Polycomb Group Protein Ezh2 Regulates Hepatic Progenitor Cell Proliferation and Differentiation in Murine Embryonic Liver

<div><p>In embryonic liver, hepatic progenitor cells are actively proliferating and generate a fundamental cellular pool for establishing parenchymal components. However, the molecular basis for the expansion of the progenitors maintaining their immature state remains elusive. Polycomb group proteins regulate gene expression throughout the genome by modulating of chromatin structure and play crucial roles in development. <i>Enhancer of zeste homolog 2</i> (<i>Ezh2</i>), a key component of polycomb group proteins, catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3), which trigger the gene suppression. In the present study, we investigated a role of <i>Ezh2</i> in the regulation of the expanding hepatic progenitor population <i>in vivo</i>. We found that Ezh2 is highly expressed in the actively proliferating cells at the early developmental stage. Using a conditional knockout mouse model, we show that the deletion of the SET domain of <i>Ezh2</i>, which is responsible for catalytic induction of H3K27me3, results in significant reduction of the total liver size, absolute number of liver parenchymal cells, and hepatic progenitor cell population in size. A clonal colony assay in the hepatic progenitor cells directly isolated from <i>in vivo</i> fetal livers revealed that the bi-potent clonogenicity was significantly attenuated by the Ezh2 loss of function. Moreover, a marker expression based analysis and a global gene expression analysis showed that the knockout of Ezh2 inhibited differentiation to hepatocyte with reduced expression of a number of liver-function related genes. Taken together, our results indicate that Ezh2 is required for the hepatic progenitor expansion <i>in vivo</i>, which is essential for the functional maturation of embryonic liver, through its activity for catalyzing H3K27me3.</p></div

Ezh2 SET domain depletion caused blockade of the cellular differentiation in embryonic liver.

<p>A: Western blot analysis for Albumin (hepatocyte marker) in the CD45⁻ TER119⁻ non-hematopoietic liver cells from control (Rosa26::CreER(T2)^−/−;Ezh2^F/F mouse) and Ezh2 KO (Ezh2 SET domain depleted mouse; Rosa26::CreER(T2)^+/−;Ezh2^F/F mouse) at ED 18.5 after 3 days TAM injection (TAM; ED 10.5–12.5). B: Expression levels of hepatocytes related genes in the CD45⁻ TER119⁻ non-hematopoietic liver cells of the control and Ezh2 KO at indicated points were measured by qRT-PCR. Data are mean ± SD (n = 3). <i>P</i> values (asterisks) are from the Mann–Whitney <i>U</i>-test. *<i>P</i>− TER119⁻ non-hematopoietic liver cells of the control and Ezh2 KO. D: Significant enrichment of metabolism related liver functional GO terms for decreased genes upon Ezh2KO. Corrected <i>P</i> values of GO terms are shown.</p

Ezh2 is highly expressed in parenchymal epithelial cells in early developing liver.

<p>A: Time course analysis of expression levels of the polycomb group (PcG) members (Ezh1, Eed, Suz12, Bmi1, Ring1B, and Ezh2) in CD45⁻ TER119⁻ non-hematopoietic liver cells of mice at nine time points between ED 9.5 and 8 weeks. Relative expression values from microarray analyses are shown. B: Western blot analysis of Ezh2 in CD45⁻ TER119⁻ non-hematopoietic liver cells of fetal mice at ED 11.5, 13.5, 15.5, and 17.5. β-actin is indicated as control. C: Immunofluorescence staining for Ezh2, CK8/18 (epithelial marker), and DAPI in liver of fetuses at ED 11.5, 13.5, 15.5, 17.5, P 0 (neonatal), and 8 weeks. Arrows indicate Ezh2 positive cells in CK8/18 positive cells. (Scale bar: 100 µm) D: Frequency of Ezh2 positive cells in CK8/18 positive cells at indicated time points. (n = 3). E: Immunofluorescence staining for histone H3K27me3, CK8/18 (epithelial marker), DAPI in the liver tissues at ED 11.5, 13.5, 15.5, and 17.5. Scale bar = 100 µm. F: Frequency of H3K27me3 expressing cells in CK8/18 positive cells at indicated points. Data are mean ± SD (n = 3). G Immunofluorescence staining for Ezh2, H3K27me3, Ck8/18 and DAPI in the liver of fetuses at ED 13.5. Upper is merge image of all colors. Bottom column shows image of Ezh2 and H3K27me3, lower right is magnified image of left. Red: Ezh2, Green: H3K27me3, Light blue: CK8/18, Blue: DAPI. Scale bar = 100 µm. H Immunofluorescence staining for AFP, Ezh2, CK8/18, and DAPI in the liver of fetuses at ED 13.5. Arrows indicate AFP and Ezh2 double positive cells in CK8/18 positive cells. (Scale bar: 50 µm) I: Frequency of Ezh2 positive cells in AFP and CK8/18 double positive cells and AFP negative and CK8/18 positive cells at indicated time points. (n = 3). <i>P</i> values (asterisks) are from the Mann–Whitney <i>U</i>-test. *<i>P</i></p

Negative regulators of cell cycle were significantly up-regulated in the Ezh2 SET domain depleted fetal livers.

<p>A: The G1_to_S_Cell_Cycle_Control pathway (WP413_41269) from analysis using the WikiPathways platform is shown, in which gene hits with more than 2-fold change up to 8-fold change (<i>P</i> = 0.0018) are highlighted in yellow and hits with more than 8-fold change (<i>P</i> = 0.0046) in red in the Ezh2-KO CD45⁻ TER119⁻ liver cells at ED 13.5 compared with WT control. B: ChIP-PCR analysis with the anti-Ezh2 antibody and the primer sets detecting promoter regions of Cdkn1a, Cdkn2a, and Cdkn2b in the CD45⁻ TER119⁻ liver cells from WT livers at ED 13.5 are shown. C: ChIP-PCR analysis with the anti-H3K27me3 antibody and the primer sets detecting promoter regions of Cdkn1a, Cdkn2a, and Cdkn2b in the CD45⁻ TER119⁻ liver cells from the WT control livers at ED 13.5 are shown. D: Immunohistochemical analysis of Cdkn1a protein in the WT control and Ezh2 KO liver tissues at ED 13.5. Scale bar = 50 µm.</p

Ezh2 SET domain depletion inhibited the proliferation of hepatic progenitor cells.

<p>A: Immunofluorescence staining for AFP (hepatic undifferentiated marker), BrdU, CK8/18 (epithelial marker) and DAPI in the liver tissues of control (Rosa26::CreER(T2)^−/−;Ezh2^F/F mouse) and Ezh2 KO (Ezh2 SET domain depleted mouse; Rosa26::CreER(T2)^+/−;Ezh2^F/F mouse) at ED 13.5 after 3 days TAM injection (ED 8.5–10.5). Arrows indicates AFP and BrdU double positive cells in CK8/18 positive cells. Scale bar = 100 µm. B: Frequency of AFP and BrdU double positive cells in CK8/18 positive cells of the control and Ezh2 KO at ED 13.5 (TAM; ED 8.5–10.5). Data are mean ± SD (n = 3). <i>P</i> values (asterisks) are from the Mann–Whitney <i>U</i>-test. *<i>P</i>− CD29⁺ CD49f⁺ CD45⁻ TER119⁻ hepatic progenitor cells. Left panels: dot plot of c-Kit and TER119/CD45. Right panels: dot plot of CD29 and CD49f. D: Absolute numbers of c-kit⁻ CD29⁺ CD49f⁺ CD45⁻ TER119⁻ hepatic progenitor cells per a liver of the control and Ezh2 KO at ED 13.5 (TAM; ED 8.5–10.5). Data are mean ± SD (n = 3). *<i>P</i>− CD29⁺ CD49f⁺ CD45⁻ TER119⁻ cells at day 5 of culture obtained from the control and Ezh2 KO livers at ED 13.5 (TAM; ED 8.5–10.5) and their immunofluorescence labeling of Albumin (hepatocyte marker) and CK7 (cholangicoyte marker) positive cells. Scale bar = 200 µm. F: Number of hepatic colony-forming units in culture (H-CFU-C) derived colonies composed of over 90 cells at day 5 of culture. The c-kit⁻ CD29⁺ CD49f⁺ CD45⁻ TER119⁻ cells were freshly isolated from the control and Ezh2 KO fetal livers at ED 13.5 (TAM; ED 8.5–10.5) and underwent a clonal density colony formation assay. Data are mean ± SD (n = 3). *<i>P</i>− CD29⁺ CD49f⁺ CD45⁻ TER119⁻ cells in the control and Ezh2 KO livers at ED 13.5 (TAM; ED 8.5–10.5). Data are mean ± SD (n = 3). *<i>P</i>− CD29⁺ CD49f⁺ CD45⁻ TER119⁻ cells in the control and Ezh2 depleted. Data are mean ± SD (n = 3). *<i>P</i>− CD29⁺ CD49f⁺ CD45⁻ TER119⁻ hepatic progenitor cells from the control and Ezh2 KO depletion livers at ED 13.5 (TAM; ED 8.5–10.5).</p