Research ArticleCELL BIOLOGY

Bach1 regulates self-renewal and impedes mesendodermal differentiation of human embryonic stem cells

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Science Advances  13 Mar 2019:
Vol. 5, no. 3, eaau7887
DOI: 10.1126/sciadv.aau7887
  • Fig. 1 Bach1 is required for maintaining hESCs identity.

    (A) Immunoblot analysis of Bach1 protein level and alkaline phosphatase (AP) staining of colonies in WT hESCs and DoxBach1-transfected Bach1-KO hESCs that had been treated with Dox (Bach1-KO + Dox) or without Dox (Bach1-KO − Dox). Scale bars, 500 μm. (B) Bars show mean percentages of differentiated (Diff.) AP-negative, mixed (some cells AP-positive, some-negative), and undifferentiated (Undiff.) uniformly AP-positive embryonic stem cell colonies in WT hESCs and Bach1-KO hESCs (n = 3). *P < 0.05; **P < 0.01 compared with WT, t test. (C) WT, Bach1-KO − Dox, and Bach1-KO + Dox hESCs were seeded into Matrigel-coated wells (3 × 104 cells per well), and proliferation was evaluated by monitoring cell counts over the ensuing 4-day culture period (n = 3). *P < 0.05; **P < 0.01 compared with WT; #P < 0.05, ##P < 0.01 compared with Bach1-KO − Dox; one-way analysis of variance. (D) Nanog, Sox2, Oct4, and Bach1 protein levels in WT, Bach1-KO − Dox, and Bach1-KO + Dox hESCs were evaluated via Western blot (left panel) and quantified (right panel); β-Actin levels were also evaluated to confirm equal loading (n = 3). **P < 0.01 compared with WT; ##P < 0.01 compared with Bach1-KO − Dox; one-way analysis of variance. (E) WT and Bach1-KO hESCs were immunofluorescently stained for Sox2 or Oct4 expression, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 μm. (F) AP staining of colonies and mean percentages of differentiated, mixed, and undifferentiated cell colonies in hESCs treated with lentivirus control shRNA (Lv-Con) or lentivirus Bach1-shRNAs (Lv-shBach1). Scale bars, 500 μm. *P < 0.05; **P < 0.01 compared with Lv-Con; t test. (G and H) Western blot analysis of pluripotent factors and quantification of cell numbers for 4 days in hESCs treated with Lv-Con or Lv-shBach1 (n = 3). *P < 0.05; **P < 0.01 compared with Lv-Con; t test. (I) Overexpression of Bach1 enhanced reprogramming of human dermal fibroblasts to pluripotency. Left: AP staining of reprogramming colonies. Right: Quantitative and statistical analysis of AP-positive colonies (n = 4). *P < 0.05 compared with adenovirus green fluorescent protein (AdGFP). Data were collected from three or four independent replicates and are shown as means ± SD.

  • Fig. 2 Bach1 interacts with and stabilizes Nanog, Sox2, and Oct4 by recruiting DUB Usp7.

    (A) DoxBach1-transfected WT hESCs that been treated with or without Dox were treated with CHX (10 μg/ml) for the indicated times. The levels of Nanog, Sox2, and Oct4 proteins were determined by immunoblotting (IB). The band intensities from immunoblots were quantified (n = 3). *P < 0.05; **P < 0.01 compared with Dox−; t test. (B) WT and Bach1-KO hESCs were treated with or without proteasome inhibitor MG132 (10 μM) for 6 hours, and the levels of Nanog, Sox2, and Oct4 proteins were determined by immunoblotting and quantified (n = 3). *P < 0.05 compared with WT, dimethyl sulfoxide (DMSO); #P < 0.05; ##P < 0.01 compared with Bach1-KO, DMSO; one-way analysis of variance. (C) DoxBach1-transfected WT hESCs that been treated with or without Dox were treated with MG132 (10 μM) for 6 hours. The ubiquitinated proteins were pulled down using an anti-ubiquitin antibody, and the ubiquitination of Nanog was detected by Western blotting using anti-Nanog antibody. Ub, ubiquitination. (D) Bach1 was immunoprecipitated from WT hESCs; then, the amount of Usp7, Nanog, Sox2, Oct4, and c-Myc present in the precipitate was evaluated via Western blot. (E and F) Usp7 mRNA (E) and protein (F) levels in WT hESCs and Bach1-KO hESCs were evaluated via qRT-PCR or Western blot and quantified; β-Actin levels were also evaluated to confirm equal loading (n = 3). *P < 0.05; **P < 0.01 compared with WT; t test. (G) Bach1 signal track for representative locus Usp7 in hESCs from published ChIP-seq data, and the binding of promoter sequences for Usp7 to Bach1 was evaluated in WT hESCs via ChIP-qPCR, and the immunoglobulin G (IgG) served as a negative control (n = 3). *P < 0.05 compared with IgG; t test. (H) Western blot analysis of pluripotent factors in DoxBach1-transfected WT hESCs that had been treated with (Dox+) or without (Dox−) Dox and transfected with or without a pool of Usp7 siRNAs (n = 3). *P < 0.05; **P < 0.01 compared with Dox−, Control siRNA (Consi); ##P < 0.01 compared with Dox+, Consi; one-way analysis of variance. (I) Ubiquitylation of Nanog in DoxBach1-transfected WT hESCs after transfection of either nonspecific or Usp7-specific siRNAs in the presence and absence of Dox. IP was performed using anti-ubiquitin antibody, and the level of Nanog was determined by immunoblotting. Data were collected from three independent replicates and are shown as means ± SD.

  • Fig. 3 Bach1-KO promotes mesendodermal gene expression in hESCs.

    (A) Heatmap illustrating the RNA expression in WT and Bach1-KO hESCs of RNA-seq analysis for selected genes of different lineages. FPKM, fragments per kilobase of transcript per million mapped reads. (B) mRNA levels of mesoderm, endoderm, and neuroectoderm markers in WT and Bach1-KO hESCs were measured via qRT-PCR at the indicated time points, as the cells spontaneously differentiated into embryoid bodies (EBs); results were normalized to measurements for WT cells at the beginning of the differentiation period (n = 3). *P < 0.05; **P < 0.01 compared with WT; t test. (C) WT and KO hESCs were immunofluorescently stained for Gata6 (red) and T (green) expression, and nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) Gata6, T, Sox2, Oct4, and Bach1 protein levels were evaluated in WT and Bach1-KO hESCs via Western blot. β-Actin levels were also evaluated to confirm equal loading. (E) mRNA levels of mesendodermal and neuroectodermal genes were measured in WT, Bach1-KO − Dox, and Bach1-KO + Dox hESCs on day 3 of EB differentiation via qRT-PCR; results were normalized to measurements in WT cells (n = 3). *P < 0.05; **P < 0.01 compared with WT; #P < 0.05; ##P < 0.01 compared with Bach1-KO; one-way analysis of variance. (F) WT or Bach1-KO hESCs were subcutaneously injected into SCID mice; 8 weeks later, teratomas were harvested, sectioned, and stained with hematoxylin and eosin for histological analysis (left). Scale bars, 100 μm. mRNA levels of lineage marker genes in teratomas from mice that had been injected with WT, or Bach1-KO hESCs were measured via qRT-PCR (right panel; n = 3). *P < 0.05; **P < 0.01 compared with WT; t test. Data were collected from three independent replicates and are shown as means ± SD.

  • Fig. 4 Bach1 suppresses mesendodermal gene expression in hESCs via the EZH2-catalyzed trimethylation of H3K27 in mesendodermal gene promoters.

    (A) The heatmap of Bach1, EZH2, and H3K27me3 at genomic regions surrounding (±5 kb) TSSs (transcription start sites) in hESCs from published ChIP-seq data. (B) Bach1, EZH2, and H3K27me3 signal tracks for representative loci T, Gata6, Wnt3, and Nodal in hESCs from published ChIP-seq data. (C) H3K27me3 and EZH2 ChIP-seq signals at differentiation genes up-regulated by loss of Bach1 within ±5 kb of TSSs in WT and Bach1-KO hESCs. (D) H3K27me3 and EZH2 signal tracks for representative loci T and Gata6 in WT and Bach1-KO hESCs. (E) The binding of promoter sequences for mesendodermal genes, Nodal, and Wnt3 to H3K27me3, EZH2, and H3K4me3 was evaluated in WT and Bach1-KO hESCs via ChIP-qPCR (n = 3). *P < 0.05; **P < 0.01 compared with WT; t test. (F) Mesendodermal gene mRNA levels were measured via qRT-PCR in DoxBach1-transfected WT hESCs that had been treated with (Dox+) or without (Dox−) Dox and with (+DZNep) or without (−DZNep) the EZH2 inhibitor DZNep (20 ng/ml) at 12 hours after EB differentiation (n = 3). *P < 0.05; **P < 0.01 compared with Dox−, DMSO; ##P < 0.01 compared with Dox+, DMSO; one-way analysis of variance. (G and H) DoxBach1-transfected WT hESCs were transfected with plasmids containing luciferase reporter constructs controlled by (G) the T or Gata6 promoter sequence or (H) WT and mutated versions of the T promoter; then, luciferase activity was measured in cells that had been treated with (Dox+) or without Dox (Dox−), and results were normalized to measurements in Dox− hESCs (n = 3). *P < 0.05; **P < 0.01 compared with Dox−; t test. Data were collected from three independent replicates and are shown as means ± SD.

  • Fig. 5 Bach1 interacts with PRC2 in hESCs.

    (A) WT hESCs were immunofluorescently stained for Bach1 (red) and EZH2 (green) expression; nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (B) Bach1 was immunoprecipitated from WT hESCs; then, the amount of EZH2, EED, and SUZ12 present in the precipitate was evaluated via Western blot. (C) EZH2, EED, and SUZ12 were immunoprecipitated from WT hESCs and then, the amount of EZH2, EED, SUZ12, and Bach1 present in the precipitate was evaluated via Western blot. (D) Co-IP between Bach1 and EZH2 was performed in WT hESCs in the presence and absence of DNAse and RNase A. (E) HEK293 cells were transfected with a vector coding for Flag-tagged Bach1 and an empty vector or with vectors coding for Flag-tagged Bach1 and HA-tagged EZH2 and lysed; then, Bach1 was immunoprecipitated with an anti-Flag antibody, and Bach1 was detected in the immunoprecipitate and cell lysate via Western blot with an anti-Flag antibody, while EZH2 was detected in the precipitate via Western blot with an anti-HA antibody. β-Actin was evaluated in the lysate to confirm equal loading. (F) Bacterially expressed His-tagged EZH2 was incubated with GST-tagged versions of the full Bach1 protein (Bach1-Full-GST) or with mutated versions of Bach1 that lacked either the C-terminal bZip domain (Bach1-N-GST) or the N-terminal BTB domain (Bach1-C-GST) then, the reaction products were precipitated with glutathione-Sepharose 4B beads, and EZH2 was detected in the precipitate via Western blot with anti-His antibodies.

  • Fig. 6 Bach1-KO promotes mesendodermal differentiation by activating Wnt/β-catenin signaling.

    (A) mRNA levels of components of the Wnt signaling pathway were measured in WT and Bach1-KO hESCs on day 3 of differentiation into EBs via qRT-PCR and normalized to measurements in WT cells (n = 3). *P < 0.05; **P < 0.01 compared with WT; t test. (B) Protein levels of Wnt3, Fzd1, active β-catenin, and total β-catenin were evaluated in WT and Bach1-KO hESCs on day 3 of differentiation via Western blot. (C) Total β-catenin and histone 3 protein levels were evaluated in the nucleus and cytoplasm of WT and Bach1-KO hESCs on day 3 of differentiation via Western blot. (D) WT and Bach1-KO hESCs were transfected with the TOPflash luciferase reporter, which contains eight copies of the binding site for Tcf/lef, a downstream target of Wnt signaling; then, the cells were treated with or without the Wnt inhibitor IWP2 (10 μM), and luciferase activity was evaluated on day 3 of differentiation and normalized to measurements in WT cells (n = 3). **P < 0.01 compared with WT (IWP2−); ##P < 0.01 compared with Bach1-KO (IWP2−); one-way analysis of variance. (E) mRNA levels of mesendodermal genes were evaluated in WT and Bach1-KO hESCs that had been treated with or without IWP2 (10 μM) on day 3 of differentiation (n = 3). **P < 0.01 compared with WT, DMSO; #P < 0.05; ##P < 0.01 compared with Bach1-KO, DMSO; one-way analysis of variance. (F) The binding of promoter sequences for mesendodermal genes and for the signaling molecules Wnt3 and Nodal to activated β-catenin was evaluated in WT and Bach1-KO hESCs (left) and in DoxBach1-transfected WT hESCs that had (Dox+) or had not (Dox−) been treated with Dox (right) on day 3 of differentiation via ChIP-qPCR; results were normalized to WT (left) or Dox− (right) cells (n = 3). *P < 0.05; **P < 0.01 compared with WT or Dox−; t test. (G) mRNA levels of mesendodermal genes and protein level of β-catenin were evaluated in WT and Bach1-KO hESCs that had been transfected with adenoviruses coding for β-catenin shRNA (Ad-shβ-catenin) or a control shRNA sequence (Ad-shCtrl) on day 3 of differentiation (n = 3). **P < 0.01 compared with WT, Ad-shCtrl; ##P < 0.01 compared with KO, Ad-shCtrl; one-way analysis of variance. (H) mRNA levels of Wnt3, Nodal, and Bach1 were evaluated in DoxBach1-transfected WT hESCs that had (Dox+) or had not (Dox−) been treated with Dox on day 3 of differentiation via qPCR (n = 3). *P < 0.05 compared with Dox−; t test. (I) DoxBach1-transfected WT hESCs were transfected with plasmids containing luciferase reporter constructs controlled by WT or mutated versions of the Wnt3 promoter; then, luciferase activity was measured in cells that had been treated with (Dox+) or without Dox (Dox−) on day 3 of differentiation, and results were normalized to measurements in Dox− cells (n = 3). *P < 0.05 compared with Dox−; t test. Data were collected from three independent replicates and are shown as means ± SD.

  • Fig. 7 Bach1-KO promotes mesendodermal differentiation by activating Nodal signaling.

    (A) mRNA levels of Nodal and the Nodal receptors ALK4 and ACVRIIB were evaluated in WT and Bach1-KO hESCs on day 3 of differentiation via qRT-PCR, and the results were normalized to measurements in WT cells (n = 3). **P < 0.01 compared with WT; t test. (B) Protein levels of Nodal, ACVRIIB, phosphorylated Smad2 and Smad3 (p-Smad2 and p-Smad3, respectively), and total Smad2/3 were evaluated in WT and Bach1-KO hESCs on day 3 of differentiation via Western blot. (C) DoxBach1-transfected WT hESCs were transfected with plasmids containing luciferase reporter constructs controlled by WT or mutated versions of the Nodal promoter; then, luciferase activity was measured in cells that had been treated with (Dox+) or without Dox (Dox−) on day 3 of differentiation, and results were normalized to measurements in Dox− cells (n = 3). *P < 0.05 compared with Dox−; t test. (D) p-Smad2, total Smad2/3, and histone 3 protein levels were evaluated in the nucleus and cytoplasm of WT and Bach1-KO hESCs on day 3 of differentiation via Western blot. (E) The binding of promoter sequences for mesendodermal genes and for the signaling molecules Wnt3 and Nodal was evaluated in WT and Bach1-KO hESCs and in DoxBach1-transfected WT hESCs that had (Dox+) or had not (Dox−) been treated with Dox on day 3 of differentiation via ChIP-qPCR; results were normalized to measurements in WT or Dox− cells (n = 3). *P < 0.05; **P < 0.01 compared with WT or Dox−; t test. (F and G) mRNA levels of mesendodermal genes were evaluated in WT and Bach1-KO hESCs and in WT and Bach1-KO hESCs that had been treated with SB-431542 (20 μM), which inhibits TGF-β receptors (F) or transfected with adenoviruses coding for β-catenin shRNA and treated with SB-431542 (G), and assessments were performed on day 3 of differentiation via qRT-PCR; results were normalized to measurements in WT cells (n = 3). **P < 0.01 compared with WT, DMSO (F) or WT, DMSO, Ad-shCtrl (G); ##P < 0.01 compared with KO, DMSO (F) or KO, DMSO, Ad-shCtrl (G); one-way analysis of variance. Data were collected from three independent replicates and are shown as means ± SD. (H) Model showing that Bach1 plays an important role in the maintenance of hESC self-renewal and suppression of mesendodermal differentiation. Bach1 interacts with Nanog, Sox2, and Oct4 and facilitates deubiquitylation of these pluripotency factors by recruiting DUB Usp7 and therefore stabilizes pluripotency factors and maintains the stem cell self-renewal. Bach1 also impedes mesendodermal differentiation via recruitment of PRC2, which leads to the deposition of H3K27me3 and gene silencing, as well as inhibiting Wnt/β-catenin and Nodal/Smad2/3 signaling.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaau7887/DC1

    Fig. S1. Protein expression of Bach1 in mouse embryos and generation of Bach1-KO and DoxBach1 hESCs.

    Fig. S2. Usp7 is required for Bach1-induced stabilization of pluripotency proteins in hESCs.

    Fig. S3. RNA-seq analysis for differential gene expression in WT and Bach1-KO hESCs.

    Fig. S4. The role of Usp7 in the mesendodermal differentiation induced by Bach1 deficiency.

    Fig. S5. The genome-wide distribution profiles of Bach1 binding sites and the Gene Ontology analysis.

    Fig. S6. Depletion of Bach1 impairs recruitment of H3K27me3 and EZH2 to the mesendodermal gene.

    Fig. S7. RNA-seq analysis for differential gene expression in WT and Bach1-KO hESCs on day 3 of differentiation.

    Table S1.1. RNA-seq analysis of DE genes in WT and Bach1-KO hESCs (day 0).

    Table S1.2. The up-regulated genes associated with cell differentiation in Bach1-KO hESCs (day 0).

    Table S1.3. RNA-seq analysis of DE genes on day 3 of EB differentiation of WT hESCs and Bach1-KO hESCs.

    Table S2.1. Primers used for CRISPR sgRNA and off-target.

    Table S2.2. Primers used for plasmids construction and reporters.

    Table S2.3. Primers used for qRT-PCR.

    Table S2.4. Primers used for Lv-Con and Lv-Bach1 shRNAs.

    Table S2.5. The sequences of siRNAs.

    Table S2.6. Primers used for ChIP-qPCR.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Protein expression of Bach1 in mouse embryos and generation of Bach1-KO and DoxBach1 hESCs.
    • Fig. S2. Usp7 is required for Bach1-induced stabilization of pluripotency proteins in hESCs.
    • Fig. S3. RNA-seq analysis for differential gene expression in WT and Bach1-KO hESCs.
    • Fig. S4. The role of Usp7 in the mesendodermal differentiation induced by Bach1 deficiency.
    • Fig. S5. The genome-wide distribution profiles of Bach1 binding sites and the Gene Ontology analysis.
    • Fig. S6. Depletion of Bach1 impairs recruitment of H3K27me3 and EZH2 to the mesendodermal gene.
    • Fig. S7. RNA-seq analysis for differential gene expression in WT and Bach1-KO hESCs on day 3 of differentiation.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1.1 (Microsoft Excel format). RNA-seq analysis of DE genes in WT and Bach1-KO hESCs (day 0).
    • Table S1.2 (Microsoft Excel format). The up-regulated genes associated with cell differentiation in Bach1-KO hESCs (day 0).
    • Table S1.3 (Microsoft Excel format). RNA-seq analysis of DE genes on day 3 of EB differentiation of WT hESCs and Bach1-KO hESCs.
    • Table S2.1 (Microsoft Excel format). Primers used for CRISPR sgRNA and off-target.
    • Table S2.2 (Microsoft Excel format). Primers used for plasmids construction and reporters.
    • Table S2.3 (Microsoft Excel format). Primers used for qRT-PCR.
    • Table S2.4 (Microsoft Excel format). Primers used for Lv-Con and Lv-Bach1 shRNAs.
    • Table S2.5 (Microsoft Excel format). The sequences of siRNAs.
    • Table S2.6 (Microsoft Excel format). Primers used for ChIP-qPCR.

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