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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

Characterization of pluripotency in iPSC clones reprogrammed upon Ezh2 inactivation.A. Images of phase contrast (left), alkaline phosphatase staining (middle) and Oct4-driven GFP fluorescence (right) of representative control (Ezh2+/ΔSET, upper row) and mutant (Ezh2ΔSET/ΔSET, lower row) iPSC colonies. B. Flow cytometric analysis of SSEA-1 and endogenous OCT4 (OCT4-GFP) levels in representative control (Ezh2+/ΔSET, upper row) and mutant (Ezh2ΔSET/ΔSET, lower row) iPSC clones (cl.1 and cl.2). Embryonic stem cells (ESC) were used as positive control for SSEA1 expression (upper left). C. Growth curve of representative control (Ezh+/+, grey) and mutant (Ezh2ΔSET/ΔSET, purple) iPSC clones cultured in 2i/LIF medium for the indicated hours. Column height represents cell number. D. Hematoxylin & eosin staining and immunohistochemical analysis of representative sections of teratomas generated from either Ezh2 control (Ezh2+/+) or mutant (Ezh2ΔSET/ΔSET) iPSC cells of two representative clones. Stainings for mesoderm (upper row), endoderm (middle row) and ectoderm (lower row) markers are displayed. Data displayed in A, B, C and D are representative of at least four independent experiments, using two iPSC clones per genotype. E. Scatter plots showing global gene expression correlation analyses between Ezh2+/+ and Ezh2ΔSET/ΔSET iPSC (left panel), Ezh2+/+ iPSCs and MEF (central panel) and Ezh2ΔSET/ΔSET iPSC and MEF (right panel). Correlation coefficients (r) reveal the degree of similarity for each comparison. Genes within red lines differ less than 1.5-fold. F. Heat map representation of expression levels of genes involved in pluripotency, stemness and differentiation in two control (Ezh2+/+, first and second column) and two mutant (Ezh2ΔSET/ΔSET, third and fourth column) iPSC clones. ESC (fifth column) and MEF (last column) were used for comparison. Colors range from yellow (low dCt, higher expression) to black (high dCt, lower expression). Hierarchical clustering of samples is also shown.
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pgen-1003292-g002: Characterization of pluripotency in iPSC clones reprogrammed upon Ezh2 inactivation.A. Images of phase contrast (left), alkaline phosphatase staining (middle) and Oct4-driven GFP fluorescence (right) of representative control (Ezh2+/ΔSET, upper row) and mutant (Ezh2ΔSET/ΔSET, lower row) iPSC colonies. B. Flow cytometric analysis of SSEA-1 and endogenous OCT4 (OCT4-GFP) levels in representative control (Ezh2+/ΔSET, upper row) and mutant (Ezh2ΔSET/ΔSET, lower row) iPSC clones (cl.1 and cl.2). Embryonic stem cells (ESC) were used as positive control for SSEA1 expression (upper left). C. Growth curve of representative control (Ezh+/+, grey) and mutant (Ezh2ΔSET/ΔSET, purple) iPSC clones cultured in 2i/LIF medium for the indicated hours. Column height represents cell number. D. Hematoxylin & eosin staining and immunohistochemical analysis of representative sections of teratomas generated from either Ezh2 control (Ezh2+/+) or mutant (Ezh2ΔSET/ΔSET) iPSC cells of two representative clones. Stainings for mesoderm (upper row), endoderm (middle row) and ectoderm (lower row) markers are displayed. Data displayed in A, B, C and D are representative of at least four independent experiments, using two iPSC clones per genotype. E. Scatter plots showing global gene expression correlation analyses between Ezh2+/+ and Ezh2ΔSET/ΔSET iPSC (left panel), Ezh2+/+ iPSCs and MEF (central panel) and Ezh2ΔSET/ΔSET iPSC and MEF (right panel). Correlation coefficients (r) reveal the degree of similarity for each comparison. Genes within red lines differ less than 1.5-fold. F. Heat map representation of expression levels of genes involved in pluripotency, stemness and differentiation in two control (Ezh2+/+, first and second column) and two mutant (Ezh2ΔSET/ΔSET, third and fourth column) iPSC clones. ESC (fifth column) and MEF (last column) were used for comparison. Colors range from yellow (low dCt, higher expression) to black (high dCt, lower expression). Hierarchical clustering of samples is also shown.

Mentions: Having determined that Ezh2 inactivation in MEF is compatible with TF-induced cell reprogramming, we performed a comprehensive functional characterization of representative control and mutant iPSC clones. Ezh2ΔSET/ΔSET and Ezh2ΔSET/+ control MEF yielded iPSC colonies: i) with distinctive iPSC/ESC morphology (Figure 2A, left panel), ii) that stained positive for AP (Figure 2A, middle panel); and iii) that had reactivated the endogenous Oct4 gene (as assessed by GFP fluorescence, Figure 2A, right panel). iPSC clones of either genotype had the same percentage of cells co-expressing the pluripotency markers OCT4 and SSEA1, as measured by flow cytometry (Figure 2B). Next we assessed control and mutant iPSC clones for the two cardinal features that define the pluripotent state: self-renewal and the ability to differentiate into cell types of the three germ layers.


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)

Characterization of pluripotency in iPSC clones reprogrammed upon Ezh2 inactivation.A. Images of phase contrast (left), alkaline phosphatase staining (middle) and Oct4-driven GFP fluorescence (right) of representative control (Ezh2+/ΔSET, upper row) and mutant (Ezh2ΔSET/ΔSET, lower row) iPSC colonies. B. Flow cytometric analysis of SSEA-1 and endogenous OCT4 (OCT4-GFP) levels in representative control (Ezh2+/ΔSET, upper row) and mutant (Ezh2ΔSET/ΔSET, lower row) iPSC clones (cl.1 and cl.2). Embryonic stem cells (ESC) were used as positive control for SSEA1 expression (upper left). C. Growth curve of representative control (Ezh+/+, grey) and mutant (Ezh2ΔSET/ΔSET, purple) iPSC clones cultured in 2i/LIF medium for the indicated hours. Column height represents cell number. D. Hematoxylin & eosin staining and immunohistochemical analysis of representative sections of teratomas generated from either Ezh2 control (Ezh2+/+) or mutant (Ezh2ΔSET/ΔSET) iPSC cells of two representative clones. Stainings for mesoderm (upper row), endoderm (middle row) and ectoderm (lower row) markers are displayed. Data displayed in A, B, C and D are representative of at least four independent experiments, using two iPSC clones per genotype. E. Scatter plots showing global gene expression correlation analyses between Ezh2+/+ and Ezh2ΔSET/ΔSET iPSC (left panel), Ezh2+/+ iPSCs and MEF (central panel) and Ezh2ΔSET/ΔSET iPSC and MEF (right panel). Correlation coefficients (r) reveal the degree of similarity for each comparison. Genes within red lines differ less than 1.5-fold. F. Heat map representation of expression levels of genes involved in pluripotency, stemness and differentiation in two control (Ezh2+/+, first and second column) and two mutant (Ezh2ΔSET/ΔSET, third and fourth column) iPSC clones. ESC (fifth column) and MEF (last column) were used for comparison. Colors range from yellow (low dCt, higher expression) to black (high dCt, lower expression). Hierarchical clustering of samples is also shown.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003292-g002: Characterization of pluripotency in iPSC clones reprogrammed upon Ezh2 inactivation.A. Images of phase contrast (left), alkaline phosphatase staining (middle) and Oct4-driven GFP fluorescence (right) of representative control (Ezh2+/ΔSET, upper row) and mutant (Ezh2ΔSET/ΔSET, lower row) iPSC colonies. B. Flow cytometric analysis of SSEA-1 and endogenous OCT4 (OCT4-GFP) levels in representative control (Ezh2+/ΔSET, upper row) and mutant (Ezh2ΔSET/ΔSET, lower row) iPSC clones (cl.1 and cl.2). Embryonic stem cells (ESC) were used as positive control for SSEA1 expression (upper left). C. Growth curve of representative control (Ezh+/+, grey) and mutant (Ezh2ΔSET/ΔSET, purple) iPSC clones cultured in 2i/LIF medium for the indicated hours. Column height represents cell number. D. Hematoxylin & eosin staining and immunohistochemical analysis of representative sections of teratomas generated from either Ezh2 control (Ezh2+/+) or mutant (Ezh2ΔSET/ΔSET) iPSC cells of two representative clones. Stainings for mesoderm (upper row), endoderm (middle row) and ectoderm (lower row) markers are displayed. Data displayed in A, B, C and D are representative of at least four independent experiments, using two iPSC clones per genotype. E. Scatter plots showing global gene expression correlation analyses between Ezh2+/+ and Ezh2ΔSET/ΔSET iPSC (left panel), Ezh2+/+ iPSCs and MEF (central panel) and Ezh2ΔSET/ΔSET iPSC and MEF (right panel). Correlation coefficients (r) reveal the degree of similarity for each comparison. Genes within red lines differ less than 1.5-fold. F. Heat map representation of expression levels of genes involved in pluripotency, stemness and differentiation in two control (Ezh2+/+, first and second column) and two mutant (Ezh2ΔSET/ΔSET, third and fourth column) iPSC clones. ESC (fifth column) and MEF (last column) were used for comparison. Colors range from yellow (low dCt, higher expression) to black (high dCt, lower expression). Hierarchical clustering of samples is also shown.
Mentions: Having determined that Ezh2 inactivation in MEF is compatible with TF-induced cell reprogramming, we performed a comprehensive functional characterization of representative control and mutant iPSC clones. Ezh2ΔSET/ΔSET and Ezh2ΔSET/+ control MEF yielded iPSC colonies: i) with distinctive iPSC/ESC morphology (Figure 2A, left panel), ii) that stained positive for AP (Figure 2A, middle panel); and iii) that had reactivated the endogenous Oct4 gene (as assessed by GFP fluorescence, Figure 2A, right panel). iPSC clones of either genotype had the same percentage of cells co-expressing the pluripotency markers OCT4 and SSEA1, as measured by flow cytometry (Figure 2B). Next we assessed control and mutant iPSC clones for the two cardinal features that define the pluripotent state: self-renewal and the ability to differentiate into cell types of the three germ layers.

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