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. 2011 Jun;21(6):911-21.
doi: 10.1038/cr.2011.47. Epub 2011 Mar 22.

Smek Promotes Histone Deacetylation to Suppress Transcription of Wnt Target Gene Brachyury in Pluripotent Embryonic Stem Cells

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Free PMC article

Smek Promotes Histone Deacetylation to Suppress Transcription of Wnt Target Gene Brachyury in Pluripotent Embryonic Stem Cells

Jungmook Lyu et al. Cell Res. .
Free PMC article

Abstract

In embryonic stem cells (ESCs), Wnt-responsive development-related genes are silenced to maintain pluripotency and their expression is activated during differentiation. Acetylation of histones by histone acetyltransferases stimulates transcription, whereas deacetylation of histones by HDACs is correlated with transcriptional repression. Although Wnt-mediated gene transcription has been intimately linked to the acetylation or deacetylation of histones, how Wnt signaling regulates this type of histone modification is poorly understood. Here, we report that Smek, a regulatory subunit of protein phosphatase 4 (PP4) complex, plays an important role in histone deacetylation and silencing of the Wnt-responsive gene, brachyury, in ESCs. Smek mediates recruitment of PP4c and HDAC1 to the Tcf/Lef binding site of the brachyury promoter and inhibits brachyury expression in ESCs. Activation of Wnt signaling during differentiation causes disruption of the Smek/PP4c/HDAC1 complex, resulting in an increase in histones H3 and H4 acetylation at the brachyury gene locus. These results suggest that the Smek-containing PP4 complex represses transcription of Wnt-responsive development-related genes through histone deacetylation, and that this complex is essential for ESC pluripotency maintenance.

Figures

Figure 1
Figure 1
Smek1 and Smek2 are required for maintenance of pluripotency in ESCs. (A) Cells expressing control shRNA, or shRNA for Smek1, Smek2, or both, were lysed and subjected to western blot analysis with anti-Smek1 and -Smek2 antibodies (a). Alkaline phosphatase (AP) staining (blue) of ESCs expressing control shRNA (b), Smek1 shRNA (c), Smek2 shRNA (d), or both (e). Notably, double knockdown for both Smek1 and Smek2 reduces AP expression and morphological change of ESCs, reflective of differentiation. ESC status was evaluated by quantifying the number of colonies consisting of AP-negative cells (differentiated), positive cells (undifferentiated), and both AP-negative and -positive cells (mixed) (f). Scale bars, 100 μm. (B) Expression of genes characteristic of ESC stemness (oct4, nanog, and rex1), endoderm (gata6 and sox17), primitive ectoderm (fgf5), and mesoderm (brachyury and goosecoid) were measured by RT-PCR in ESC lines cultured in the presence of LIF. Gapdh was used as an internal control. Two independent ESC lines were examined for each shRNA construct. EB was used as a positive control for differentiation of ESC. (C) ESC lines expressing control or Smek1/2 shRNA were stained for brachyury. Scale bar, 100 μm. (D) Quantitative real-time PCR analysis of early mesodermal and cardiac progenitor markers during differentiation of ESCs expressing control shRNA or Smek1/2 shRNA. (E) Cells dissociated from EB expressing control or Smek1/2 shRNA were cultured on gelatin-coated plates and subjected to immunostaining with antibodies against a cardiac progenitor marker islet1. Hoechst dye was used as a counterstaining. Scale bar, 100 μm. Depletion of Smek facilitates differentiation into islet1-positive cardiac progenitors from mesoderm.
Figure 2
Figure 2
Smek represses the transcription of Wnt target genes. (A) Expression of Wnt/β-catenin target genes, brachyury, pitx2, islet1, axin2, lef1, and sox2, was analyzed by quantitative real-time PCR in undifferentiated ESC lines. (B) ESC lines were transfected with the TOPFLASH reporter and were cultured under ESC conditions for 2 days. Luciferase activity in each sample was measured as described in the methods. Data are presented as mean ± S.D. of three independent experiments. (C) Smek1, Smek2, Wnt3a, β-catenin, or control vectors were transfected into HEK293T cells with TOPFLASH reporter. Data are presented as mean ± S.D. of four independent experiments. (D) The plasmid encoding Flag-tagged Smek1 was transiently transfected into HEK293T cells with TOPFLASH or FOPFLASH. Cells were lysed 48 h after transfection and were subjected to ChIP with anti-Flag antibody. Quantitative real-time PCR using a primer set, which is specific for −20 bp upstream and +170 bp downstream of the luciferase transcriptional start site, show that enrichment of Smek at Tcf/Lef binding sites of TOPFLASH DNA compared with that of mutated Tcf/Lef binding sites of FOPFLASH DNA. (E) HEK293T cells transfected with TOPFLASH and plasmids expressing control, Flag-Smek1, and HA-β-catenin, were subjected to western blot analysis (top) and ChIP with anti-HA or Flag antibodies, and the precipitated DNAs were analyzed by quantitative real-time PCR using a primer set as in D (bottom).
Figure 3
Figure 3
The brachyury promoter is occupied by the Smek/PP4c complex containing HDAC1. (A) Quantitative real-time PCR was performed using ChIP DNA with anti-Smek1 and -Smek2 antibodies and the primer set specific for regions of the brachyury promoter as indicated at the top of the figure. Data are presented as fold change as compared to cell lysates precipitated with control antibody. (B) PCR products amplified with the −317/−61 primer from control input DNA and ChIP DNA with control IgG, anti-Smek1, Smek2, PP4c, and HDAC1 antibodies show that PP4c and HDAC1 are associated with promoter of brachyury gene occupied by Smek1 and Smek2. (C) Depletion of Smeks inhibits the occupancy of PP4c and HDAC1, and increases the acetylation of histones H3 and H4. ChIP DNA precipitated with the indicated antibodies from ESCs expressing control or Smek1/2 shRNA and analyzed by quantitative real-time PCR with the −317/−61 primer set as in panel (A). (D) Binding of HDAC1 to Smek/PP4c complex. Nuclear extracts from ESCs were immunoprecipitated with anti-Smek1 (left) and Smek2 (right) antibodies and subjected to western blot analysis using anti-HDAC1 or PP4c antibody. (E) Western blot analysis of anti-Smek1 and anti-PP4c immunoprecipitates in ESCs knocked down for PP4c (left) and both Smek1 and Smek2 (right). (F, G) HDAC activities in immunoprecipitates from panels (D and E), respectively, were measured using a fluorescence-producing substrate in the presence or absence of TSA. Data were normalized to the HDAC activity of the anti-HDAC1 immunoprecipitate, and are presented as mean ± S.D. of three independent experiments. (H) ESCs expressing control and PP4c shRNA were subjected to ChIP using the indicated antibodies and analyzed by quantitative real-time PCR with the −317/−61 primer set.
Figure 4
Figure 4
Wnt, but not β-catenin, disrupts the Smek/PP4c/HDAC1 complex to activate transcription of the brachyury gene. ESC lines expressing control shRNA or double shRNA were incubated with vehicle, Wnt3a (50 ng/ml), or BIO (5 μM) for 6 h (A, C, E, and F), or were cultured in hanging drops of medium in the presence of vehicle or Dkk1 (100 ng/ml) for 3 days (B, D, G, and H). (A) Wnt3a or BIO activates brachyury but not Smek1 or Smek2 expression. ESC lines were treated with vehicle, Wnt3a (50 ng/ml), or BIO (5 μM) for 6 h. Transcript levels of brachyury, Smek1, and Smek2 were determined buy quantitative real-time PCR. (B) Dkk1 inhibits brachyury expression during ESC differentiation. ESCs were cultured in hanging drops of medium for EB formation in the presence of vehicle or Dkk1 (100 ng/ml) for 3 days. Transcript levels of brachyury, Smek1, and Smek2 were determined by quantitative real-time PCR. (C) Activation of Wnt signaling decreases the occupancy of HDAC1 and PP4c to the brachyury promoter, and increases histones H3 and H4 acetylation. Smek1 occupancy was not affected. The occupancy of Smek1, PP4c, HDAC1 and histones H3 and H4 acetylation was measured by ChIP followed by quantitative real-time PCR analysis with primer set of −317/−61 site of the brachyury promoter. (D) PP4c and HDAC1 occupancy at the brachyury promoter decreased during ESC differentiation, while histones H3 and H4 acetylation is increased. Dkk1 inhibits these changes during ESC differentiation. Smek1 occupancy was not affected. (E) Treatment with Wnt3a or BIO in ESCs decreases interaction of HDAC1 and PP4c proteins with Smek1. Lysates from ESCs were immunoprecipitated with anti-Smek1 and then subjected to western blot analysis using anti-Smek1, HDAC1, and PP4c antibodies. (F) HDAC activities in Smek1-immunoprecipitates from (E) were measured by a HDAC-activity assay in the presence or absence of TSA. Data are presented as mean ± S.D. of three independent experiments. (G) HDAC1 and PP4c associated with Smek1 decreased during ESC differentiation, while addition of Dkk1 inhibits the decrease of association of HDAC1 and PP4c with Smek1. (H) HDAC activities in immunoprecipitates from panel (G) were measured by a HDAC-activity assay in the presence or absence of TSA. Data are presented as mean ± S.D. of three independent experiments. (I) β-catenin does not affect the occupancy of PP4c and HDAC1 at the brachyury promoter. ESCs were transiently transfected with control vector or HA-β-catenin construct, and were then subjected to ChIP with the indicated antibodies followed by quantitative real-time PCR analysis with −317/−61 primer set at the brachyury promoter (top). Data are presented as mean ± S.D. from three independent experiments. Western blot analysis from nuclear extracts reveals the protein level of β-catenin (top).

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