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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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Inhibition of H3K27me3 activity impairs mesenchymal to epithelial transition during iPSC generation.(a) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in hFibs treated with H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 a-c. (b) Relative expression of p53 and p21 mRNA in DMSO control and GSK treated hFibs. (c) Representative bright field and β- gal staining images upon treatment of hFibs with DMSO and GSK-126. (d) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in H9 hESC treated with DMSO or H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 d–f. (e) Tra 1-60 DAB staining in IMR-90 iPSC, H9 hESC cultures treated with DMSO and GSK-126. (f) Schematic representation of reprogramming protocol and time points of addition of GSK to the reprogramming cultures. (g) Total number of reprogrammed colonies generated three weeks post OSKM transduced hFibs and upon GSK treatment at indicated time points in schema (h) Tra 1-60 DAB staining on day 21 post addition of reprogramming factors and GSK treatment (i) E-cad DAB staining on day 10 post addition of reprogramming factors and GSK treatment as indicated in schema.
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f2: Inhibition of H3K27me3 activity impairs mesenchymal to epithelial transition during iPSC generation.(a) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in hFibs treated with H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 a-c. (b) Relative expression of p53 and p21 mRNA in DMSO control and GSK treated hFibs. (c) Representative bright field and β- gal staining images upon treatment of hFibs with DMSO and GSK-126. (d) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in H9 hESC treated with DMSO or H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 d–f. (e) Tra 1-60 DAB staining in IMR-90 iPSC, H9 hESC cultures treated with DMSO and GSK-126. (f) Schematic representation of reprogramming protocol and time points of addition of GSK to the reprogramming cultures. (g) Total number of reprogrammed colonies generated three weeks post OSKM transduced hFibs and upon GSK treatment at indicated time points in schema (h) Tra 1-60 DAB staining on day 21 post addition of reprogramming factors and GSK treatment (i) E-cad DAB staining on day 10 post addition of reprogramming factors and GSK treatment as indicated in schema.

Mentions: While there are several reports indicating removal of Ezh2 from somatic cells leads to senescence by induction of p53, p21 and ARF proteins2627, it is not clear if inhibition of its H3K27me3 activity would have the same senescence-inducing effect on hFibs. Thus we studied the effect of GSK-126 a small molecule inhibitor of Ezh2 on hFib cultures. Treatment of GSK126 inhibited H3K27me3 activity without altering Ezh2 protein levels in human fibroblasts (Fig. 2a). Similar to the effect of shEzh2, GSK increased the expression of p53 and p21, albeit at lower levels without affecting the cell shape or proliferation (Fig. 2b,c). Furthermore, GSK treated cells did not undergo cell senescence, which was detected by β-galactosidase staining (Fig. 2c). Collectively, these results suggest that the loss of Ezh2 versus abrogation of methyltransferase activity alone seems to have a differential effect on cell proliferation in somatic fibroblast cells. This was particularly surprising since transduction of shRNA or treatment with inhibitors ultimately reduced overall H3K27me3 levels.


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)

Inhibition of H3K27me3 activity impairs mesenchymal to epithelial transition during iPSC generation.(a) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in hFibs treated with H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 a-c. (b) Relative expression of p53 and p21 mRNA in DMSO control and GSK treated hFibs. (c) Representative bright field and β- gal staining images upon treatment of hFibs with DMSO and GSK-126. (d) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in H9 hESC treated with DMSO or H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 d–f. (e) Tra 1-60 DAB staining in IMR-90 iPSC, H9 hESC cultures treated with DMSO and GSK-126. (f) Schematic representation of reprogramming protocol and time points of addition of GSK to the reprogramming cultures. (g) Total number of reprogrammed colonies generated three weeks post OSKM transduced hFibs and upon GSK treatment at indicated time points in schema (h) Tra 1-60 DAB staining on day 21 post addition of reprogramming factors and GSK treatment (i) E-cad DAB staining on day 10 post addition of reprogramming factors and GSK treatment as indicated in schema.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Inhibition of H3K27me3 activity impairs mesenchymal to epithelial transition during iPSC generation.(a) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in hFibs treated with H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 a-c. (b) Relative expression of p53 and p21 mRNA in DMSO control and GSK treated hFibs. (c) Representative bright field and β- gal staining images upon treatment of hFibs with DMSO and GSK-126. (d) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in H9 hESC treated with DMSO or H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 d–f. (e) Tra 1-60 DAB staining in IMR-90 iPSC, H9 hESC cultures treated with DMSO and GSK-126. (f) Schematic representation of reprogramming protocol and time points of addition of GSK to the reprogramming cultures. (g) Total number of reprogrammed colonies generated three weeks post OSKM transduced hFibs and upon GSK treatment at indicated time points in schema (h) Tra 1-60 DAB staining on day 21 post addition of reprogramming factors and GSK treatment (i) E-cad DAB staining on day 10 post addition of reprogramming factors and GSK treatment as indicated in schema.
Mentions: While there are several reports indicating removal of Ezh2 from somatic cells leads to senescence by induction of p53, p21 and ARF proteins2627, it is not clear if inhibition of its H3K27me3 activity would have the same senescence-inducing effect on hFibs. Thus we studied the effect of GSK-126 a small molecule inhibitor of Ezh2 on hFib cultures. Treatment of GSK126 inhibited H3K27me3 activity without altering Ezh2 protein levels in human fibroblasts (Fig. 2a). Similar to the effect of shEzh2, GSK increased the expression of p53 and p21, albeit at lower levels without affecting the cell shape or proliferation (Fig. 2b,c). Furthermore, GSK treated cells did not undergo cell senescence, which was detected by β-galactosidase staining (Fig. 2c). Collectively, these results suggest that the loss of Ezh2 versus abrogation of methyltransferase activity alone seems to have a differential effect on cell proliferation in somatic fibroblast cells. This was particularly surprising since transduction of shRNA or treatment with inhibitors ultimately reduced overall H3K27me3 levels.

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