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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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Effect of PRC2 inactivation on established Ezh2ΔSET/ΔSET iPSC clones and TF–induced reprogramming.A. Western blot analysis of EED protein levels in two Ezh2ΔSET/ΔSET iPSC clones infected with control virus (empty) or lentiviruses expressing independent short hairpin (sh) RNAs targeting Eed (#19 and #21)(left). Quantification of EED protein levels in infected cells after normalization based on Vinculin levels (right). B. H3K27me3 status (left panel) and expression levels (right panel) measured respectively by ChIP-qPCR and qRT-PCR, of 4 representative genes up regulated in MEF relative to iPSC, in two Ezh2ΔSET/ΔSET iPSC clones. Ezh2-mutant iPSC were infected with viruses expressing two independent hairpins for Eed or with a control virus. Status of H3K27me3 (±SEM) is represented as enrichment relative to input, after normalization for H3 density within the same amplicon. Expression levels are shown as fold change relative to iPSC infected with the empty vector. Error bars refer to qPCR triplicates. C. Western blot analysis of EED and H3K27me3 protein levels at day-6 of puromycin selection on Cdkn2a−/− Ezh2-proficient TTF expressing three independent Eed hairpins (lines 1, 2 and 3) or infected with a control virus (line 4). Quantification of protein levels relative to Vinculin or Histone H3 are shown in the right panel. D. AP staining of primary iPSC colonies obtained upon reprogramming of 1×103 (upper panel) Cdkn2a−/− Ezh2-proficient TTF expressing three independent Eed hairpins (lines 1, 2 and 3) or infected with an empty lentiviral vector (line 4) used as control. E. Quantification of AP+ iPSC colonies. Column height represents number of AP+ iPSC colonies obtained from 1×103 TTF expressing either one of the three independent Eed hairpins (lines 1, 2 and 3) or infected with an empty lentivirus vector (line 4) as control. Data are representative of two independent experiments performed using three different shRNAs.
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pgen-1003292-g005: Effect of PRC2 inactivation on established Ezh2ΔSET/ΔSET iPSC clones and TF–induced reprogramming.A. Western blot analysis of EED protein levels in two Ezh2ΔSET/ΔSET iPSC clones infected with control virus (empty) or lentiviruses expressing independent short hairpin (sh) RNAs targeting Eed (#19 and #21)(left). Quantification of EED protein levels in infected cells after normalization based on Vinculin levels (right). B. H3K27me3 status (left panel) and expression levels (right panel) measured respectively by ChIP-qPCR and qRT-PCR, of 4 representative genes up regulated in MEF relative to iPSC, in two Ezh2ΔSET/ΔSET iPSC clones. Ezh2-mutant iPSC were infected with viruses expressing two independent hairpins for Eed or with a control virus. Status of H3K27me3 (±SEM) is represented as enrichment relative to input, after normalization for H3 density within the same amplicon. Expression levels are shown as fold change relative to iPSC infected with the empty vector. Error bars refer to qPCR triplicates. C. Western blot analysis of EED and H3K27me3 protein levels at day-6 of puromycin selection on Cdkn2a−/− Ezh2-proficient TTF expressing three independent Eed hairpins (lines 1, 2 and 3) or infected with a control virus (line 4). Quantification of protein levels relative to Vinculin or Histone H3 are shown in the right panel. D. AP staining of primary iPSC colonies obtained upon reprogramming of 1×103 (upper panel) Cdkn2a−/− Ezh2-proficient TTF expressing three independent Eed hairpins (lines 1, 2 and 3) or infected with an empty lentiviral vector (line 4) used as control. E. Quantification of AP+ iPSC colonies. Column height represents number of AP+ iPSC colonies obtained from 1×103 TTF expressing either one of the three independent Eed hairpins (lines 1, 2 and 3) or infected with an empty lentivirus vector (line 4) as control. Data are representative of two independent experiments performed using three different shRNAs.

Mentions: Previous reports have highlighted partial redundancy between Ezh2 and Ezh1 in several cell types [27], [39]. To determine whether an alternative PRC2 complex is responsible for H3K27me3 of target genes also in Ezh2-mutant iPSC, we knocked down the essential PRC2 component EED in Ezh2-mutant iPSC. Infection of two Ezh2-mutant iPSC clones with lentiviruses expressing two independent shRNAs against Eed promoted a substantial reduction of EED protein levels (Figure 5A). Ezh2-mutant iPSC stably interfered for Eed (or infected with a control virus) were subjected to ChIP-qPCR assays on a representative set of target genes retaining the H3K27me3 mark in mutant iPSC. As shown in Figure 5B, levels of H3K27me3 decreased substantially in two independent Ezh2-mutant iPSC clones upon Eed knock-down. Importantly, loss of H3K27me3 led to the reactivation of target gene expression (Figure 5B, right panel). These results indicate that an alternative EZH1-containing PRC2 complex deposits H3K27me3 on a selected subset of Polycomb targets during reprogramming of Ezh2-mutant iPSC, to promote stable gene repression.


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)

Effect of PRC2 inactivation on established Ezh2ΔSET/ΔSET iPSC clones and TF–induced reprogramming.A. Western blot analysis of EED protein levels in two Ezh2ΔSET/ΔSET iPSC clones infected with control virus (empty) or lentiviruses expressing independent short hairpin (sh) RNAs targeting Eed (#19 and #21)(left). Quantification of EED protein levels in infected cells after normalization based on Vinculin levels (right). B. H3K27me3 status (left panel) and expression levels (right panel) measured respectively by ChIP-qPCR and qRT-PCR, of 4 representative genes up regulated in MEF relative to iPSC, in two Ezh2ΔSET/ΔSET iPSC clones. Ezh2-mutant iPSC were infected with viruses expressing two independent hairpins for Eed or with a control virus. Status of H3K27me3 (±SEM) is represented as enrichment relative to input, after normalization for H3 density within the same amplicon. Expression levels are shown as fold change relative to iPSC infected with the empty vector. Error bars refer to qPCR triplicates. C. Western blot analysis of EED and H3K27me3 protein levels at day-6 of puromycin selection on Cdkn2a−/− Ezh2-proficient TTF expressing three independent Eed hairpins (lines 1, 2 and 3) or infected with a control virus (line 4). Quantification of protein levels relative to Vinculin or Histone H3 are shown in the right panel. D. AP staining of primary iPSC colonies obtained upon reprogramming of 1×103 (upper panel) Cdkn2a−/− Ezh2-proficient TTF expressing three independent Eed hairpins (lines 1, 2 and 3) or infected with an empty lentiviral vector (line 4) used as control. E. Quantification of AP+ iPSC colonies. Column height represents number of AP+ iPSC colonies obtained from 1×103 TTF expressing either one of the three independent Eed hairpins (lines 1, 2 and 3) or infected with an empty lentivirus vector (line 4) as control. Data are representative of two independent experiments performed using three different shRNAs.
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Related In: Results  -  Collection

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pgen-1003292-g005: Effect of PRC2 inactivation on established Ezh2ΔSET/ΔSET iPSC clones and TF–induced reprogramming.A. Western blot analysis of EED protein levels in two Ezh2ΔSET/ΔSET iPSC clones infected with control virus (empty) or lentiviruses expressing independent short hairpin (sh) RNAs targeting Eed (#19 and #21)(left). Quantification of EED protein levels in infected cells after normalization based on Vinculin levels (right). B. H3K27me3 status (left panel) and expression levels (right panel) measured respectively by ChIP-qPCR and qRT-PCR, of 4 representative genes up regulated in MEF relative to iPSC, in two Ezh2ΔSET/ΔSET iPSC clones. Ezh2-mutant iPSC were infected with viruses expressing two independent hairpins for Eed or with a control virus. Status of H3K27me3 (±SEM) is represented as enrichment relative to input, after normalization for H3 density within the same amplicon. Expression levels are shown as fold change relative to iPSC infected with the empty vector. Error bars refer to qPCR triplicates. C. Western blot analysis of EED and H3K27me3 protein levels at day-6 of puromycin selection on Cdkn2a−/− Ezh2-proficient TTF expressing three independent Eed hairpins (lines 1, 2 and 3) or infected with a control virus (line 4). Quantification of protein levels relative to Vinculin or Histone H3 are shown in the right panel. D. AP staining of primary iPSC colonies obtained upon reprogramming of 1×103 (upper panel) Cdkn2a−/− Ezh2-proficient TTF expressing three independent Eed hairpins (lines 1, 2 and 3) or infected with an empty lentiviral vector (line 4) used as control. E. Quantification of AP+ iPSC colonies. Column height represents number of AP+ iPSC colonies obtained from 1×103 TTF expressing either one of the three independent Eed hairpins (lines 1, 2 and 3) or infected with an empty lentivirus vector (line 4) as control. Data are representative of two independent experiments performed using three different shRNAs.
Mentions: Previous reports have highlighted partial redundancy between Ezh2 and Ezh1 in several cell types [27], [39]. To determine whether an alternative PRC2 complex is responsible for H3K27me3 of target genes also in Ezh2-mutant iPSC, we knocked down the essential PRC2 component EED in Ezh2-mutant iPSC. Infection of two Ezh2-mutant iPSC clones with lentiviruses expressing two independent shRNAs against Eed promoted a substantial reduction of EED protein levels (Figure 5A). Ezh2-mutant iPSC stably interfered for Eed (or infected with a control virus) were subjected to ChIP-qPCR assays on a representative set of target genes retaining the H3K27me3 mark in mutant iPSC. As shown in Figure 5B, levels of H3K27me3 decreased substantially in two independent Ezh2-mutant iPSC clones upon Eed knock-down. Importantly, loss of H3K27me3 led to the reactivation of target gene expression (Figure 5B, right panel). These results indicate that an alternative EZH1-containing PRC2 complex deposits H3K27me3 on a selected subset of Polycomb targets during reprogramming of Ezh2-mutant iPSC, to promote stable gene repression.

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