With the goal of achieving insights into the mechanisms of PRC2-dependent transcriptional regulation, we decided to investigate if the trimethylation of H3K27 influences other posttranslational modifications of the histones. To do this, we used Suz12
KO mouse ES cells as a model system for global loss of H3K27me3 and quantified by SILAC mass spectrometry, the relative changes of a large number of other histone modifications. We grew Suz12
KO ES cells (12
) for 6 days (equal to approximately 15 population doublings) in a media that contained heavy-isotope-labeled lysine (Lys8) and Suz12
WT ES cells in media containing light-isotope-labeled lysine (Lys0) (A, left panel). The histones were purified from these cells and analyzed by nanoLC-tandem mass spectrometry to determine the relative abundance of all histone modifications in the two cell lines. The technical aspect and the overall results of this analysis are described in a separate manuscript (32
). Interestingly, this analysis showed that the most significant posttranslational change of the histones in the Suz12
KO ES cells, apart from the global loss of H3K27me2 and H3K27me3 [A and (32
)], was a significant increase of H3K27Ac (A, right panel). This observation could suggest the existence of a posttranslational switch between the acetylation and methylation of H3K27 controlled by PRC2.
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 (more ...)
To validate the mass spectrometry results, we performed western blot analysis using antibodies specific for H3K27me3 and H3K27Ac on histones purified from WT and Suz12
KO ES cells. In agreement with the mass spectrometry data, loss of Suz12 results in a significant increase of global H3K27Ac and a loss of H3K27me3 (B). This result was confirmed in an independently isolated Suz12
KO ES cell line (12
) (C), strongly suggesting that the increased H3K27Ac levels are a specific consequence of Suz12 loss.
These findings highlight the possibility that the switch between H3K27 methylation and acetylation may play a role in the transcriptional activation that follows displacement of PcG proteins from promoters. Furthermore, they may suggest that preventing H3K27 acetylation could be part of the mechanism by which PRC2 controls transcription. In order to obtain further evidence that this switch occurs specifically at the promoters of PRC2 target genes, we tested the H3K27Ac levels at the Olig1
) in WT and Suz12
KO ES cells by ChIP analysis. Consistent with the results presented in B and C, loss of Suz12 results in a specific increase of H3K27Ac of the Olig1
The global increase of H3K27Ac in Suz12
KO ES cells suggests that the PcG proteins antagonize an H3K27 acetyltransferase activity. Thus, we tested if re-expression of Suz12 in the KO ES cells could restore wild type levels of H3K27 posttranslational modifications. To do this, we inactivated the gene-trap cassette (12
) by CRE-mediated excision of the splice-acceptor site situated upstream of the β-galactosidase-neomycin cassette. Western blot analysis of Suz12
KO ES cells before and after CRE expression demonstrated that the inactivation of the gene-trap cassette restores physiological level of Suz12 expression (E). Importantly, western blot analyses on histones purified from the same cells demonstrated that the re-expression of Suz12 restores global H3K27me3 levels and, at the same time, decreases H3K27Ac to levels comparable to those observed in WT ES cells (E). Taken together, these results strongly suggest that the PcG proteins prevent H3K27 acetylation of target genes.
Next, we wanted to analyze if loss of other components of the PRC2 complex also leads to increased global levels of H3K27Ac. To do this, we analyzed the H3K27me3 and H3K27Ac levels in ES cell lines lacking different components of PRC2. Western blot analyses of histones purified from either WT or Suz12, Eed and Ezh2 KO ES cells demonstrated that all the PcG subunits of the PRC2 complex are essential for H3K27 trimethylation (A). Moreover, loss of H3K27me3 leads to a global increase of H3K27Ac in all the different PRC2 KO ES cell lines (A). Finally, ChIP analyses in Suz12 and Eed KO ES cells showed that H3K27Ac is specifically increased at the Olig2 promoter when compared to WT ES cells (B), confirming the results presented in B and D.
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 (more ...)
To directly analyze if PRC2 recruitment to target genes excludes H3K27Ac, we took advantage of a reporter system developed in our laboratory that combines the integration of a heterologous luciferase reporter construct containing five Gal4 DNA binding sites with the stable expression of a Gal4-EZH2 fusion protein (27
). As previously reported (27
), Gal4-EZH2 expression leads to a strong repression of luciferase activity (C). Moreover, ChIP analysis using Gal4-, EZH2- and SUZ12-specific antibodies demonstrated that EZH2 binding to the artificial promoter recruits endogenous components of the PRC2 complex (D, upper panel). Importantly, PRC2 recruitment to the luciferase promoter correlates with increased H3K27me3 levels and a significant decrease of H3K27Ac (D, lower panel). Moreover, EZH2 recruitment to the luciferase promoter does not lead to an enrichment of H3K9me3, but to a loss of H3K9 acetylation and possibly of other acetylated H3 residues as indicated by the decrease in global H3 acetylation (see D; Ac-H3 ChIP measuring K14/K9 acetyl H3). Consistent with previous publications (27,33
), EZH2 recruitment also correlates with a strong decrease of H3K4me3 (D). In order to understand the contribution of lysine de-acetylation in EZH2-mediated transcriptional repression, we treated the cells presented in C with the HDAC inhibitor Trichostatin-A (TSA). As shown in E, 6 h treatment with TSA abolished the repressive activity of EZH2. All together, these data show that PRC2-mediated trimethylation of H3K27 is sufficient to displace and/or prevent acetylation of histone H3 at PcG target genes.
The differentiation of ES cells to neural precursor cells (NPC) leads to the displacement of the PcG proteins from ~50% of their target genes and to their recruitment to a similar number of other target genes (35
). In both situations, binding of PcG proteins correlates with repressed transcription, whereas loss of PcG binding correlates with transcriptional activation (35
). To analyze if H3K27 acetylation is involved in the transcriptional activation of PcG target genes, we differentiated ES cells into NPCs, and characterized PcG binding and H3K27 modifications by ChIP analyses. We focused on two genes whose expression changes in opposite direction during differentiation. One gene, Hoxa5
, is repressed in ES cells and transcribed in NPC cells (A, left panel). The other gene, Fgf4
, is expressed at high levels in ES cells and silenced in NPC cells (B, left panel). To analyze PRC2 recruitment and the modification status of H3K27 in these two conditions, we performed ChIP analysis using Suz12-, H3K27me3- and H3K27Ac-specific antibodies in ES and NPC cells. Consistent with Hoxa5
-specific NPC expression, the Suz12 binding and the H3K27me3 levels at the Hoxa5
promoter are strongly reduced in NPC cells (A). Moreover, loss of PRC2 activity in NPC is associated with a strong increase of H3K27Ac levels that correlates with Hoxa5
transcriptional activation (A). In contrast, Suz12 and H3K27me3 are not found associated with the Fgf4
promoter in ES cells (B). Importantly, lack of PcG activity at Fgf4
promoter correlates with a strong enrichment of H3K27Ac and with high expression of Fgf4
in ES cells (B). Differentiation of ES cells to NPC leads to a strong repression of Fgf4
transcription that correlates with the recruitment of Suz12, the loss of H3K27Ac and the enrichment of H3K27me3 (B). Together, these data provide strong evidence for a competition between H3K27me3 and H3K27Ac in regulating gene expression during ES cell differentiation.
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 (more ...)
Homeotic genes (HOX
) are the best-characterized PcG target genes. HOX
genes play an essential role in the regulation of normal development (36
). Moreover, deregulation of HOX
expression has been linked to the development of different forms of human cancer (37
). For example, HOXA9
overexpression in HSPC is important for HSPC immortalization (38
), and the specific activation of HOXA9
expression is a feature of several leukemic fusion proteins including MLL-AF9 (39–41
). To investigate if the increased expression of Hoxa9
in immortalized HSPC involves transcriptional mechanism similar to the one described above for ES cell differentiation, we compared MLL-AF9 immortalized c-kit+
HSPC with a multipotent hematopoietic progenitor cell line FDCP-mix [the FDCP-mix cells were chosen to allow the expansion in TC of normal hematopoietic progenitors (31
)]. As previously reported (28,42
), MLL-AF9 immortalized HSPCs are blocked at the progenitor stage of the granulocytic differentiation pathways as confirmed by the expression of the granulocytic marker Lipocalin
expression is silenced in the FDCP-mix cells and is activated to similar levels as in MLL-AF9 expressing cells when induced to differentiate into granulocytes (A). Importantly, Hoxa9 expression was specifically detected in the MLL-AF9 expressing cells, but not in the differentiating FDCP-mix cells demonstrating the direct role of MLL-AF9 in Hoxa9
transcriptional activation (A).
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. (more ...)
To compare the effect of MLL-AF9 on the posttranslational modification of H3K27 on the Hoxa9 promoter, we performed ChIP analyses using H3K27me3- and H3K27Ac-specific antibodies in undifferentiated FDCP-mix cells and in MLL-AF9 expressing HSPC. In agreement with our previous observations, MLL-AF9 expression correlates with a specific loss of H3K27me3 from the Hoxa9 promoter (B). Importantly, this loss correlates with a strong increase of H3K27Ac and, consistent with the data presented in D, with an increase in H3K9Ac (B). Taken together, these data show that MLL-AF9 can compete for PcG binding to the Hoxa9 promoter and suggests that the molecular switch between H3K27me3 and H3K27Ac might play a role in Hoxa9 expression.
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 A, 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 B showing that siRNAs to Hat1
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 (C and D) as further confirmed by the quantification presented in E. Importantly, the siRNA oligonucleotide that induced the most efficient downregulation of p300 correlates with the strongest reduction of H3K27Ac (C). Taken together, these results suggest that p300 and Cbp are the major H3K27 HATs in ES cells.
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 (more ...)
To obtain independent evidence that Cbp and p300 are regulating H3K27 acetylation, we took advantage of the ability of anacardic acid (AA) to inhibit the in vitro
and in vivo
acetyltransferase activity of p300 and CBP (44,45
). Thus, we analyzed the H3K27Ac levels in Suz12
KO ES cells cultured in the presence of AA for 72 h by western blot analysis. As shown in A, the treatment of two independent Suz12
KO ES cell lines with AA led to a strong reduction of H3K27Ac. Moreover, overexposure of the same western blots showed that, in WT ES, the physiological levels of H3K27Ac are also reduced upon AA treatment. Importantly, while H3K27Ac was strongly reduced upon AA treatment, the acetylation of other histone H3 lysine residues was only mildly (H3K9) or not affected (H3K14) (B). Although we cannot exclude that AA treatment could also inhibit the activity of other acetyltransferases, these data, together with the siRNA results presented in , strongly support that p300 and Cbp are H3K27 acetyltransferases and further suggest a competition between PRC2, p300 and Cbp in the posttranslational modification of H3K27.
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 (more ...)