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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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Establishment of iPSC clones upon genome-wide erasure of H3K27me3 at the onset of reprogramming.A. Western blot analysis of EZH2 and H3K27me3 protein levels respectively at onset or 48 hr after reprogramming in Ezh2+/ΔSET and Ezh2ΔSET/ΔSET MEFs. Data are representative of two experiments. Quantification of protein levels at the indicated time points is shown in the right panel (controls in grey, mutants in purple). B. Strategy to induce reprogramming of tail tip fibroblasts (TTFs) lacking H3K27me3 at the onset of reprogramming. C. Western blot analysis of EZH2 and H3K27me3 protein levels in Cdkn2a−/− TTF carrying either one (+/ΔSET) or both (ΔSET/ΔSET) Ezh2 mutant alleles. As comparison, representative Ezh2-proficient (+/+) and -deficient (ΔSET/ΔSET) iPSC clones were analyzed. The band corresponding to H3K27me3 in mutant TTF has the same intensity of that from a mutant iPSC clone for which mass-spectrometry did not detect the presence of H3K27me3. D. Quantification of AP-positive primary iPSC colonies obtained from infection of 2.5×103Cdkn2a−/− TTF, respectively proficient (grey bar) or deficient (purple) for Ezh2, assessed in three independent experiments.
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pgen-1003292-g003: Establishment of iPSC clones upon genome-wide erasure of H3K27me3 at the onset of reprogramming.A. Western blot analysis of EZH2 and H3K27me3 protein levels respectively at onset or 48 hr after reprogramming in Ezh2+/ΔSET and Ezh2ΔSET/ΔSET MEFs. Data are representative of two experiments. Quantification of protein levels at the indicated time points is shown in the right panel (controls in grey, mutants in purple). B. Strategy to induce reprogramming of tail tip fibroblasts (TTFs) lacking H3K27me3 at the onset of reprogramming. C. Western blot analysis of EZH2 and H3K27me3 protein levels in Cdkn2a−/− TTF carrying either one (+/ΔSET) or both (ΔSET/ΔSET) Ezh2 mutant alleles. As comparison, representative Ezh2-proficient (+/+) and -deficient (ΔSET/ΔSET) iPSC clones were analyzed. The band corresponding to H3K27me3 in mutant TTF has the same intensity of that from a mutant iPSC clone for which mass-spectrometry did not detect the presence of H3K27me3. D. Quantification of AP-positive primary iPSC colonies obtained from infection of 2.5×103Cdkn2a−/− TTF, respectively proficient (grey bar) or deficient (purple) for Ezh2, assessed in three independent experiments.

Mentions: TAT-Cre-mediated inactivation of Ezh2 resulted in a threefold decrease in global H3K27me3 levels by the onset of the reprogramming process (Figure 3A). We therefore asked whether the unexpected possibility to reprogram despite inactivation of Ezh2 was due to residual H3K27me3 that could have still ensured, during the very first days of reprogramming, a sufficient degree of repression of lineage specific genes. To this end, we aimed at erasing the H3K27me3 mark completely by dilution, through serial passage of TAT-Cre treated MEF before the start of reprogramming. In MEF, however, PRC2 is a direct repressor of Cdkn2a, a locus encoding three key cell cycle regulators (p16, p19 and p15) whose activation promotes senescence [35]. Importantly, expression of this locus, and in particular of p19/Arf, was shown to hinder iPSC reprogramming [9], Thus, to prevent senescence driven by Cdkn2a de-repression following Ezh2 inactivation (Figure S3A), we resorted to compound primary tail tip fibroblasts (TTF) harboring both the conditional Ezh2 allele and the Ink4/Arf knock-out allele [36]. We subjected TTF to two sequential rounds of TAT-Cre transduction and passaged them 5 times before infection with STEMCCA and doxycycline administration (Figure 3B). Following confirmation that H3K27me3 was undetectable by Western blot on the day of infection with the reprogramming lentivirus (Figure 3C), and that efficiency of infection was equivalent for control and mutant TTF (Figure S3B), we went on to measure the efficiency of TF-dependent reprogramming under these most stringent conditions. As shown in Figure 3D, AP staining revealed that TTF starting off with undetectable H3K27me3 and controls yielded iPSC colonies with similar efficiency. This indicates that bulk levels of H3K27me3 are not required to prime silencing during the first days of reprogramming.


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)

Establishment of iPSC clones upon genome-wide erasure of H3K27me3 at the onset of reprogramming.A. Western blot analysis of EZH2 and H3K27me3 protein levels respectively at onset or 48 hr after reprogramming in Ezh2+/ΔSET and Ezh2ΔSET/ΔSET MEFs. Data are representative of two experiments. Quantification of protein levels at the indicated time points is shown in the right panel (controls in grey, mutants in purple). B. Strategy to induce reprogramming of tail tip fibroblasts (TTFs) lacking H3K27me3 at the onset of reprogramming. C. Western blot analysis of EZH2 and H3K27me3 protein levels in Cdkn2a−/− TTF carrying either one (+/ΔSET) or both (ΔSET/ΔSET) Ezh2 mutant alleles. As comparison, representative Ezh2-proficient (+/+) and -deficient (ΔSET/ΔSET) iPSC clones were analyzed. The band corresponding to H3K27me3 in mutant TTF has the same intensity of that from a mutant iPSC clone for which mass-spectrometry did not detect the presence of H3K27me3. D. Quantification of AP-positive primary iPSC colonies obtained from infection of 2.5×103Cdkn2a−/− TTF, respectively proficient (grey bar) or deficient (purple) for Ezh2, assessed in three independent experiments.
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pgen-1003292-g003: Establishment of iPSC clones upon genome-wide erasure of H3K27me3 at the onset of reprogramming.A. Western blot analysis of EZH2 and H3K27me3 protein levels respectively at onset or 48 hr after reprogramming in Ezh2+/ΔSET and Ezh2ΔSET/ΔSET MEFs. Data are representative of two experiments. Quantification of protein levels at the indicated time points is shown in the right panel (controls in grey, mutants in purple). B. Strategy to induce reprogramming of tail tip fibroblasts (TTFs) lacking H3K27me3 at the onset of reprogramming. C. Western blot analysis of EZH2 and H3K27me3 protein levels in Cdkn2a−/− TTF carrying either one (+/ΔSET) or both (ΔSET/ΔSET) Ezh2 mutant alleles. As comparison, representative Ezh2-proficient (+/+) and -deficient (ΔSET/ΔSET) iPSC clones were analyzed. The band corresponding to H3K27me3 in mutant TTF has the same intensity of that from a mutant iPSC clone for which mass-spectrometry did not detect the presence of H3K27me3. D. Quantification of AP-positive primary iPSC colonies obtained from infection of 2.5×103Cdkn2a−/− TTF, respectively proficient (grey bar) or deficient (purple) for Ezh2, assessed in three independent experiments.
Mentions: TAT-Cre-mediated inactivation of Ezh2 resulted in a threefold decrease in global H3K27me3 levels by the onset of the reprogramming process (Figure 3A). We therefore asked whether the unexpected possibility to reprogram despite inactivation of Ezh2 was due to residual H3K27me3 that could have still ensured, during the very first days of reprogramming, a sufficient degree of repression of lineage specific genes. To this end, we aimed at erasing the H3K27me3 mark completely by dilution, through serial passage of TAT-Cre treated MEF before the start of reprogramming. In MEF, however, PRC2 is a direct repressor of Cdkn2a, a locus encoding three key cell cycle regulators (p16, p19 and p15) whose activation promotes senescence [35]. Importantly, expression of this locus, and in particular of p19/Arf, was shown to hinder iPSC reprogramming [9], Thus, to prevent senescence driven by Cdkn2a de-repression following Ezh2 inactivation (Figure S3A), we resorted to compound primary tail tip fibroblasts (TTF) harboring both the conditional Ezh2 allele and the Ink4/Arf knock-out allele [36]. We subjected TTF to two sequential rounds of TAT-Cre transduction and passaged them 5 times before infection with STEMCCA and doxycycline administration (Figure 3B). Following confirmation that H3K27me3 was undetectable by Western blot on the day of infection with the reprogramming lentivirus (Figure 3C), and that efficiency of infection was equivalent for control and mutant TTF (Figure S3B), we went on to measure the efficiency of TF-dependent reprogramming under these most stringent conditions. As shown in Figure 3D, AP staining revealed that TTF starting off with undetectable H3K27me3 and controls yielded iPSC colonies with similar efficiency. This indicates that bulk levels of H3K27me3 are not required to prime silencing during the first days of reprogramming.

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