The polycomb group protein Suz12 is required for embryonic stem cell differentiation

Abstract

Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12(-/-) ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation.

FIG. 1.

Analysis of Suz12^−/− ES cells. (A) Phase-contrast pictures of growing Suz12^+/− (clone SBE4) and Suz12^−/− (SBE8 8) ES cell clones growing on feeders cells. (B) Phase-contrast pictures (top) of growing Suz12^+/− (clone SBE4) and Suz12^−/− (SBE1 and SBE8) ES cell clones on gelatin-coated plates. Genotype and sex determination PCRs (middle) show that both male (M) and female (F) Suz12^−/− ES cells can be derived. Immunoblots (bottom) using antibodies to Suz12, Ezh2, and β-tubulin are shown. β-Tubulin served as a loading control. KO, knockout; WT, wild type. (C) RNA expression levels of the ES cell markers Oct4 and Nanog in Suz12^+/− and Suz12^−/− ES clones compared to MEFs. (D) Metaphase spreads showing a normal karyotype for Suz12^+/− and Suz12^−/− ES clones. At the top the average numbers of counted chromosomes per cell are given. The bottom panels are representative pictures of metaphase spreads from Suz12^+/− and Suz12^−/− ES clones. (E) Immunoblots of different histone H3 lysine (K) modifications using specific antibodies to the indicated proteins and their modifications.

FIG. 2.

Expression of Nanog and Oct4 in Suz12^−/− ES cells. Immunostaining of Suz12^+/− and Suz12^−/− ES cells growing on feeders cells shown with anti-Nanog-specific (A) and anti-Oct4-specific (B) antibodies reveals expression of both ES cell markers in Suz12^+/− and Suz12^−/− ES cells but not in feeder cells (arrows).

FIG. 3.

Suz12 is required for the regulation of a large number of genes involved in development, differentiation, and homeostasis. (A) Functional clustering of gene expression changes between Suz12^+/− and Suz12^−/− ES cells. Expression downregulation refers to the functional clustering of genes whose expression was downregulated in Suz12^−/− compared to Suz12^+/− ES cells. Expression upregulation refers to the functional clustering of genes whose expression was upregulated in Suz12^−/− compared to Suz12^+/− ES cells. (B) Expression (top) and ChIP analysis using the indicated antibodies (bottom) were determined by real-time qPCR. HA, hemagglutinin. (C) Expression levels of H19 in Suz12^+/− and Suz12^−/− male ES clones.

FIG. 4.

Suz12^−/− ES cells fail to undergo proper differentiation. (A) Phase-contrast pictures of neuronal differentiation of Suz12^+/− and Suz12^−/− ES cells. Arrows in the left panels show neuron formation in Suz12^+/− cells, while the right panels show lack of neuron formation in Suz12^−/− cells. (B) Expression levels of two specific neuronal markers GluR6 and Gad65 showing strong activation in differentiated Suz12^+/− cells and no activation in Suz12^−/− cells. Undiff, undifferentiated; diff, differentiated. (C) Hematoxylin and eosin staining of 7-day EBs formed by Suz12^+/− and Suz12^−/− ES cells. Top panels show normal morphology of Suz12^+/− EBs. High-magnification fields highlight outer endodermal layers and epithelium-like cavities. Bottom panels show Suz12^−/− EBs that lack forms of organized structures and that are often smaller.

FIG. 5.

Suz12^−/− ES cells fail to repress ES cells markers and to activate differentiation-specific genes upon induction of differentiation. (A) Expression levels of Oct4, Nanog, Fgf4, Fgf17, and Pou2f3 in ES cells and in 9-day differentiated EBs determined by real-time qPCR. Right panels highlight the expression differences between Suz12^+/− and Suz12^−/− in 9-day differentiated EBs. (B) Expression levels of gastrulation markers in ES cells and in EBs at 3, 6, and 9 days after induction of differentiation. (C) Immunoblotting for Oct4 and Nanog during Suz12^+/− and Suz12^−/− ES cell differentiation showing repression of Oct4 and Nanog expression in Suz12^+/− but not Suz12^−/− ES cells. (D) Expression levels of Sox1, Nestin, Mausashi, and Calib2 during Suz12^+/− and Suz12^−/− ES cell differentiation showing the lack of activation of the neuronal precursor marker in Suz12^−/− cells.

FIG. 6.

PcG binding does not prevent transcriptional activation. ChIP analyses performed on promoters of genes that are activated during differentiation of ES cells. Real-time qPCR was used to determine the expression levels of the genes, and values were normalized as described in Materials and Methods. Antibodies specific for Ezh2, Suz12, Cbx8, Bmi1, H3K27me3, and the hemagglutinin (HA) epitope (negative control) were used for ChIPs. Enrichment is given as a percentage of input. Black bars, Suz12^+/− cells; red bars, Suz12^−/− cells; d, day; Ab, antibody.

FIG. 7.

PRC2 is actively recruited to repress gene transcription during ES cell differentiation. (A) ChIP analysis of the promoter regions of repressed genes during ES cell differentiation using antibodies against Ezh2, Suz12, Cbx8, Bmi1, and histone H3K27me3. Results of ChIPs and qPRCs in Suz12^+/− (black bars) and Suz12^−/− ES cells (red bars) are shown. The expression profile for each gene during differentiation is also presented (far left). HA, hemagglutinin; Ab, antibody. (B) Immunoblots of Suz12, Ezh2, Eed, Cbx8, and Bmi1 in Suz12^+/− and Suz12^−/− ES cells and in proliferating (P3) and senescent (P7) MEFs. β-Tubulin served as a loading control. (C) Different models for how the PcG proteins regulate transcription during differentiation. In the derepression model (1) the PcGs repress the expression of differentiation-specific genes in proliferating ES cells. The loss of PcG binding during differentiation leads to the activation or derepression of transcription. In the repression model (2) the PcGs are specifically recruited to target genes that undergo transcriptional repression during differentiation. In the activation model (3) PcGs (PRC2) accumulate on a subset of target genes during differentiation despite their transcriptional activation. In this model we propose that the binding of transcriptional activators is sufficient to overcome the PcGs. The binding of the PcGs could be important for the repression of the target genes during terminal differentiation and in this way preprogram the target genes during early development.