Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes

Abstract

Polycomb group (PcG) proteins are transcriptional repressors, which regulate proliferation and cell fate decisions during development, and their deregulated expression is a frequent event in human tumours. The Polycomb repressive complex 2 (PRC2) catalyzes trimethylation (me3) of histone H3 lysine 27 (K27), and it is believed that this activity mediates transcriptional repression. Despite the recent progress in understanding PcG function, the molecular mechanisms by which the PcG proteins repress transcription, as well as the mechanisms that lead to the activation of PcG target genes are poorly understood. To gain insight into these mechanisms, we have determined the global changes in histone modifications in embryonic stem (ES) cells lacking the PcG protein Suz12 that is essential for PRC2 activity. We show that loss of PRC2 activity results in a global increase in H3K27 acetylation. The methylation to acetylation switch correlates with the transcriptional activation of PcG target genes, both during ES cell differentiation and in MLL-AF9-transduced hematopoietic stem cells. Moreover, we provide evidence that the acetylation of H3K27 is catalyzed by the acetyltransferases p300 and CBP. Based on these data, we propose that the PcG proteins in part repress transcription by preventing the binding of acetyltransferases to PcG target genes.

Figure 1.

Loss of Suz12 induces H3K27 hyperacetylation. (A) (left panel) Coomassie-blue staining of SILAC-labeled histones purified from light-isotope-labeled (Lys-0) WT ES cells and heavy-isotope-labeled (Lys-8) Suz12 KO ES cells. Nanolc-tandem mass spectrometry quantification of the K27 methylation and acetylation levels of H3.2 and H3.3 in WT and Suz12 KO ES cells (right panel). (B) Western blot analyses of histones purified from WT and Suz12 KO ES cells using the indicated antibodies. H3 is presented as loading control. (C) Western blot analyses of histones purified from WT and two independent Suz12 KO ES cell lines using the indicated antibodies. H3 is presented as loading control. (D) ChIP analysis of the Olig1 and Olig2 promoter in WT and Suz12 KO ES cells using an H3K27Ac-specific antibody. H3K27Ac signal is normalized to histone density using an H3-specific antibody. (E) Western blot analyses of total protein extracts obtained from WT and Suz12 KO ES cell before and after CRE expression using the indicated antibodies. β-tubulin, H3 and Ponceau staining are presented as loading controls.

Figure 2.

PRC2 activity regulates H3K27Ac levels. (A) Western blot analyses of histones purified from WT, Eed^−/−, Suz12^−/−, Ezh2 conditional (Ezh2 loxP/loxP) and Ezh2^−/− ES cells using the indicated antibodies. H3 is presented as loading control. (B) ChIP analysis of the Olig2 promoter in WT, Suz12 and Eed KO ES cells using the indicated antibodies. Signals are normalized to histone density using an H3-specific antibody. (C) Western blot analyses using the indicated antibodies and luciferase activity of 293T cells containing a stable integration of a heterologous Gal4/luciferase reporter construct before and after Gal4-EZH2 expression. (D) ChIP analysis of the luciferase TSS in the cells presented in (C) using the indicated antibodies. Gal4, EZH2 and SUZ12 enrichments are presented as percentage of input while the different histone modifications signals are normalized to histone density using an H3-specific antibody.

Figure 3.

Regulation of H3K27me3 and H3K27Ac during ES cell differentiation. (A and B) qPCR expression (left panels) and ChIP analyses (right panels) of the Hoxa5 and Fgf4 promoters in ES and NPC cells using the indicated antibodies. Suz12 enrichments are presented as percentage of input while H3K27Ac and H3K27me3 signals are normalized to histone density using an H3-specific antibody.

Figure 4.

Regulation of H3K27me3 and H3K27Ac target gene binding in MLL-AF9 HSPCs and FDCP-mix cells. (A) β-Globin, Lipocalin and Hoxa9 qPCR expression analyses in FDCP-mix cells before and after granulocytic differentiation and in MLL-AF9-expressing HSPC. (B) ChIP analyses of Hoxa9 and Olig1 promoters in FDCP-mix cells and MLL-AF9-expressing HSPCs using the indicated antibodies. H3K27Ac, H3K9Ac and H3K27me3 signals are normalized to histone density using an H3-specific antibody.

Figure 5.

p00 and Cbp are required for efficient H3K27 acetylation in Suz12 KO ES cells. (A) qPCR expression analyses of the indicated genes in Suz12 KO ES cells transfected for 48 h with the indicated siRNA oligos. ‘U’ indicates the control siRNA oligo carrying a scrambled oligoribonucleotide sequence. (B) Western blot analyses of histones purified from Suz12 KO ES cells transfected with the indicated siRNA oligos using the indicated antibodies. H3 is presented as loading control. Quantification of the H3/H3K27Ac signal is indicated above each lane. A scrambled siRNA oligo (SCR) was used as negative control. (C and D) Western blot analyses of protein extracts and of purified histones from Suz12 KO ES cells transfected with the indicated siRNA oligos using the indicated antibodies. Vinculin, Ponceau staining and H3 are presented as loading controls. A scrambled siRNA oligo (SCR) was used as negative control. Quantification of the H3/H3K27Ac signal of western blots presented in ‘C’ is indicated above each lane. (E) Average quantification of the H3/H3K27Ac signals between the two independent siRNA experiments presented in C and D.

Figure 6.

Inhibition of p300 and Cbp activities leads to downregulation of H3K27 acetylation. (A) Western blot analyses of histones purified from WT and two independent Suz12 KO ES cell lines cultured in the presence (+) or absence (−, DMSO) of AA using the indicated antibodies. Ponceau staining of histones is presented as loading control. (B) Western blot analyses of histones purified from Suz12 KO ES cell lines cultured in the presence (+) or absence (−, DMSO) of AA using the indicated antibodies. H3 is presented as loading control. (C) Schematic representation of a model for the competition between PcG and p300/Cbp activities during ES cell differentiation and HSPC immortalization. TF, transcription factor.