Figure 1.
TSA induces early differentiation of ESCs and promotes mesodermal lineage differentiation.
(A) Bright-field images, alkaline phosphatase staining of ESCs and representative immunofluorescence images of Oct4 staining in control or TSA-treated ESCs (10 and 20 ng/ml) in the presence of LIF. (B) Western blotting verification of H3, acetyl-H3, H4, and acetyl-H4 in control or TSA-treated ESCs (10 and 20 ng/ml). GAPDH was used as a loading control. (C) The relative expression levels of Oct4, Nanog, and Rex1 mRNA in control or TSA-treated ESCs (10 and 20 ng/ml). (D, E) QRT-PCR analysis for marker genes of three germ layers (endoderm, mesoderm and ectoderm) in control or TSA-treated ESCs (10 and 20 ng/ml), under the monolayer differentiation condition without LIF. The cells were treated by TSA after removing LIF for 24h and collected mRNA for QRT-PCR analysis at day 3 of monolayer differentiation. (F, G) QRT-PCR analysis for marker genes of the three germ layers in control or TSA-treated ESCs (10 and 20 ng/ml) during EB differentiation. The EBs was treated by TSA from day 2 to 6 of EB differentiation. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P<0.001; **, P<0.01; *, P<0.05.
Figure 2.
The expression levels of HDAC1 and 3 are decreased during differentiation.
(A) QRT-PCR for genes characteristic of undifferentiated stem cells (Oct4, Nanog) was performed as indicated on mRNA collected at days 0, 3, 6, and 10 during EB differentiation. (B) The relative expression levels of marker genes for three germ layers (endoderm, Gata6; mesoderm, T, Mixl1; primitive ectoderm, Fgf5) at days 0, 3, 6, and 10 during EB differentiation. (C) Western blotting verification for genes characteristic of undifferentiated stem cells (Oct4, Nanog) was performed as indicated on protein samples collected at days 0, 3, 6, and 10 during EB differentiation. The expression level of global acetyl-H4 was increasing during EB differentiation. GAPDH and H4 were used as loading controls. (D) Western blotting verification for class I HDAC members (HDAC1, 2, 3, and 8) at the indicated days 0, 3, 6, and 10 during EB differentiation. GAPDH was used as a loading control. (E, F) QRT-PCR analysis for the expression levels of class I HDAC members (HDAC1, 2, 3, and 8) at the indicated days 0, 3, 6, and 10 during EB differentiation. (G, H) Western blotting and QRT-PCR analysis for the expression levels of HDAC members (HDAC1, 2, 3, and 8) at days 0, 2, 3, and 4 during differentiation without LIF.
Figure 3.
Loss of HDAC1 or 3 enhances mesodermal lineage differentiation.
(A) Bright-field images and alkaline phosphatase staining of ESCs in shHDAC1 and shHDAC3 ESCs. (B) Western blotting verification and QRT-PCR analysis of the knockdown of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. (C) QRT-PCR analysis of mesoderm genes in shHDAC1 ESCs and control cells at the days 0, 3, 6, and 10 during EB differentiation. (D) QRT-PCR analysis of mesoderm genes in shHDAC3 ESCs and control cells during EB differentiation. (E) Representative immunofluorescence images for the GATA4 expression level in control, shHDAC1, and shHDAC3 cells after 9 days of EB formation. Green, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P<0.001; **, P<0.01; *, P<0.05.
Figure 4.
Ectopic expression of HDAC1 and 3 inhibits the differentiation into the mesodermal lineage in EBs.
(A) Bright-field images and alkaline phosphatase staining of ESCs in control, HDAC1-overexpression (HDAC1-OE), and HDAC3-overexpression (HDAC3-OE) ESCs. (B) Western blotting verification and QRT-PCR analysis of the overexpression of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. (C) QRT-PCR analysis for the mRNA levels of mesoderm genes in HDAC1-OE ESCs, HDAC3-OE ESCs and control cells during EB differentiation. (D) Western blotting analysis of the Gata4 and α-SMA protein levels in HDAC3-OE ESCs and control cell lines during EB differentiation. (E) Representative immunofluorescence images for the GATA4 expression level in control, HDAC1-OE, and HDAC3-OE cells after 9 days of EB formation. Red, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P<0.001; **, P<0.01; *, P<0.05.
Figure 5.
The histone deacetylase activity of HDACs is required for the regulation of mesoderm gene.
(A) Western blotting verification of acetyl-H4 and H4 expression levels in control, HDAC1-OE (H1 OE), and TSA-treated H1-OE cells. GAPDH was used as a loading control. (B, C) QRT-PCR analysis of the three germ layer genes at day 6 of EB differentiation in control, H1-OE, and TSA-treated H1-OE cells. (D) Western blotting verification of acetyl-H4 and H4 expression levels in control, HDAC3-OE (H1 OE), and TSA-treated H3-OE cells. GAPDH was used as a loading control. (E, F) QRT-PCR analysis of the three germ layer genes at day 6 of EB differentiation in control, H3-OE, and TSA-treated H3-OE cells. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P<0.001; **, P<0.01; *, P<0.05.
Figure 6.
HDAC can repress the transcriptional activity of T/Bry via physical interaction.
(A) HDAC1 and HDAC3 interact with the T-box transcription factor T/Bry. Co-immunoprecipitation (Co-IP) was performed using control IgG or T/Bry antibody, followed by western blot analysis for HDAC1 and HDAC3. 5% Input (v/v) indicated that the ratio between the loading sample and precipitation is one to twenty. (B) Co-IP was performed using control IgG or HDAC1 antibody, followed by western blot analysis for T/Bry. (C) Co-IP was performed using control IgG or HDAC3 antibody, followed by western blot analysis for T/Bry. (D) HDAC3 does not interact with Gata4. Co-IP was performed using control IgG or HDAC3 antibody, followed by western blot analysis for Gata4. (E) A summary model shows the mechanism of HDACs in regulating the expression of mesodermal genes.