 and Oct4 proteins (n ═ 3) were determined by WB and quantified. P <0.01 compared to WT, GFP; # P <0.05, # P <0.01 vs Bach1-KO, GFP; and (4) carrying out one-way analysis of variance.

FIG. 5

A: heat map, analysis of expression of the designated genes of the different germ layers in WT and Bach1-KO hESC.

B: EB differentiation mRNA levels of mesodermal, endodermal and neuroectodermal markers in WT and Bach1-KO hESC were measured by qRT-PCR at the indicated time points, and the results were normalized to WT, day0 ═ 1(n ═ 3). P < 0.05; p <0.01 compared to WT; t test.

C: EB differentiated Day3, immunofluorescent staining of WT and KO hESC with Gata6 and T, and counterstaining of nuclei with DAPI. Scale bar, 100 μm.

Evaluation of the levels of Gata6, T, Sox2, Oct4 and Bach1 protein by WB in WT and Bach1-KO hESC levels β -actin indicate the same loading.

E: mRNA levels of mesendoderm and neuroectoderm genes were measured by qRT-PCR in WT, Bach1-KO-Dox and Bach1-KO + Dox hESC on day3 of EB differentiation; results were normalized to the measurement in WT cells (n-3). P < 0.05; p <0.01 compared to WT; # P <0.05, # P <0.01 vs Bach 1-KO; and (4) carrying out one-way analysis of variance.

F: injecting WT or Bach1-KOhESC subcutaneously into SCID mice; after 8 weeks teratomas were harvested, sectioned and stained with hematoxylin and eosin for histological analysis (left). Scale bar, 100 μm. mRNA levels of lineage marker genes were measured by qRT-PCR in teratomas of mice of WT or Bach1-KOhESC (right panel, n ═ 3). P < 0.05; p <0.01 compared to WT; t test.

Data were collected from three independent replicates and shown as mean ± SD.

FIG. 6

A: in hescs treated with lentival control-shRNA (Lv-Con) or lentival Bach1-shRNA (Lv-shBach1), mRNA levels of mesendoderm and neuroectoderm genes were measured by qRT-PCR on day3 of EB differentiation; the results were normalized to the measurement in Lv-Con cells (n-3). P < 0.05; p <0.01 compared to Lv-Con; ttest.

B spontaneous differentiation of cells into embryoid bodies, mRNA levels of markers for cardiac progenitors (Isl1), chondroprogenitors (Sox9, Col2a1), erythroid cells (β -globin), pancreas-like cells (Pdx1) and hepatocytes (Alb) in WT and Bach1-KO cells normalized to the measured values for WT, day0 cells (n-3) P < 0.05;. P <0.01, compared to WT, t test at the indicated time points, measured by qRT-PCR.

C-D: in WT and Bach1-KO hescs, with or without Usp7 sirna (c) or Usp7 plasmid (D), hescs were evaluated for mRNA levels of mesendoderm genes by qRT-PCR on day3 of differentiation and the results were normalized to the measurement in WT cells (n ═ 3). P <0.05, P <0.01 compared to WT, Consi or GFP; # P < 0.05; # P <0.01 compared to Bach1-KO, Consi or GFP; and (4) carrying out one-way analysis of variance.

FIG. 7

A: heatmaps of Bach1, EZH2 and H3K27me3 from published ChIP-seq data in hescs, genomic regions surrounding (± 5kb) TSS (transcription start site).

B: signal traces from Bach1, EZH2 and H3K27me3 ChIP-seq data published in hESC, representative loci T, Gata6, Wnt3 and Nodal.

C: in knock-out Bach1 up-regulated genes, H3K27me3 and EZH2 ChIP-seq signals within WT and Bach1-KO hESC + -5 Kb TSS were analyzed.

D: H3K27me3 and EZH2 signals of representative genes T and Gata6 in WT and Bach1-KO hESC.

E: mesendoderm genes were assessed by ChIP-qPCR (n-3) in WT and Bach1-KO hESC. The binding of Nodal and Wnt3 promoter sequences to H3K27me3, EZH2 and H3K4me 3. P < 0.05; p <0.01, compared to WT; ttest.

F: mesendoderm gene mRNA levels were measured by qRT-PCR in WT hescs transfected with DoxBach1, which had been treated with (Dox +) or without (Dox-) Dox and with (+ dzneep) or without (-dzneep) EZH2 inhibitor dzneep (20ng/mL), 12 hours after EB differentiation (n ═ 3). P < 0.05; p <0.01 vs Dox-, DMSO, # # P <0.01 vs Dox +, DMSO; and (4) carrying out one-way analysis of variance.

G-H: transfection with plasmids containing the T or Gata6 promoter sequence luciferase reporter (WT hESC transfected with DoxBach1, T or Gata6 promoter sequence (G) or wild-type and mutant form of the T promoter (H), followed by measurement of luciferase activity in cells treated with (Dox +) or without Dox (Dox-), and normalization of the results to measurements in Dox-hESC (n-3). P < 0.05; P <0.01 x vs Dox-; T test.

Data were collected from three independent replicates and shown as mean ± SD.

FIG. 8

A: WT hESC were immunofluorescent stained for Bach1 and EZH2 expression; nuclei were counterstained with DAPI. Scale bar, 50 μm.

B: bach1 immunoprecipitated from WT hESC; the amount of EZH2, EED and SUZ12 present in the pellet was then assessed by western blot.

C: EZH2, EED and SUZ12 immunoprecipitates from WT hescs; the amount of EZH2, EED, SUZ12 and Bach1 present in the precipitate was then assessed by western blotting.

D: Co-IP between Bach1 and EZH2 was performed in WT hESC in the presence and absence of DNase and RNase A.

E. transfection of HEK293 cells with Bach1 encoding Flag tag and EZH2 plasmid labelled with HA and no load, then Bach1 was immunoprecipitated with anti-Flag antibody, Bach1 was detected in immunoprecipitates and cell lysates by western blotting with anti-Flag antibody and EZH2 was detected in the pellet by western blotting with anti-HA antibody β -actin in lysates indicated the same loading.

F: bacterially expressed His-tagged EZH2 was incubated with GST-tagged intact Bach1 protein (Bach1-Full-GST) or Bach1 mutant form lacking a C-terminal bzip domain (Bach1-N-GST), or an N-terminal BTB domain (Bach 1-C-GST). Then, the reaction product was precipitated with glutathione-Sepharose 4B beads, and EZH2 was detected in the precipitate by Western blotting with an anti-His antibody.

FIG. 9

A: protein levels of H3K27me3, EZH2 and Bach1 in WT and Bach1-KO hESC were evaluated by WB, as well as protein levels of histone 3 lysine 4-trimethylation (H3K4me3), lysine 9-dimethylation (H3K9me2), lysine 27-acetylation (H3K27ac), lysine 9-acetylation (H3K9ac), SUZ12 and EED.

B-C: enrichment of H2AK119ub (B) and Ring1B (C) in the promoter regions of the mesendoderm gene, Nodal and Wnt3 was assessed by ChIP-qPCR in WT and Bach1-KO hESC (n-3).

D: ChIP-qPCR assessed H3K27me3 enriched in the promoter regions of the mesendoderm gene, Nodal and Wnt3 in WT hescs transfected with or without Usp7 siRNA (n ═ 3). P <0.05, P <0.01 compared to Consi, ttest.

E: the enrichment of H3K27me3 in the promoter regions of the mesendoderm gene, Nodal and Wnt3 was assessed by ChIP-qPCR with WT and Bach1-KO hESC, with or without Usp7 plasmid transfection (n-3). P < 0.05; p <0.01 compared to WT, GFP; one-way analysis of variance

FIG. 10 shows a schematic view of a

Heat maps of EB differentiation day3 in WT and Bach1-KO hESC illustrate gene expression in mesodermal, endodermal, Wnt and TGF- β signaling pathways.

B: EB differentiation day3, RNA-seq data of WT and Bach1-KO hESCs were analyzed using Kyoto Encyclopedia of Genes and genoms to identify signaling pathways upregulated in Bach1-KO hESCs.

Transfection of WT and Bach1-KO hESCs with TOPflash luciferase reporter genes, followed by treatment of cells with or without Wnt/β -catenin inhibitors IWR1-e (10. mu.M) or Wnt3a (10ng/mL), and normalization of luciferase activity to measurements in WT cells on day3 of differentiation (n. gtoreq.3). P < 0.05;. P <0.01 vs. WT, IWR 1-e-or Wnt3 a;. # # P <0.01 vs. Bach1-KO, IWR 1-e-or Wnt3a-, one-way anova.

D: WT and Bach1-KO hESC, qRT-PCR, treated with or without Wnt3a (10ng/mL) on day3 of differentiation, assessed the mRNA levels of the mesendoderm gene and normalized the results to measurements in WT cells (n-3). P < 0.05; p <0.01 vs WT, Wnt3a-, # P <0.01 vs Bach1-KO, Wnt3 a-; and (4) carrying out one-way analysis of variance.

FIG. 11

A: on day3 of differentiation into embryoid bodies, mRNA levels of components of the Wnt signaling pathway were measured by qRT-PCR in WT and Bach1-KO hESC and normalized to the measurements in WT cells (n ═ 3). P <0.05, P < 0.01; comparing to WT; t test.

Protein levels of Wnt3, Fzd1, the activated form of β -catenin and total β -catenin were assessed by WB in WT and Bach1-KO hESC on day3 of differentiation.

Protein levels of activated forms of β -catenin and total β -catenin in the nucleus and cytoplasm were determined by WB in WT and Bach1-KO hESC on day3 of differentiation.

D: WT and Bach1-KO hESC were transfected with TOPflash luciferase reporter gene, and then cells were treated with or without Wnt inhibitor IWP2(10 μ M) and luciferase activity was assessed on day3 of differentiation and normalized to the measurement in WT cells (n-3). P <0.01, compared to WT (IWP 2-); # P <0.01 compared to Bach1-KO (IWP 2-); and (4) carrying out one-way analysis of variance.

E: mRNA levels of the mesendoderm genes were assessed in WT and Bach1-KO hESC treated with or without IWP2(10 μ M) on day3 of differentiation (n ═ 3). P <0.01 compared to WT, DMSO; # P < 0.05; # P <0.01 compared to Bach1-KO, DMSO; and (4) carrying out one-way analysis of variance.

Mesendoderm genes and signal molecules Wnt3 and Nodal were assessed for binding to the promoter sequence of activated β -catenin on day3 of differentiation by ChIP-qPCR in WT and Bach1-KO hescs (left) and DoxBach1 with or without (Dox +).

G day3 of differentiation, mRNA levels of mesendoderm genes and protein levels of β -catenin (n 3) were assessed in adenovirus-infected WT and Bach1-KO hESCs encoding β -catenin shRNA (Ad-sh β -catenin) or control shRNA sequences (Ad); # P <0.01 vs. WT, Ad-shCtrl, # P <0.01 vs. KO, Ad-shCtrl; one-way anova.

H: on day3 of differentiation, Wnt3, Nodal and Bach1 mRNA levels (n-3) were assessed by qPCR with or without Dox (Dox +) treatment or (Dox-) treatment in DoxBach1 transfected WT hescs. P <0.05 compared to Dox-; t test.

I: WT hescs transfected with DoxBach1 were transfected with plasmids containing luciferase reporter genes, which contained the Wnt3 promoter in wild-type or mutant form, and then luciferase activity was measured in cells treated with (Dox +) or without Dox (Dox-) on day3 of differentiation and the results were normalized to that measured in Dox-cells (n-3). P <0.05 vs Dox-; ttest. Data were collected from three independent replicates and shown as mean ± SD

FIG. 12

A: on day3 of differentiation, mRNA levels of Nodal and receptors ALK4 and ACVRIIB were assessed by qRT-PCR in WT and Bach1-KO hESC and the results were normalized to measurements in WT cells. (n-3). P <0.01 vs WT, ttest.

B: protein levels of Nodal, ACVRIIB, phosphorylated Smad2 and Smad3 (p-Smad 2 and p-Smad3, respectively) and total Smad2/3 were assessed by WB on day3 of differentiation in WT and Bach1-KO hESC.

C: transfection of DoxBach 1-transfected WT hescs with a plasmid containing a luciferase reporter gene controlled by the wild-type or mutated form of the Nodal promoter; then, luciferase activity was measured in cells treated with Dox (Dox +) or without Dox (Dox-) on day3 of differentiation, and the results were normalized to the measurement in Dox-cells (n ═ 3). P <0.05 compared to Dox-; ttest.

D: p-Smad2, total Smad2/3 and histone 3 protein levels were assessed by WB in the nuclei and cytoplasm of WT and Bach1-KO hESCs examined on day3 of differentiation.

E: on day3 of differentiation, ChIP-qPCR was performed on WT and Bach1-KO hescs (left), and WT hescs transfected with DoxBach1, with (Dox +) or without (Dox-) treatment (right), and the binding of the mesendoderm gene and the promoter sequences of the signal molecules Wnt3 and Nodal was evaluated, with the results normalized to the measurements in WT or Dox-cells (n ═ 3). P < 0.05; p <0.01 as compared to WT or Dox-; ttest.

F-G assessment of mesendoderm gene mRNA levels in WT and Bach1-KO hESCs and WT and Bach1-KO hESCs treated with SB-431542(20 μ M), SB-431542 as TGF- β receptor inhibition (F) or infecting shRNA encoding β -catenin with adenovirus and treated with SB-431542(G) and assessed by qRT-PCR on day3 of differentiation, the results were normalized to the measured values in WT cells (n 3). P <0.01 vs. WT, DMSO (F) or WT, DMSO, Ad-shrl (G); # P <0.01 vs. KO, DMSO (F) or KO, DMSO, Ad-shrl (G); single factor analysis of variance.

Data were collected from three independent replicates and shown as mean ± SD.

Bach1 interacts with Nanog, Sox2 and Oct4 to stabilize pluripotency factors and maintain stem cell self-renewal Bach1 also prevents mesendoderm differentiation by recruiting PRC2, which results in deposition and gene silencing of H3K 3527 me3, which in turn inhibits Wnt/68-catenin and Nodal/Smad2/3 signaling pathways.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

In each of the following examples, the major equipment and materials were obtained from several companies as indicated below:

1. experimental Material

1.1 test cells: the human embryonic stem cell in this experiment was H7(Wicell Research Institute).

1.2 Experimental instruments: a

General PCR instrument (Thermo Co.); infinite TM 200 series multifunctional microplate reader (Tecan corporation); real-time quantitative PCR instrument (BIO-RAD Co.); a developing apparatus (Tanon Corp.); leica IM50 image acquisition system, Leica Dmi8 fluorescence microscope (Leica).

1.3 antibodies

Bach1 (RND/Santa Cruz Co.), EZH2, SUZ12, Non-p- β -catenin, smad2/3, Histone 3(CST Co.), EED, GST antibody (Proteintetech), T, Msx2, gat 6, gat 4(RND Co.), Sox2, Oct4, Nanog, Total β -catenin, ACVRIIB (Santa Cruz Co.), Wnt3, Nodal, H3k9me2, H3k27AC, H3k9AC (Abcam Co.), β -actin (Affiniy Co.), p-smad2, p-smad 3(Abway Co.), H3k27me3, H3k4me3 (Ablipore, Sigma).

2. Statistical analysis

The experimental data are expressed as Mean ± standard deviation (Mean ± SD). Two sets of data were tested using unpaired t test (Non-pair test) and multiple sets of data were analyzed using One-way ANOVA. The significance is shown by P < 0.05. The plots were generated using GraphPad Prism 5 mapping software.

Example 1: preparation of Bach1 knockout hESC

1. Construction of sgRNA

Bach1-KO hESC was generated by CRISPR/Cas9 gene editing technology. Sgrnas (http:// criprpr. mit. edu /) targeting genomic regions of interest were designed using CRISPR design tools, gene targeted against Bach1 exon 2, screened, and primer synthesized by shanghai sony corporation. sgRNA-1 target site sequence (SEQ ID NO: 1): 5'-GAC AGC GGT TCC GCG CTC AC-3' and its reverse complement are (SEQ ID NO: 2): 5'-GTG AGCGCG GAA CCG CTG TC-3' are provided.

The targeting strategy is shown in FIG. 1D.

2. Cloning of Guide RNA into the vector of Lenti criprpr V2(Addgene, Watertown, MA, #52961)

2.1 digestion of the Lenti criprpr V2 vector

The Lenti criprpr V2 vector was cleaved at 37 ℃ for 2h

TABLE 1.120. mu.l digestion system

2.2 purification and recovery of the cleaved Lenti criprpr V2 vector

The universal DNA purification and recovery kit for the Tiangen is utilized to purify and recover the Lenti criispr V2 vector after enzyme digestion, and the purification and recovery steps are shown in the specification of the kit.

2.3 upstream and downstream Oligo phosphorylating and annealing Guide RNA sequences

Annealing the Oligo's in the PCR instrument according to the system in the following table,

the procedure is as follows: 30min at 37 DEG C

Cooling at 95 deg.C for 5min to 25 deg.C at a cooling rate of 5 deg.C/min

TABLE 1.2oligo information

TABLE 1.320. mu.l digestion system

2.4 ligation reaction overnight

And (3) performing a ligation reaction on the enzyme-digested vector purified and recovered in the step 2.1 and the annealed Oligo in the step 2.3, wherein the system is as follows:

TABLE 1.420. mu.l of digestion system

2.5 conversion Panels

The above connection system is transformed and paved according to the following operation

1) Preparation of selection medium: ampicillin 120 ng/. mu.l, kanamycin 60 ng/. mu.l;

2) thawing DH5 α on ice;

3) slightly mixing;

4) adding all the DNA samples after connection;

5) standing in ice for 30 min;

6) activating the thallus at 42 ℃ for 60 sec;

7) standing in ice for 1-2 min;

8) 500ul of LB liquid without antibiotics is added;

9) shaking culture at 37 ℃ for 45min (160 and 225 rpm);

10) spread on selection plates containing antibiotics. 100mm large dish 500. mu.l:

11) the culture was carried out overnight at 37 ℃.

2.6 cloning, sequencing and identification

And (3) selecting 3-5 clones from the plate for sequencing, and extracting plasmids from positive clones for later use.

3. Transfection of hESCs

Transient transfection into hescs was performed by electroporation (Life technologies, Carlsbad, CA, # MPK5000) and screened with puromycin (Invivogen, San Diego, CA, # ant-pr-5 b).

Transfection by electroporation

1) Digestion of H7 cells:

the H7 cells to be transfected were cultured using six-well plates until the cells grew substantially to the petri dish. The culture medium was discarded, the cells were washed with 1 × PBS wash, and 1ml of the digesting enzyme Accutase was added to each well to digest the cells from adherent state to suspension. Rapidly neutralizing, centrifuging 20 μ l of cell suspension at 4 deg.C for 5min at 640g, collecting cell precipitate, and adding 20 μ l of R buffer to resuspend the cells;

2) adding 3 mu g of plasmid into the cell sediment, and uniformly mixing;

3) sucking the mixed liquid by using a 10-microliter electric transfer gun head;

4) electric conversion: adding 3ml of E buffer into the electric rotating cup;

5) and (3) electrotransfer conditions:

1100V Voltage

10ms Width

1pulses Pulses

pressing a Start key, and waiting for 2 seconds;

6) transferring the cells in the head of the electrotransfer gun to a culture dish of 10cm for culture;

7) if the cell amount is less, repeating the steps of 3-5;

8) cleaning the electric rotating gun head and the electric rotating cup by using PBS;

9) after the cells are cultured for 48h, Puromycin antibiotics are added for screening to obtain Bach1 knockout hESC (Bach1-KO hESC). After one week of selection culture, cell monoclonals were picked.

As shown in FIG. 2A, no BACHI protein band could be detected in Bach1-KO hESC cells as detected by West-Blot, whereas a BACHI protein band could be detected in WT cells.

Example 2: dox inducible Bach1 hESC1 is generated by PiggyBac transposon system, and hBach1 overexpression plasmid is constructed to Piggy Bac transposon system

1.1 amplification of hBach1 target fragment

1) Primers and KOD high-performance fidelity enzyme for amplifying hBach1 target fragments, which are designed according to the following table 2.1, are amplified according to the PCR system in the following table 2.2, and the amplification PCR program is shown in the following table 2.3.

TABLE 2.1 amplification details

TABLE 2.250. mu.l PCR System

TABLE 2.3 PCR amplification conditions

2) All PCR products were mixed with 6 × Loading Buffer, all loaded, subjected to 120V electrophoresis for 45min, and the band of interest was cut out of the agarose gel according to its size, and weighed in a new 1.5ml EP tube.

3) And then, purifying and recovering the hBach1 target fragment according to the steps of the Tiangen universal DNA purification and recovery kit.

2. The enzyme digestion PB-TRE3G-eGFP-PGK-Neo plasmid

The PB-TRE3G-eGFP-PGK-Neo plasmid in the Piggy Bac transposon system is subjected to enzyme digestion reaction according to the system in the following table 2.4, is kept overnight at 37 ℃, and is purified and recovered.

TABLE 2.4 digestion of 50. mu.l

3. Ligation of the Gene fragment of interest to the vector

The purified and recovered vector and the target fragment after enzyme digestion are connected for 3 hours at 37 ℃ according to a connection system shown in Table 2.5.

TABLE 2.5 ligation 10. mu.l system

4. Conversion and planking

The procedure for transformation of the planks was the same as in example 1.

5. Selecting clone and sequencing identification

And (3) selecting 3-5 monoclonals from the bacterial plate cultured overnight, shaking bacteria, sequencing, selecting clones with positive sequencing, and extracting plasmids for later use.

6. Electrotransfection of embryonic stem cells

Hescs and Bach1 knockout hescs obtained in example 1 (Bach1-KO hESC) were transiently transfected by electroporation (Life technologies, Carlsbad, CA, # MPK5000) and screened with puromycin (Invivogen, san diego, CA, # ant-pr-5 b). (see example 1 for specific methods), and finally, the inducible Bach1 hESC line (DoxBach1) and Bach1-KO hESC (Bach1-KO + Dox) transfected by DoxBach1 are obtained.

FIG. 1E is a schematic diagram of the expression system of Bach1 induced by Dox. DOX-induced WT hESC overexpression Bach1 As shown in FIG. 1G, BACH1 protein expression was increased after DOX induction. Results of DOX-induced Bach1-KO hESC are shown in FIG. 2A, and BACH1 protein bands were detected by both WT and Bach1-KO + Dox after Dox induction, while BACH1-KO (Bach1-KO-Dox) without Dox induction failed to detect BACH1 protein.

Example 3: bach1 maintains the characteristics of its stem cells in hESCs

The growth of the cells, the Alkaline Phosphatase (AP) activity, the protein levels of the pluripotency factors Sox2, Oct4 and/or Nanog were determined by Western Blot, Alkaline Phosphatase (AP) activity assay, lentivirus infection, RT-PCR, cytometry, flow, immunofluorescence.

The results show that: bach1 was expressed in both the Inner Cell Mass (ICM) and trophectoderm cells, and suggested that Bach1 co-localized with Oct4 in ICM cells of mouse blastocysts (fig. 1B, top panel). Bach1 is also widely expressed in endoderm, mesoderm and ectoderm of E7.5 embryos, while T is predominantly expressed in the primitive streak region (FIG. 1B, bottom panel).

Bach1 expression was observed in both the nucleus and cytoplasm of undifferentiated hescs (fig. 1C). Generating Bach1 knockout hESC (Bach1-KO hESC) by CRISPR-Cas9 genome editing (fig. 1D) and generating a doxycycline inducible Bach1 hESC line (DoxBach1) schematic using PiggyBac transposon system (fig. 1E); WB demonstrated Bach1 null expression in Bach1-KO hESC, Bach1 knock-out did not affect Bach2 expression profile (fig. 1F), and Bach1 expression was restored in Bach1-KO hESC transfected with Dox 1 after treatment with Dox (fig. 2A).

The clone of Bach1KO-hESCs was flatter than that of wild-type hESCs (WT hESCs), and Alkaline Phosphatase (AP) activity was lower in Bach 1-KOhESCs than WT hESCs, and Dox was added to Bach1-KO hESCs transfected with DoxBach1 to restore them to near WT levels (FIG. 2A). Bach1-KO hESC contains a greater proportion of differentiated or mixed clones (where the proportion of mixed and differentiated state clones in WT hESC is 6% and 2%, respectively, and the proportion of mixed and differentiated state clones in Bach1-KO hESC is 25% and 15%, respectively) (fig. 2B), Bach1-KOhESC proliferates more slowly (fig. 2C, the lowest line is Bach1-KO-Dox, the uppermost line is Bach1-KO + Dox, the middle line is WT), the expression of protein levels of the pluripotency factors Sox2, Oct4 and/or Nanog is reduced (fig. 2D-2E) (where in the right graph of fig. 2D, the left bar corresponding to each protein is WT, the middle bar is Bach1-KO-Dox, and the right bar is Bach1-KO + Dox); notably, Bach1 knock-out did not affect the mRNA expression levels of Nanog, Sox2 and Oct4 (fig. 1F), indicating that Bach1 promotes protein levels of pluripotency factors that occur post-transcriptionally.

In Bach1-KO hESCs transfected with DoxBach1, Dox treatment restored WT-like colony morphology and increased proliferation and expression of pluripotency factors (FIG. 2A and FIGS. 2C-2D). Expression of pluripotency factors was also increased in Dox-Bach 1-transfected WT hESCs after treatment with Dox (fig. 1G) (where Nanog was increased 1.7 fold, Sox2 was increased 1.8 fold, Oct4 was increased 2.3 fold), and cell cycle analysis showed that Bach1-KO was at G1 stage (fig. 1H) in greater proportion than WT hESCs (WT 26.09%, Bach1-KO 51.24%), consistent with the lower proliferation rate observed in Bach1-KO cells, while knockout Bach1 had no significant difference in the effect on apoptosis (fig. 1I) (WT 2.01%, Bach1-KO 1.88%). Furthermore, infection of hESCs with lentiviral Bach1-shRNA was found to be similar to that observed in Bach1-KO hESCs (fig. 2F-2H), reprogramming human fibroblasts with the transcription factors Oct4, Klf4, Sox2 and c-Myc, and overexpression of Bach1 was found to promote the efficiency of reprogramming, increasing the number of hESC-like (i.e., AP-positive) clones (fig. 2I) (WT 19.75, Bach1-KO 34 per 5 million cells). Taken together, these results indicate that Bach1 helps to maintain stem cell pluripotency and self-renewal, facilitating reprogramming of cells.

Example 4: bach1 interacted with Nanog, Sox2 and Oct4 and promoted its stability by recruiting the deubiquitinase Usp 7.

The protein levels of the pluripotency factors Sox2, Oct4 and/or Nanog and the effect of the deubiquitinase Usp7 were determined by Western Blot, Immunoprecipitation (IP) and Mass Spectrometry (MS), Chip-qPCR method.

Since knockout of Bach1 in hESCs resulted in a decrease in Nanog, Sox2 and Oct4 protein levels without affecting their mRNA levels, we investigated whether Bach1 helped to maintain the protein stability of the pluripotency factor of stem cells by preventing the rate of pluripotency factor degradation. When Dox-induced Bach1 overexpression significantly prolonged its half-life and delayed the degradation of Nanog, Sox2 and Oct4 proteins (fig. 3A) (Dox-group Nanog, Sox2 and Oct4 degraded to 7.9%, 12.2% and 32.0%, respectively, after CHX 9h addition), while Bach1 overexpression group (Dox +) Nanog, Sox2 and Oct4 degraded to 29.2%, 86.0% and 74.1%, respectively), naog, Sox2 and Oct4 protein levels (fig. 3B) in Bach1-KO sc could be restored with proteasome inhibitor MG132 at the same time when Dox-Bach 1 transfected WT hESC was treated with Cycloheximide (CHX) to block the synthesis of new proteins (where fig. 3B lower panel, each protein corresponds to the first bar of Bach + WT, the second bar of Bach 62-KO 1, the third bar of Bach + MG 632 + koch 685 132). Bach1 overexpression also significantly reduced ubiquitination of Nanog, Sox2 and Oct4 (FIG. 3C and FIG. 4A), suggesting that Bach1 promotes the stability of these pluripotency factors by disrupting ubiquitin-dependent proteasome degradation. Additionally, 293HEK was transfected with Flag-Bach1 plasmid, Usp7 could be detected by Immunoprecipitation (IP) and Mass Spectrometry (MS), endogenous IP was made in hescs where Bach1 and Usp7 were bound, and Bach1 also interacted with Nanog, Sox2 and Oct4, but not c-Myc (fig. 3D). Whereas the mRNA and protein levels of Usp7 were significantly lower in Bach1-KO hESCs than in WT hESCs (FIGS. 3E-3F) (where the mRNA for Usp7 was reduced to 34.2% and the protein level was reduced to 49.0%), the expression of Usp7 was increased in hESCs overexpressing Bach1 (FIGS. 4C-4D) (where the mRNA for Usp7 was increased 2.6-fold and the protein level was increased 1.5-fold). Chip-qPCR assay confirmed that Bach1 occupied the Usp7 promoter region (fig. 3G), indicating that Usp7 is a directly regulated target by Bach1, when Usp7 activity was down-regulated by siRNA transfection and/or treatment with Usp7 inhibitor P5091, Nanog ubiquitination was increased in WT hescs (fig. 4E), expression of pluripotency gene protein levels was decreased (fig. 4F), and the increase in pluripotency gene protein levels caused by Bach1 overexpression could be reversed (fig. 3H and fig. 4G) (in the lower panel of fig. 3H, the first bar on the left for each protein was Dox- (Bach1 off) + Consi, the second bar was Dox- (Bach1 off) + Usp7si, the third bar was Dox- (Bach1on) + Consi, and the fourth bar was Dox- (Bach1on 397) + 7 si). Furthermore, overexpression of Usp7 in Bach1-KO hESC can partially reverse the decrease in the levels of their pluripotency gene proteins (fig. 4H) (where in the right panel of fig. 4H the first bar to the left for each protein is WT + GFP, the second bar is Bach1-KO + GFP, the third bar is WT + Usp7, and the fourth bar is Bach1-KO + Usp7), and inhibition of Usp7 can reverse the decrease in Nanog ubiquitination caused by Bach1 overexpression (fig. 3I). Taken together, these results indicate that the stability of Bach 1-induced pluripotency proteins is mediated by the deubiquitinating activity of Usp 7.

Example 5: bach1 knockout promoted the differentiation of mesendoderm of hescs.

We investigated whether loss or overexpression of Bach1 altered germ layer marker expression in hescs spontaneously differentiating into Embryoid Bodies (EBs). The detection method comprises the following steps: and detecting cell markers of mesoderm, endoderm and ectoderm by using methods such as immunofluorescence, Western Blot, lentivirus transfection, qRT-PCR, teratoma formation experiment and the like.

During EB differentiation, most of the mesodermal, endodermal and ectodermal cell markers peaked at Day4 or Day6 expression, consistent with previous reports; therefore, as the purpose of these experiments was to study the role of Bach1 in the early stages of hESC differentiation, our analysis was performed on days 0 to 6 of hESC-EB differentiation. In WT hescs, mRNA levels of mesodermal markers (T and Msx2), endodermal markers (Gata4 and Gata6) and neuroectodermal markers (Otx2 and Pax6) increased gradually four days before spontaneous EB differentiation (fig. 5B), whereas the time-dependence of marker expression was approximately similar in WT-and Bach1-KO hescs, endodermal mesodermal marker expression was consistently higher in Bach1-KO hescs, while neuroectodermal marker expression was consistently lower than WT hescs (fig. 5B). When assessed by immunofluorescence (FIG. 5C) and/or Western Blot (FIG. 5D), the expression of Gata6 and T in Bata1-KO hESCs was also significantly higher, and Sox2 and Oct4 expression were lower than WTHESCs, while Bach1 expression was up-regulated on

days

2 and 4 of hESC differentiation and decreased on day6 (FIG. 5D). Similar changes in germ layer marker expression were detected on day3 of hESC differentiation when Bach1 was knocked down by infection with lentiviral Bach1-shRNA (fig. 6A), and qRT-PCR measurements showed that these changes could reverse the changes in both ectodermal and endodermal marker genes caused by Bach1 knock-out upon induction of Bach1 overexpression with Dox (fig. 5E).

EB differentiated for 12 days, expression of markers for cardiac progenitors (Isl1), chondrocytes (Sox9, Col2a1), erythroid cells (β -globin) and pancreas-like cells (Pdx1) was higher in Bach1-KO hESCs than in WT hESCs, whereas expression of hepatic marker gene (Alb) was unchanged (FIG. 6B), whereas WT and Bach1-KO hESCs formed teratomas containing all three germ layers (endoderm, mesoderm, ectoderm) upon injection into adult SCID mice, mesoderm markers were higher in Bach1-KO hESCs and lower in neuroderm markers (FIG. 5F) (6.8-fold increase in mesoderm T, 2.6-fold increase in Gata, 1.9-fold increase in Gata6, 8.2-fold increase in a Gata, ectoderm gene until the expression of the right stem cell protein of Bach 638 + KO 19. WT, 8-fold decrease in a consistent with increasing amounts of left-KO mRNA expression of the Bach 638 + KO gene, 8 + KO β 9 + 7. WT 367, and 7 + 7. WT 367 + 8 + WT 3. WT + WT.

Example 6: bach1 inhibited mesendoderm gene expression in hESCs by H3K27 trimethylation catalyzed by EZH2 in the mesendoderm gene promoter.

By using methods such as ChIP-seq, ChIP-qPCR, co-immunoprecipitation, western blotting, RT-PCR, luciferase reporter gene and the like, changes of Bach1, EZH2 and H3K27me3 in the enrichment of mesendoderm differentiation gene promoter regions are detected, and whether the interaction between Bach1, EZH2 and H3K27me3 is involved in regulating the expression of hESC differentiation genes is researched.

Analysis of Bach1 knockdown induced upregulated differentiation genes, and found that H3K27me3 and EZH2 in Bach1-KO hESCs had decreased enrichment at ± 5kb of TSS (fig. 7C), and also decreased enrichment in the promoter regions of mesendoderm development regulators (T, Gata6, Wnt3, Nodal, Mesp1, Msx2, and Gata4) (fig. 7D), ChIP-qPCR further confirmed that H3K27me3 and EZH2 in Bach1-KO hESC had decreased enrichment in the promoter regions of T, Gata6, and many other mesendoderm differentiation genes (fig. 7E) (about 50% decrease in enrichment of H3K27me3 and EZH2 in Bach1-KO hESC). Notably, binding of H3K4me3 to these promoters was elevated in Bach1-KO hESCs (fig. 7E). In WT and Bach1-KO hESC, the levels of H3K27me3, EZH2, H3K4me3, H3K9me2, H3K9ac and H3K27ac proteins were similar (FIG. 9A).

After Dox treatment, the mesendoderm gene mRNA levels in the WT hescs transfected with DoxBach1 decreased, but the EZH2 inhibitor dzneep partially abolished this decrease (fig. 7F), indicating that EZH2 is involved in the inhibition of mesendoderm gene expression by Bach 1. The binding site of Bach1 was present in the T and Gata6 promoter regions (FIG. 7B), so we constructed the luciferase reporter of the T and Gata6 promoters containing the binding site of Bach1, and the transcriptional activity of the T or Gata6 luciferase reporter gene was significantly reduced by Dox-induced overexpression of Bach1 (FIG. 7G) (reduction of Gata6 to 21.2% and reduction of T to 44.1%), whereas the transcriptional activity was not affected by the overexpression of Bach1 after mutation of the binding site of Bach1 (FIG. 7H). Bach1 also co-localized with EZH2 in the nucleus (fig. 8A), and co-immunoprecipitation experiments confirmed that Bach1 bound to EZH2, as well as the other two subunits EED and SUZ12 in the PRC2 complex (fig. 8B and 8C). Notably, the binding between Bach1 and EZH2 persists in the presence of DNase or RNaseA (fig. 8D), indicating that no DNA or RNA molecules are required for the interaction between Bach1 and EZH 2. Transfection of Flag-tagged Bach1 and HA-tagged EZH2 in 293FT demonstrated that Bach1 and EZH2 bound exogenously (FIG. 8E), GST-pull down experiments constructed Full-length Bach1 with GST-tag (Bach1-Full-GST), GST-Bach1 deleted C-terminal (Bach1-N-GST), GST-Bach1 deleted N-terminal (Bach1-C-GST), and His-tagged EZH2(EZH2-His) were co-incubated and passed through glutathione-agarose beads, and as a result, it was confirmed that EZH2 co-precipitated with the Full-length sequence most strongly (FIG. 8F). Taken together, these observations indicate that the N-and C-terminal regions of Bach1 interact directly with EZH2, and Bach1 recruits EZH2 to the Bach1 binding site in the promoter regions of several mesendoderm genes, promoting trimethylation of H3K27 and inhibiting mesendoderm gene expression.

Although the enrichment of H3K27me3 in some mesendoderm genes was slightly reduced when WT hescs were transfected with Usp7 siRNA (fig. 9D), the reduction of H3K27me3 in Bach1-KO hescs could not be reversed by overexpression of Usp7 (fig. 9E). Furthermore, although there is evidence that PRC1 was recruited to mesoderm genes, Bach1 knockout did not affect E3 ligase Ring1B and H2AK119ub in mesendoderm gene enrichment (fig. 9B and 9C), where Ring1B is a component of PRC 1.

Example 7 Bach1 knockout promotes mesendoderm differentiation by activating the Wnt/β -catenin signaling pathway.

By using methods such as ChIP-qPCR, western blotting, RT-PCR, luciferase reporter gene and the like, expression changes of Wnt/β -catenin signal channel ligand, receptor and downstream effector molecules after Bach1 knockout are detected, and whether the interaction between Bach1 and the Wnt/β -catenin signal channel participates in the promotion of mesendoderm differentiation or not is researched.

Mesendoderm differentiation of hESCs was at least partially mediated by activation of Wnt/β -catenin signaling pathway, RNA-seq analysis at day3 of EB differentiation showed higher expression of endoderm and Wnt signaling genes in Bach1-KO hESCs (fig. 10A-10B).

1) Several ligands for Wnt (including Wnt3, Wnt2B, Wnt5B), receptors (Frizzled-1, -2, -10), and downstream target genes (LEF1) (fig. 11A-11B) were elevated in expression (with Wnt3 elevated 51.0 fold, Wnt2B elevated 5.0 fold, Wnt5B elevated 55.2 fold, Fzd1 elevated 4.1 fold, Fad2 elevated 5.4 fold, Fzd10 elevated 6.6 fold, LEF1 elevated 47.3 fold);

2) promoting protein expression of the activated form of β -catenin and total β -catenin (FIGS. 11B-11C);

3) promote the transcriptional activity of the TOPflash luciferase reporter (FIG. 11D) (3.4 fold increase in Bach1-KO hESC) which contains 8 copies of the Tcf/lef binding site however, when cells were treated with either IWP2 (Wnt-producing inhibitor (FIG. 11D)) or IWR1-e which inhibits β -catenin (FIG. 10C) (WT-and Bach1-KO hESC decreased to 0.39 and 0.43 fold respectively after addition of IWP2 and 0.45 and 0.53 fold respectively after addition of IWR 1-e), the TOPflash activity was similar in WT-and Bach1-KO hESC, and the expression of the TOPflash and mesendoderm genes was significantly higher in Bach1-KO cells than in WT cells (FIG. 10C-10D) when stimulation of signaling was added to cultured cells in TOP 3 Wnt3 a.

ChIP-qPCR analysis showed that in Bach1-KO hESC, the activated form of β -catenin binds more to the mesendoderm promoter than to the WT hESC, whereas when DOxBach1 transfected WT hESC was treated with Dox, its binding was inhibited (FIG. 11F) (β -catenin enrichment in different promoter regions was increased 10-30 fold in Bach1-KO hESC, overexpression of Bach1 inhibited β -catenin enrichment at these sites to around 0.5 fold). on day3 of EB differentiation of Bach1-KO hESC, mesendoderm differentiation gene expression was increased, and when Ad- β -catenin was infected or Bach1-KO hESC or treated with IWP2, expression of mesendoderm could be significantly decreased (FIGS. 11G and 11E), whereas when analyzed for ChIP 1 in Bach hESC, the expression of the mesendoderm could be analyzed for the expression of the Wnt 1-seq ID Wnt gene, thus the expression of the gene binding of the mesendoderm gene could be shown to be at least the expression inhibition of the binding of its binding to the expression of the gene binding of the promoter region of the gene 587-BACh cDNA 5828-CD 587-CD 7-CD (FIG. 7-CD) observed by the binding of Bach cDNA reported in Bach cDNA reported by the binding of Bach promoter region reported in Bach 1-CD 7-CD 8-KOSC, thus the binding was at least the binding of the gene found in Bach promoter region of the binding map 7-CD 95-CD 7-CD 8-KO-CD 7 gene (FIG. 7-CD 8-KOSC, the binding was inhibited by the binding to the binding of the.

Example 8: bach1 knockdown promoted mesendoderm differentiation by activating Nodal signaling pathway.

By using methods such as ChIP-qPCR, co-immunoprecipitation, western blot, RT-PCR, luciferase reporter gene and the like, expression changes of Nodal/Smad2/3 signal channel ligand, receptor and downstream effector molecule after Bach1 knockout are detected, and whether the interaction between Bach1 and the Nodal signal channel participates in promotion of mesendoderm differentiation regulation or not is researched.

During mesendoderm differentiation, Wnt and Nodal, a member of the TGF- β superfamily, generally have a synergistic effect, and analysis of RNA-seq results at day3 of EBs differentiation shows that Nodal and TGF- β signal transduction genes in Bach β -KO hESCs express more than WT hESCs (FIGS. 10A-10B). accordingly, we investigated whether Bach β effects in inhibiting mesendoderm differentiation are mediated in part by modulation of Nodal signaling. analysis of Bach β ChIP data identifies Bach β binding sites in the vicinity of Nodal TSS in hESCs, and at day3 of EB differentiation, Bach β -KO hESCs, Nodal mRNA (FIG. 12A) (increased by 2.78 times) and protein (FIG. 12B) expression levels are increased when Nodal-CD 3 CD + CD 3 + CD 7 and the expression level of Nodal receptor ACVRIIB (see that the expression level of Nodal receptor ACVRIIB and the expression level of Nodal receptor BAC is not changed at day3 of Bach β, and the expression of Nodal-CD + CD 3-CD + CD 7. C + CD) shows that the expression of Bach CD + CD is increased when the expression of Bach CD 3 CD + CD, the Nodal-CD, the expression of Bach CD 3. C + CD, the expression of Bach CD + CD 3. C + CD, the expression of Bach CD + CD 3 CD, and the cDNA is increased, and the cDNA, and the expression of the cDNA + CD 3 CD + CD 3 CD, thus the expression of the cDNA is increased in the CD, the expression of the cDNA, and the CD-CD, the expression of the CD-CD 3 CD-CD shows that the expression of the CD, the expression of the CD 3 CD, and the CD-CD shows that the CD-CD shows that the expression of the cDNA is increased in the CD, when the CD, the expression of the CD-CD, the expression of the CD-CD, the expression of the CD-CD, the CD-CD, the expression of the CD-CD shows that the expression of the CD-CD, the CD-CD, the expression of the CD shows that the expression of the CD-CD shows that the CD stem cell CD shows that the CD-CD shows that the CD-CD is increased in the CD-CD, when the CD-CD, the binding sites of the CD, when the CD, the binding sites of the CD, the CD-CD, the CD-CD.

Bach1 is highly expressed at the blastocyst stage of mouse development, and mice with all Bach1 full exon knockouts are sublethal, but their underlying molecular mechanisms have not been fully elucidated. Our data indicate that Bach1 maintains the sternness of hescs by stabilizing pluripotency factors and inhibiting expression of lineage-specific genes (fig. 12H). We also show that deletion of Bach1 in hESCs results in activation of mesendoderm gene expression and differentiation; thus, Bach1 activity appears to be a key determinant of cell fate and lineage specification in hESCs.

The regulation of pluripotency factor in embryonic stem cells is in part via ubiquitination and deubiquitination, for example Oct4 and Sox2 protein levels are regulated by E3 ligase WWP2, the deubiquitinase Usp21 stabilizes Nanog, maintaining pluripotency, whereas FBXW8, an E3 ligase, ubiquitinates Nanog, and destabilizes it, thus promoting ESC differentiation however, although Usp7 maintains pluripotency of neural progenitor/stem cells by deubiquitinating and stabilizing REST and c-Myc, it is largely unknown whether Usp7 activity contributes to the role of Bach1 in hESC stems. In this study, we demonstrated that Bach1 directly targets and increases expression of Usp7 in hESCs, and Bach1 interacts with Nanog, Sox2 and Oct4 and recruits Usp7 to their promoters, facilitating pluripotency and maintenance of hESC stem cells. We also found that the increase in mesendoderm gene expression associated with the Bach1 knockout could be partially abolished by up-regulation of Usp7 expression, suggesting that Usp7 may be a component of the mechanism by which Bach1 lacks promotion of hESC differentiation, with secondary consequences probably due to decreased levels of pluripotency proteins.

Previous reports show that Bach1 regulates the expression of many genes involved in cell cycle and apoptosis, and promotes apoptosis while inhibiting proliferation of human umbilical vein endothelial cells; however, Bach1 knockout in hESCs did not alter apoptosis, and Bach1-KO hESCs had a lower proliferation rate than WT hESCs. Taken together, these observations suggest that the effects of Bach1on apoptosis and cell proliferation may vary greatly among different cell types.

It is noteworthy that although Bach is known to mediate cancer metastasis, as well as oxidative stress, heme oxidation and angiogenesis, and knocking down Bach can reduce the level of H3K27me enrichment for specific gene promoters in breast cancer cells, the enrichment of H3K27me in the promoter region of mesendoderm differentiation genes is reduced and not only due to reduced expression of Usp, Usp overexpression cannot restore such reduction, PRC is also essential for inhibitory H3K methylation, and is closely related to stem cell pluripotency and cell lineage differentiation during development, EZH subunits of PRC catalyze dimethylation and trimethylation of H3K, and defects in EZH reduce pluripotency and promote mesendoderm differentiation, while the mechanism of EZH transcription inhibiting mesendoderm genes is still confirmed to inhibit the effects of the mesendoderm differentiation and promote the recruitment of Wnt in cells by the reciprocal inhibition of Wnt/endoderm 3 and recruitment of Wnt signaling, the inhibitory action of Wnt signaling in the promoter, and repression the transcriptional repression the stem cell lineage, the transcriptional repression of the stem cell proliferation of Smad stem cell, no receptor, no-homing factor, no-t-s, no-t-s, and inhibit the effects of the transcriptional repression the proliferation of stem cell-stem cell, no-t-s, and/s-s, and promote the effects of the activity of stem-t-s, and the activity of stem-s, and the transcriptional-t-s, and/s, and promote the effects of stem-s, and/s, and the effects of stem-s, and the effects of the activity of stem-s, and the effects of stem-s, and the activity of stem-s, and the effects of stem-s, and/s, including the effects of stem-s, and the activity of stem-s, and the effects of stem-s, and the activation of stem-s, and the activity of stem-s, and the activity of stem-s, and the inhibitory action of stem-s, and the activity of stem-s, and the inhibitory action of stem-s, and the inhibitory.

Sequence listing

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Claims

1. A modified transcription factor Bach1 gene, wherein the transcription factor Bach1 gene is modified such that the activity or expression of Bach1 protein of a cell is inhibited or the activity or expression of Bach1 protein of a cell is increased.

2. Use of a modified transcription factor Bach1 gene for modulating stem cell pluripotency, self-renewal, proliferation, and/or reprogramming.

3. Use of a regulatory agent of the transcription factor Bach1 for modulating stem cell pluripotency, self-renewal, proliferation and/or reprogramming.

4. A method of modulating pluripotency, self-renewal, proliferation and/or reprogramming of a stem cell, wherein the Bach1 gene of the stem cell is modified such that the Bach1 protein activity or expression of the stem cell is inhibited or the Bach1 protein activity or expression of the stem cell is increased.

5. The use or method as claimed in any one of claims 2 to 4 wherein the transcription factor Bach1 interacts with the deubiquitinating protease Usp7, the pluripotency factor Nanog, Sox2 and/or Oct4, the PRC2 complex, the Wnt/β -catenin signalling pathway, and/or the Nodal/Smad2/3 signalling pathway to modulate stem cell pluripotency, self-renewal, proliferation and/or reprogramming.

6. A sgRNA sequence, characterized in that the sgRNA sequence targets the transcription factor Bach1 gene of a cell, while the sgRNA is unique on the target sequence on the transcription factor Bach1 gene to be altered and complies with the sequence arrangement rule of 5 ' -NNN (20) -NGG3 ' or 5 ' -CCN-N (20) -3 ', and the sequence of the 5 ' end target site targeted by the sgRNA is as set forth in SEQ ID NO: 1, the sequence of the sgRNA-targeted 3' end target site is shown as SEQ ID NO: 2, respectively.

7. A method for culturing a stem cell line is characterized in that BACH1 protein is added in the method to culture the stem cell line.

8. A stem cell line comprising the modified transcription factor Bach1 gene of claim 1 or obtained by the method of claim 7.

9. Use of the stem cell line of claim 8 in the preparation of a pharmaceutical composition, wherein the pharmaceutical composition modulates stem cell pluripotency, self-renewal, proliferation, and/or reprogramming.

10. A pharmaceutical composition comprising the stem cell line of claim 8.