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Ezh2 mediated H3K27me3 activity facilitates somatic transition during human pluripotent reprogramming.

Rao RA, Dhele N, Cheemadan S, Ketkar A, Jayandharan GR, Palakodeti D, Rampalli S - Sci Rep (2015)

Bottom Line: At the epigenetic level, it results in drastic chromatin changes to erase the existing somatic "memory" and to establish the pluripotent state.We also demonstrate that the Ezh2 negatively regulates the expression of pro-EMT miRNA's such as miR-23a locus during MET.Collectively, our findings provide a mechanistic understanding by which Ezh2 restricts the somatic programme during early phase of cellular reprogramming and establish the importance of Ezh2 dependent H3K27me3 activity in transcriptional and miRNA modulation during human iPSC generation.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre For Inflammation and Tissue Homeostasis, Institute for Stem Cell Biology and Regenerative Medicine (inStem), National Centre for Biological Sciences, GKVK Campus, Bellary Road, Bangalore 560065, Karnataka, India [2] Sastra University, Tirumalaisamudram, Thanjavur - 613 401, TamilNadu, India.

ABSTRACT
Factor induced reprogramming of fibroblasts is an orchestrated but inefficient process. At the epigenetic level, it results in drastic chromatin changes to erase the existing somatic "memory" and to establish the pluripotent state. Accordingly, alterations of chromatin regulators including Ezh2 influence iPSC generation. While the role of individual transcription factors in resetting the chromatin landscape during iPSC generation is increasingly evident, their engagement with chromatin modulators remains to be elucidated. In the current study, we demonstrate that histone methyl transferase activity of Ezh2 is required for mesenchymal to epithelial transition (MET) during human iPSC generation. We show that the H3K27me3 activity favors induction of pluripotency by transcriptionally targeting the TGF-β signaling pathway. We also demonstrate that the Ezh2 negatively regulates the expression of pro-EMT miRNA's such as miR-23a locus during MET. Unique association of Ezh2 with c-Myc was required to silence the aforementioned circuitry. Collectively, our findings provide a mechanistic understanding by which Ezh2 restricts the somatic programme during early phase of cellular reprogramming and establish the importance of Ezh2 dependent H3K27me3 activity in transcriptional and miRNA modulation during human iPSC generation.

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Enhanced expression of PRC2 components in human fibroblast reprogramming.(a) Bright field images of hFibs undergoing reprogramming upon OSKM transduction. Cells within the white dashed line at day 7 indicate the appearance of epithelial morphology in hFib cultures. These cells grew to make bigger colony structure by day 14, eventually generating the iPSC like colony at day 21. picking the colony allowed us to establish the iPSC line. (b) Expression profile of indicated mRNAs at various time courses of hFib reprogramming. (c) Expression profile of PRC2 components (Ezh2, Suz12 and Eed) in human fibroblasts, day 21 reprogramming cultures and various human pluripotent cells.
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f1: Enhanced expression of PRC2 components in human fibroblast reprogramming.(a) Bright field images of hFibs undergoing reprogramming upon OSKM transduction. Cells within the white dashed line at day 7 indicate the appearance of epithelial morphology in hFib cultures. These cells grew to make bigger colony structure by day 14, eventually generating the iPSC like colony at day 21. picking the colony allowed us to establish the iPSC line. (b) Expression profile of indicated mRNAs at various time courses of hFib reprogramming. (c) Expression profile of PRC2 components (Ezh2, Suz12 and Eed) in human fibroblasts, day 21 reprogramming cultures and various human pluripotent cells.

Mentions: We first decided to verify the kinetics of PRC2 expression during human iPSC (hiPS) generation. Towards this hFibs (human fibroblasts) were transduced with OSKM and cultures were monitored for phenotypic changes by phase contrast microscopy along with molecular analysis. Consistent with previous reports7, clusters of cells with epithelial morphology indicative of mesenchymal to epithelial transition (MET) appeared at day 7, which eventually matured to generate iPS colonies by three weeks (Fig. 1a). These colonies were picked to establish iPSC lines. In addition, we assayed for Nanog expression, a key pluripotency marker required for the acquisition of complete reprogramming as positive control. We did not observe detectable Nanog transcripts in hFibs at day 4, 7 or 14. Nanog was expressed by day 21 and in an established iPSC line (Fig. 1b) demonstrating that our reprogramming timelines were in agreement with that reported by other groups. iPSC colonies generated from hFibs exhibited typical ESC morphology and expressed the cell surface marker Tra 1-60 well as the pluripotency transcription factors Oct4 and Nanog (Supplementary Fig. 1a–c). H9-hESC obtained from WiCell Research Institute were used as a positive control for Tra 1-60, Oct4 and Nanog staining.


Ezh2 mediated H3K27me3 activity facilitates somatic transition during human pluripotent reprogramming.

Rao RA, Dhele N, Cheemadan S, Ketkar A, Jayandharan GR, Palakodeti D, Rampalli S - Sci Rep (2015)

Enhanced expression of PRC2 components in human fibroblast reprogramming.(a) Bright field images of hFibs undergoing reprogramming upon OSKM transduction. Cells within the white dashed line at day 7 indicate the appearance of epithelial morphology in hFib cultures. These cells grew to make bigger colony structure by day 14, eventually generating the iPSC like colony at day 21. picking the colony allowed us to establish the iPSC line. (b) Expression profile of indicated mRNAs at various time courses of hFib reprogramming. (c) Expression profile of PRC2 components (Ezh2, Suz12 and Eed) in human fibroblasts, day 21 reprogramming cultures and various human pluripotent cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4316165&req=5

f1: Enhanced expression of PRC2 components in human fibroblast reprogramming.(a) Bright field images of hFibs undergoing reprogramming upon OSKM transduction. Cells within the white dashed line at day 7 indicate the appearance of epithelial morphology in hFib cultures. These cells grew to make bigger colony structure by day 14, eventually generating the iPSC like colony at day 21. picking the colony allowed us to establish the iPSC line. (b) Expression profile of indicated mRNAs at various time courses of hFib reprogramming. (c) Expression profile of PRC2 components (Ezh2, Suz12 and Eed) in human fibroblasts, day 21 reprogramming cultures and various human pluripotent cells.
Mentions: We first decided to verify the kinetics of PRC2 expression during human iPSC (hiPS) generation. Towards this hFibs (human fibroblasts) were transduced with OSKM and cultures were monitored for phenotypic changes by phase contrast microscopy along with molecular analysis. Consistent with previous reports7, clusters of cells with epithelial morphology indicative of mesenchymal to epithelial transition (MET) appeared at day 7, which eventually matured to generate iPS colonies by three weeks (Fig. 1a). These colonies were picked to establish iPSC lines. In addition, we assayed for Nanog expression, a key pluripotency marker required for the acquisition of complete reprogramming as positive control. We did not observe detectable Nanog transcripts in hFibs at day 4, 7 or 14. Nanog was expressed by day 21 and in an established iPSC line (Fig. 1b) demonstrating that our reprogramming timelines were in agreement with that reported by other groups. iPSC colonies generated from hFibs exhibited typical ESC morphology and expressed the cell surface marker Tra 1-60 well as the pluripotency transcription factors Oct4 and Nanog (Supplementary Fig. 1a–c). H9-hESC obtained from WiCell Research Institute were used as a positive control for Tra 1-60, Oct4 and Nanog staining.

Bottom Line: At the epigenetic level, it results in drastic chromatin changes to erase the existing somatic "memory" and to establish the pluripotent state.We also demonstrate that the Ezh2 negatively regulates the expression of pro-EMT miRNA's such as miR-23a locus during MET.Collectively, our findings provide a mechanistic understanding by which Ezh2 restricts the somatic programme during early phase of cellular reprogramming and establish the importance of Ezh2 dependent H3K27me3 activity in transcriptional and miRNA modulation during human iPSC generation.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre For Inflammation and Tissue Homeostasis, Institute for Stem Cell Biology and Regenerative Medicine (inStem), National Centre for Biological Sciences, GKVK Campus, Bellary Road, Bangalore 560065, Karnataka, India [2] Sastra University, Tirumalaisamudram, Thanjavur - 613 401, TamilNadu, India.

ABSTRACT
Factor induced reprogramming of fibroblasts is an orchestrated but inefficient process. At the epigenetic level, it results in drastic chromatin changes to erase the existing somatic "memory" and to establish the pluripotent state. Accordingly, alterations of chromatin regulators including Ezh2 influence iPSC generation. While the role of individual transcription factors in resetting the chromatin landscape during iPSC generation is increasingly evident, their engagement with chromatin modulators remains to be elucidated. In the current study, we demonstrate that histone methyl transferase activity of Ezh2 is required for mesenchymal to epithelial transition (MET) during human iPSC generation. We show that the H3K27me3 activity favors induction of pluripotency by transcriptionally targeting the TGF-β signaling pathway. We also demonstrate that the Ezh2 negatively regulates the expression of pro-EMT miRNA's such as miR-23a locus during MET. Unique association of Ezh2 with c-Myc was required to silence the aforementioned circuitry. Collectively, our findings provide a mechanistic understanding by which Ezh2 restricts the somatic programme during early phase of cellular reprogramming and establish the importance of Ezh2 dependent H3K27me3 activity in transcriptional and miRNA modulation during human iPSC generation.

Show MeSH
Related in: MedlinePlus