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Cell reprogramming requires silencing of a core subset of polycomb targets.

Fragola G, Germain PL, Laise P, Cuomo A, Blasimme A, Gross F, Signaroldi E, Bucci G, Sommer C, Pruneri G, Mazzarol G, Bonaldi T, Mostoslavsky G, Casola S, Testa G - PLoS Genet. (2013)

Bottom Line: Transcription factor (TF)-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications.Here, we dissected the functional role of H3K27me3 in TF-induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming.Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers.

View Article: PubMed Central - PubMed

Affiliation: European Institute of Oncology, IFOM-IEO Campus, Milan, Italy.

ABSTRACT
Transcription factor (TF)-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications. Polycomb-mediated histone H3 lysine-27 trimethylation (H3K27me3) has been proposed as a defining mark that distinguishes the somatic from the iPSC epigenome. Here, we dissected the functional role of H3K27me3 in TF-induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming. Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, the genome-wide analysis of H3K27me3 in Ezh2 mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF-induced reprogramming. These results indicate that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TF-dependent cell reprogramming.

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Related in: MedlinePlus

Genome-wide distribution of H3K27me3 in Ezh2ΔSET/ΔSET iPSC revealed through ChIP–seq.A. Pie chart showing partition of Polycomb targets based on H3K27 methylation status in Ezh2ΔSET/ΔSET iPSCs. B. Venn diagram displaying overlap between H3K27me3+ genes in Ezh2+/+ (grey) and Ezh2ΔSET/ΔSET (purple) iPSC. C. Venn diagram showing overlap between H3K27me3+ genes in Ezh2+/+ iPSC (grey), H3K27me2+/H3K27me3− genes in Ezh2ΔSET/ΔSET iPSC (orange) and H3K27me2+/H3K27me3− in Ezh2+/+ iPSC (light blue). D. Analysis of transcript levels measured by qRT-PCR, and status of SUZ12 binding, H3K27me2 and H3K27me1 enrichment revealed by ChIP q-PCR at promoters of 7 genes overexpressed in MEF relative to iPSC and representative of groups of genes marked by either H3K27me2+/H3K27me3+ or H3K27me2+/H3K27me3− in Ezh2ΔSET/ΔSET iPSC. For all analyses, two Ezh2 control (+/+; grey) iPSC clones were compared to two mutant (ΔSET/ΔSET; purple) counterparts. Levels of expression are shown as ddCt (log2 scale) relative to MEF. Status of a particular histone modification (±SEM) is represented as fold change of enrichment relative to input, after normalization for H3 density within the same amplicon. SUZ12 enrichment at promoters of indicated genes was determined comparing it to that of an unrelated IgG. Error bars refer to qPCR triplicates. E. Average reads distribution of H3K27me3 around the TSS in Ezh2+/+ iPSC. Genes were divided in two classes based on H3K27me3 status in Ezh2ΔSET/ΔSET iPSC (H3K27me3+ genes in green, H3K27me3− genes in red) and compared to all genes (black) F. Gene ontology analysis of genes belonging to the three main groups based on H3K27 methylation status in Ezh2ΔSET/ΔSET iPSC depicted in panel A. Bars represent P-values in –Log2 scale of the corresponding biological processes. Dashed lines identify the significance threshold. G. Pie chart of H3K27 methylation status in Ezh2ΔSET/ΔSET iPSC of MEF specific genes marked by H3K27me3 in Ezh2+/+ iPSC. Color code is shown in panel A.
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pgen-1003292-g004: Genome-wide distribution of H3K27me3 in Ezh2ΔSET/ΔSET iPSC revealed through ChIP–seq.A. Pie chart showing partition of Polycomb targets based on H3K27 methylation status in Ezh2ΔSET/ΔSET iPSCs. B. Venn diagram displaying overlap between H3K27me3+ genes in Ezh2+/+ (grey) and Ezh2ΔSET/ΔSET (purple) iPSC. C. Venn diagram showing overlap between H3K27me3+ genes in Ezh2+/+ iPSC (grey), H3K27me2+/H3K27me3− genes in Ezh2ΔSET/ΔSET iPSC (orange) and H3K27me2+/H3K27me3− in Ezh2+/+ iPSC (light blue). D. Analysis of transcript levels measured by qRT-PCR, and status of SUZ12 binding, H3K27me2 and H3K27me1 enrichment revealed by ChIP q-PCR at promoters of 7 genes overexpressed in MEF relative to iPSC and representative of groups of genes marked by either H3K27me2+/H3K27me3+ or H3K27me2+/H3K27me3− in Ezh2ΔSET/ΔSET iPSC. For all analyses, two Ezh2 control (+/+; grey) iPSC clones were compared to two mutant (ΔSET/ΔSET; purple) counterparts. Levels of expression are shown as ddCt (log2 scale) relative to MEF. Status of a particular histone modification (±SEM) is represented as fold change of enrichment relative to input, after normalization for H3 density within the same amplicon. SUZ12 enrichment at promoters of indicated genes was determined comparing it to that of an unrelated IgG. Error bars refer to qPCR triplicates. E. Average reads distribution of H3K27me3 around the TSS in Ezh2+/+ iPSC. Genes were divided in two classes based on H3K27me3 status in Ezh2ΔSET/ΔSET iPSC (H3K27me3+ genes in green, H3K27me3− genes in red) and compared to all genes (black) F. Gene ontology analysis of genes belonging to the three main groups based on H3K27 methylation status in Ezh2ΔSET/ΔSET iPSC depicted in panel A. Bars represent P-values in –Log2 scale of the corresponding biological processes. Dashed lines identify the significance threshold. G. Pie chart of H3K27 methylation status in Ezh2ΔSET/ΔSET iPSC of MEF specific genes marked by H3K27me3 in Ezh2+/+ iPSC. Color code is shown in panel A.

Mentions: The finding that bulk H3K27me3 was apparently dispensable for reprogramming even when erased at the onset of the process was at odds with its purported role as the critical mark that distinguishes MEF from iPSC epigenomes as well as, more broadly, with its pivotal role in the maintenance of gene repression through embryogenesis and adulthood [17]. We therefore asked whether, upon Ezh2 inactivation, residual levels of H3K27me3 below the threshold of Western blot and mass spectrometry sensitivity, could still be deposited on selected targets. To address this point we performed chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq). We generated ChIP-seq profiles from two independent control and mutant iPSC clones for both H3K27me3 and H3K27me2 with highly specific monoclonal antibodies. Consistently with our prediction, the higher sensitivity of ChIP-seq did reveal the presence of residual H3K27me3 in mutant iPSC clones. Specifically, the mark was retained on 2477 genes (with an enriched region overlapping a +/−5kb region interval around the transcriptional start site, TSS), comprising close to half of all H3K27me3 targets retrieved from wild type iPSC. Mutant clones showed a preferential retention of H3K27me3 proximal to the TSS of target genes. In comparison to the full complement of PRC2 targets in control iPSC, mutant clones displayed a clear tripartition in the genome-wide distribution of H3K27me3 and H3K27me2 marks (Figure 4A). 47% of genes retained both H3K27me3 and H3K27me2, 39% of genes were marked only by H3K27me2 and 13,7% of genes lost both marks. Importantly, the complement of genes enriched for H3K27me3 in mutant iPSC clones was almost entirely comprised within the group of H3K27me3 targets found in control iPSC cells, thus excluding a significant redistribution of the mark to new targets in cells reprogrammed in the absence of functional Ezh2 (Figure 4B). Furthermore, we found only a small overlap in the distribution of H3K27me2-only targets between control and mutant iPSC clones. Instead, the complement of genes marked only by H3K27me2 in mutant iPSC clones was to a good extent comprised within the subset of genes that are H3K27 trimethylated in control iPSC (Figure 4C). Thus, we conclude that during reprogramming in the absence of Ezh2, i) H3K27me3 is selectively retained on a subset of the targets that are normally H3K27 trimethylated in iPSC, where it coexists with H3K27me2; ii) H3K27me2 is lost at targets that are normally carrying only this mark in iPSC; and iii) H3K27me2 is retained in 86% of the targets that are normally H3K27 trimethylated in iPSC, coexisting, in half of these, with residual H3K27me3. We validated these findings through individual ChIP-qPCR on genes selected among those that were downregulated in the MEF to iPSC transition (Figure 4D and Table S2). We confirmed the sharp distinction between a group of genes that retained both H3K27me3 and H3K27me2 and those that only retained H3K27me2, irrespective of the level of transcriptional repression that was equivalent for the two groups between control and mutant iPSC (Figure 4D and Table S2). Interestingly, we found a stronger enrichment for PRC2 on the genes that selectively retained the H3K27me3 mark, likely reflecting its ability to act as docking site for the EED subunit of PRC2.


Cell reprogramming requires silencing of a core subset of polycomb targets.

Fragola G, Germain PL, Laise P, Cuomo A, Blasimme A, Gross F, Signaroldi E, Bucci G, Sommer C, Pruneri G, Mazzarol G, Bonaldi T, Mostoslavsky G, Casola S, Testa G - PLoS Genet. (2013)

Genome-wide distribution of H3K27me3 in Ezh2ΔSET/ΔSET iPSC revealed through ChIP–seq.A. Pie chart showing partition of Polycomb targets based on H3K27 methylation status in Ezh2ΔSET/ΔSET iPSCs. B. Venn diagram displaying overlap between H3K27me3+ genes in Ezh2+/+ (grey) and Ezh2ΔSET/ΔSET (purple) iPSC. C. Venn diagram showing overlap between H3K27me3+ genes in Ezh2+/+ iPSC (grey), H3K27me2+/H3K27me3− genes in Ezh2ΔSET/ΔSET iPSC (orange) and H3K27me2+/H3K27me3− in Ezh2+/+ iPSC (light blue). D. Analysis of transcript levels measured by qRT-PCR, and status of SUZ12 binding, H3K27me2 and H3K27me1 enrichment revealed by ChIP q-PCR at promoters of 7 genes overexpressed in MEF relative to iPSC and representative of groups of genes marked by either H3K27me2+/H3K27me3+ or H3K27me2+/H3K27me3− in Ezh2ΔSET/ΔSET iPSC. For all analyses, two Ezh2 control (+/+; grey) iPSC clones were compared to two mutant (ΔSET/ΔSET; purple) counterparts. Levels of expression are shown as ddCt (log2 scale) relative to MEF. Status of a particular histone modification (±SEM) is represented as fold change of enrichment relative to input, after normalization for H3 density within the same amplicon. SUZ12 enrichment at promoters of indicated genes was determined comparing it to that of an unrelated IgG. Error bars refer to qPCR triplicates. E. Average reads distribution of H3K27me3 around the TSS in Ezh2+/+ iPSC. Genes were divided in two classes based on H3K27me3 status in Ezh2ΔSET/ΔSET iPSC (H3K27me3+ genes in green, H3K27me3− genes in red) and compared to all genes (black) F. Gene ontology analysis of genes belonging to the three main groups based on H3K27 methylation status in Ezh2ΔSET/ΔSET iPSC depicted in panel A. Bars represent P-values in –Log2 scale of the corresponding biological processes. Dashed lines identify the significance threshold. G. Pie chart of H3K27 methylation status in Ezh2ΔSET/ΔSET iPSC of MEF specific genes marked by H3K27me3 in Ezh2+/+ iPSC. Color code is shown in panel A.
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Show All Figures
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pgen-1003292-g004: Genome-wide distribution of H3K27me3 in Ezh2ΔSET/ΔSET iPSC revealed through ChIP–seq.A. Pie chart showing partition of Polycomb targets based on H3K27 methylation status in Ezh2ΔSET/ΔSET iPSCs. B. Venn diagram displaying overlap between H3K27me3+ genes in Ezh2+/+ (grey) and Ezh2ΔSET/ΔSET (purple) iPSC. C. Venn diagram showing overlap between H3K27me3+ genes in Ezh2+/+ iPSC (grey), H3K27me2+/H3K27me3− genes in Ezh2ΔSET/ΔSET iPSC (orange) and H3K27me2+/H3K27me3− in Ezh2+/+ iPSC (light blue). D. Analysis of transcript levels measured by qRT-PCR, and status of SUZ12 binding, H3K27me2 and H3K27me1 enrichment revealed by ChIP q-PCR at promoters of 7 genes overexpressed in MEF relative to iPSC and representative of groups of genes marked by either H3K27me2+/H3K27me3+ or H3K27me2+/H3K27me3− in Ezh2ΔSET/ΔSET iPSC. For all analyses, two Ezh2 control (+/+; grey) iPSC clones were compared to two mutant (ΔSET/ΔSET; purple) counterparts. Levels of expression are shown as ddCt (log2 scale) relative to MEF. Status of a particular histone modification (±SEM) is represented as fold change of enrichment relative to input, after normalization for H3 density within the same amplicon. SUZ12 enrichment at promoters of indicated genes was determined comparing it to that of an unrelated IgG. Error bars refer to qPCR triplicates. E. Average reads distribution of H3K27me3 around the TSS in Ezh2+/+ iPSC. Genes were divided in two classes based on H3K27me3 status in Ezh2ΔSET/ΔSET iPSC (H3K27me3+ genes in green, H3K27me3− genes in red) and compared to all genes (black) F. Gene ontology analysis of genes belonging to the three main groups based on H3K27 methylation status in Ezh2ΔSET/ΔSET iPSC depicted in panel A. Bars represent P-values in –Log2 scale of the corresponding biological processes. Dashed lines identify the significance threshold. G. Pie chart of H3K27 methylation status in Ezh2ΔSET/ΔSET iPSC of MEF specific genes marked by H3K27me3 in Ezh2+/+ iPSC. Color code is shown in panel A.
Mentions: The finding that bulk H3K27me3 was apparently dispensable for reprogramming even when erased at the onset of the process was at odds with its purported role as the critical mark that distinguishes MEF from iPSC epigenomes as well as, more broadly, with its pivotal role in the maintenance of gene repression through embryogenesis and adulthood [17]. We therefore asked whether, upon Ezh2 inactivation, residual levels of H3K27me3 below the threshold of Western blot and mass spectrometry sensitivity, could still be deposited on selected targets. To address this point we performed chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq). We generated ChIP-seq profiles from two independent control and mutant iPSC clones for both H3K27me3 and H3K27me2 with highly specific monoclonal antibodies. Consistently with our prediction, the higher sensitivity of ChIP-seq did reveal the presence of residual H3K27me3 in mutant iPSC clones. Specifically, the mark was retained on 2477 genes (with an enriched region overlapping a +/−5kb region interval around the transcriptional start site, TSS), comprising close to half of all H3K27me3 targets retrieved from wild type iPSC. Mutant clones showed a preferential retention of H3K27me3 proximal to the TSS of target genes. In comparison to the full complement of PRC2 targets in control iPSC, mutant clones displayed a clear tripartition in the genome-wide distribution of H3K27me3 and H3K27me2 marks (Figure 4A). 47% of genes retained both H3K27me3 and H3K27me2, 39% of genes were marked only by H3K27me2 and 13,7% of genes lost both marks. Importantly, the complement of genes enriched for H3K27me3 in mutant iPSC clones was almost entirely comprised within the group of H3K27me3 targets found in control iPSC cells, thus excluding a significant redistribution of the mark to new targets in cells reprogrammed in the absence of functional Ezh2 (Figure 4B). Furthermore, we found only a small overlap in the distribution of H3K27me2-only targets between control and mutant iPSC clones. Instead, the complement of genes marked only by H3K27me2 in mutant iPSC clones was to a good extent comprised within the subset of genes that are H3K27 trimethylated in control iPSC (Figure 4C). Thus, we conclude that during reprogramming in the absence of Ezh2, i) H3K27me3 is selectively retained on a subset of the targets that are normally H3K27 trimethylated in iPSC, where it coexists with H3K27me2; ii) H3K27me2 is lost at targets that are normally carrying only this mark in iPSC; and iii) H3K27me2 is retained in 86% of the targets that are normally H3K27 trimethylated in iPSC, coexisting, in half of these, with residual H3K27me3. We validated these findings through individual ChIP-qPCR on genes selected among those that were downregulated in the MEF to iPSC transition (Figure 4D and Table S2). We confirmed the sharp distinction between a group of genes that retained both H3K27me3 and H3K27me2 and those that only retained H3K27me2, irrespective of the level of transcriptional repression that was equivalent for the two groups between control and mutant iPSC (Figure 4D and Table S2). Interestingly, we found a stronger enrichment for PRC2 on the genes that selectively retained the H3K27me3 mark, likely reflecting its ability to act as docking site for the EED subunit of PRC2.

Bottom Line: Transcription factor (TF)-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications.Here, we dissected the functional role of H3K27me3 in TF-induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming.Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers.

View Article: PubMed Central - PubMed

Affiliation: European Institute of Oncology, IFOM-IEO Campus, Milan, Italy.

ABSTRACT
Transcription factor (TF)-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications. Polycomb-mediated histone H3 lysine-27 trimethylation (H3K27me3) has been proposed as a defining mark that distinguishes the somatic from the iPSC epigenome. Here, we dissected the functional role of H3K27me3 in TF-induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming. Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, the genome-wide analysis of H3K27me3 in Ezh2 mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF-induced reprogramming. These results indicate that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TF-dependent cell reprogramming.

Show MeSH
Related in: MedlinePlus