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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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Derivation and biochemical analysis of iPSC upon conditional Ezh2 inactivation.A. Diagram of the reprogramming protocol of MEF. B. Alkaline phosphatase staining of control (lower row) and mutant (upper row) primary iPSC colonies one week following doxycycline withdrawal. C. Number of AP-positive primary iPSC colonies obtained upon infection of, respectively, 2×103, 5×103, 1×104 or 6×104 MEF in two experiments performed with two biological replicates per genotype. D. EZH2, H3K27me1, H3K27me2 and H3K27me3 protein levels assessed by Western blot in two representative Ezh2 control (+/+) and mutant (ΔSET/ΔSET) iPSC clones. Vinculin and Histone H3 were used as loading controls for, respectively, EZH2 and methylated forms of H3K27. E. Relative abundance in control (upper row) and mutant (lower row) iPSC clones of the six possible methylation isoforms of the Histone H3 peptide spanning amino acids 27–40, as determined by mass spectrometry.
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pgen-1003292-g001: Derivation and biochemical analysis of iPSC upon conditional Ezh2 inactivation.A. Diagram of the reprogramming protocol of MEF. B. Alkaline phosphatase staining of control (lower row) and mutant (upper row) primary iPSC colonies one week following doxycycline withdrawal. C. Number of AP-positive primary iPSC colonies obtained upon infection of, respectively, 2×103, 5×103, 1×104 or 6×104 MEF in two experiments performed with two biological replicates per genotype. D. EZH2, H3K27me1, H3K27me2 and H3K27me3 protein levels assessed by Western blot in two representative Ezh2 control (+/+) and mutant (ΔSET/ΔSET) iPSC clones. Vinculin and Histone H3 were used as loading controls for, respectively, EZH2 and methylated forms of H3K27. E. Relative abundance in control (upper row) and mutant (lower row) iPSC clones of the six possible methylation isoforms of the Histone H3 peptide spanning amino acids 27–40, as determined by mass spectrometry.

Mentions: In order to investigate the function of H3K27 trimethylation (H3K27me3) in TF-induced cell reprogramming, we derived embryonic fibroblasts (MEF) from mutant mice harboring a conditional Ezh2 knock-out allele carrying loxP sites (Ezh2fl) flanking exons coding for the catalytic SET domain [30] (Figure 1 and Table 1). Experimental mice carried also an Oct4-GFP knock-in reporter gene [31] to monitor endogenous Oct4 gene re-activation during reprogramming. We expressed Oct4, Klf4, c-Myc and Sox2 reprogramming factors from a single doxycycline-regulated bicistronic lentiviral vector (STEMCCA), in which each cistron codes for a fusion of two factors which are then released through, respectively, F2A and E2A self-cleaving peptide signals [32]. As depicted in Figure 1A, our reprogramming protocol entailed: i) treatment of experimental (Ezh2fl/fl; Oct4-GFP) and control (Ezh2+/fl; Oct4-GFP) MEF with cell-permeable TAT-Cre recombinase to inactivate Ezh2; ii) infection with STEMCCA and reverse tetracycline transactivator (rtTA) lentiviruses, followed by replating at clonal density and switch to a chemically-defined embryonic stem cell (ESC) medium (KSR) and administration of doxycycline for 23 days; iii) withdrawal of doxycycline to select cells that re-expressed the endogenous pluripotency factors; and iv) isolation, clonal propagation and molecular and functional characterization of individual iPSC clones. As controls, we used interchangeably iPSC from Ezh2fl/+ MEF treated with TAT-Cre (referred to as Ezh2ΔSET/+ iPSC) as well as iPSC from Ezh2+/+or Ezh2fl/fl MEF that were not exposed to Tat-cre (referred to as Ezh2+/+ and Ezh2fl/fl iPSC). Upon verification of comparable efficiency in the infection with STEMCCA of control and mutant MEF (Figure S1A), we analyzed the efficiency of reprogramming following Ezh2 inactivation by comparing the number of primary iPSC colonies assessed through staining with alkalyne phosphatase (AP) 7 days after doxycycline removal (Figure 1B). Starting from two different batches of MEF, we found no significant difference in the number of iPSC colonies generated from mutant (Ezh2ΔSET/ΔSET) and control (Ezh2ΔSET/+) fibroblasts (Figure 1B, 1C). As genome-wide chromatin analyses have postulated a critical role for H3K27me3 in the resetting of transcriptional programs during iPSC derivation [17], we tested whether this unexpected finding resulted from selective reprogramming of Ezh2fl/fl MEF that had escaped Cre-mediated recombination, thereby preserving H3K27me3-dependent reprogramming proficiency. We ruled out this possibility as the majority of iPSC clones analyzed carried the SET-deleted Ezh2 allele regardless of the genotype (Figure S1B).


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)

Derivation and biochemical analysis of iPSC upon conditional Ezh2 inactivation.A. Diagram of the reprogramming protocol of MEF. B. Alkaline phosphatase staining of control (lower row) and mutant (upper row) primary iPSC colonies one week following doxycycline withdrawal. C. Number of AP-positive primary iPSC colonies obtained upon infection of, respectively, 2×103, 5×103, 1×104 or 6×104 MEF in two experiments performed with two biological replicates per genotype. D. EZH2, H3K27me1, H3K27me2 and H3K27me3 protein levels assessed by Western blot in two representative Ezh2 control (+/+) and mutant (ΔSET/ΔSET) iPSC clones. Vinculin and Histone H3 were used as loading controls for, respectively, EZH2 and methylated forms of H3K27. E. Relative abundance in control (upper row) and mutant (lower row) iPSC clones of the six possible methylation isoforms of the Histone H3 peptide spanning amino acids 27–40, as determined by mass spectrometry.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585017&req=5

pgen-1003292-g001: Derivation and biochemical analysis of iPSC upon conditional Ezh2 inactivation.A. Diagram of the reprogramming protocol of MEF. B. Alkaline phosphatase staining of control (lower row) and mutant (upper row) primary iPSC colonies one week following doxycycline withdrawal. C. Number of AP-positive primary iPSC colonies obtained upon infection of, respectively, 2×103, 5×103, 1×104 or 6×104 MEF in two experiments performed with two biological replicates per genotype. D. EZH2, H3K27me1, H3K27me2 and H3K27me3 protein levels assessed by Western blot in two representative Ezh2 control (+/+) and mutant (ΔSET/ΔSET) iPSC clones. Vinculin and Histone H3 were used as loading controls for, respectively, EZH2 and methylated forms of H3K27. E. Relative abundance in control (upper row) and mutant (lower row) iPSC clones of the six possible methylation isoforms of the Histone H3 peptide spanning amino acids 27–40, as determined by mass spectrometry.
Mentions: In order to investigate the function of H3K27 trimethylation (H3K27me3) in TF-induced cell reprogramming, we derived embryonic fibroblasts (MEF) from mutant mice harboring a conditional Ezh2 knock-out allele carrying loxP sites (Ezh2fl) flanking exons coding for the catalytic SET domain [30] (Figure 1 and Table 1). Experimental mice carried also an Oct4-GFP knock-in reporter gene [31] to monitor endogenous Oct4 gene re-activation during reprogramming. We expressed Oct4, Klf4, c-Myc and Sox2 reprogramming factors from a single doxycycline-regulated bicistronic lentiviral vector (STEMCCA), in which each cistron codes for a fusion of two factors which are then released through, respectively, F2A and E2A self-cleaving peptide signals [32]. As depicted in Figure 1A, our reprogramming protocol entailed: i) treatment of experimental (Ezh2fl/fl; Oct4-GFP) and control (Ezh2+/fl; Oct4-GFP) MEF with cell-permeable TAT-Cre recombinase to inactivate Ezh2; ii) infection with STEMCCA and reverse tetracycline transactivator (rtTA) lentiviruses, followed by replating at clonal density and switch to a chemically-defined embryonic stem cell (ESC) medium (KSR) and administration of doxycycline for 23 days; iii) withdrawal of doxycycline to select cells that re-expressed the endogenous pluripotency factors; and iv) isolation, clonal propagation and molecular and functional characterization of individual iPSC clones. As controls, we used interchangeably iPSC from Ezh2fl/+ MEF treated with TAT-Cre (referred to as Ezh2ΔSET/+ iPSC) as well as iPSC from Ezh2+/+or Ezh2fl/fl MEF that were not exposed to Tat-cre (referred to as Ezh2+/+ and Ezh2fl/fl iPSC). Upon verification of comparable efficiency in the infection with STEMCCA of control and mutant MEF (Figure S1A), we analyzed the efficiency of reprogramming following Ezh2 inactivation by comparing the number of primary iPSC colonies assessed through staining with alkalyne phosphatase (AP) 7 days after doxycycline removal (Figure 1B). Starting from two different batches of MEF, we found no significant difference in the number of iPSC colonies generated from mutant (Ezh2ΔSET/ΔSET) and control (Ezh2ΔSET/+) fibroblasts (Figure 1B, 1C). As genome-wide chromatin analyses have postulated a critical role for H3K27me3 in the resetting of transcriptional programs during iPSC derivation [17], we tested whether this unexpected finding resulted from selective reprogramming of Ezh2fl/fl MEF that had escaped Cre-mediated recombination, thereby preserving H3K27me3-dependent reprogramming proficiency. We ruled out this possibility as the majority of iPSC clones analyzed carried the SET-deleted Ezh2 allele regardless of the genotype (Figure S1B).

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