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

Pasini D, Malatesta M, Jung HR, Walfridsson J, Willer A, Olsson L, Skotte J, Wutz A, Porse B, Jensen ON, Helin K - Nucleic Acids Res. (2010)

Bottom Line: We show that loss of PRC2 activity results in a global increase in H3K27 acetylation.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.

View Article: PubMed Central - PubMed

Affiliation: Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.

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.

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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.
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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.

Mentions: In mammals, 17 different HATs have been characterized so far and several of these have been reported to acetylate different lysine residues of histone H3 (43). To identify the HAT that could be involved in H3K27 acetylation, we generated a library containing three different siRNA oligonucleotides for each of the 17 HATs. First, we tested the efficiency of the different oligonucleotides to reduce the expression of each gene by real-time qPCR analysis of RNA extracted from Suz12 KO ES cells transfected with control (scrambled) or the specific siRNA oligonucleotide for 48 h. As shown in Figure 5A, the qPCR analysis showed that at least one oligonucleotide per gene reduced the expression of the target gene by at least 80%. Next, we picked the most efficient siRNA oligonucleotide for each gene and tested the effects of siRNA knockdown on H3K27Ac by western blot analysis. An example is presented in Figure 5B showing that siRNAs to Hat1, Kat2b, Cbp and p300 led to a significant reduction of H3K27Ac. While we were unable to further validate the effects of Hat1 and Kat2b downregulation (data not shown), independent experiments using different oligonucleotides to Cbp and p300 led to a loss of H3K27Ac levels in Suz12 KO cells (Figure 5C and D) as further confirmed by the quantification presented in Figure 5E. Importantly, the siRNA oligonucleotide that induced the most efficient downregulation of p300 correlates with the strongest reduction of H3K27Ac (Figure 5C). Taken together, these results suggest that p300 and Cbp are the major H3K27 HATs in ES cells.Figure 5.


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

Pasini D, Malatesta M, Jung HR, Walfridsson J, Willer A, Olsson L, Skotte J, Wutz A, Porse B, Jensen ON, Helin K - Nucleic Acids Res. (2010)

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.
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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.
Mentions: In mammals, 17 different HATs have been characterized so far and several of these have been reported to acetylate different lysine residues of histone H3 (43). To identify the HAT that could be involved in H3K27 acetylation, we generated a library containing three different siRNA oligonucleotides for each of the 17 HATs. First, we tested the efficiency of the different oligonucleotides to reduce the expression of each gene by real-time qPCR analysis of RNA extracted from Suz12 KO ES cells transfected with control (scrambled) or the specific siRNA oligonucleotide for 48 h. As shown in Figure 5A, the qPCR analysis showed that at least one oligonucleotide per gene reduced the expression of the target gene by at least 80%. Next, we picked the most efficient siRNA oligonucleotide for each gene and tested the effects of siRNA knockdown on H3K27Ac by western blot analysis. An example is presented in Figure 5B showing that siRNAs to Hat1, Kat2b, Cbp and p300 led to a significant reduction of H3K27Ac. While we were unable to further validate the effects of Hat1 and Kat2b downregulation (data not shown), independent experiments using different oligonucleotides to Cbp and p300 led to a loss of H3K27Ac levels in Suz12 KO cells (Figure 5C and D) as further confirmed by the quantification presented in Figure 5E. Importantly, the siRNA oligonucleotide that induced the most efficient downregulation of p300 correlates with the strongest reduction of H3K27Ac (Figure 5C). Taken together, these results suggest that p300 and Cbp are the major H3K27 HATs in ES cells.Figure 5.

Bottom Line: We show that loss of PRC2 activity results in a global increase in H3K27 acetylation.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.

View Article: PubMed Central - PubMed

Affiliation: Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.

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.

Show MeSH
Related in: MedlinePlus