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Aberrant DNA methylation reprogramming during induced pluripotent stem cell generation is dependent on the choice of reprogramming factors.

Planello AC, Ji J, Sharma V, Singhania R, Mbabaali F, Müller F, Alfaro JA, Bock C, De Carvalho DD, Batada NN - Cell Regen (Lond) (2014)

Bottom Line: Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs.Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs.These findings may provide clues into how to produce human iPSCs with fewer DNA methylation abnormalities.

View Article: PubMed Central - PubMed

Affiliation: Campbell Family Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2 M9 Canada ; Department of Morphology, Piracicaba Dental School, University of Campinas, Piracicaba, SP Brazil.

ABSTRACT
The conversion of somatic cells into pluripotent stem cells via overexpression of reprogramming factors involves epigenetic remodeling. DNA methylation at a significant proportion of CpG sites in induced pluripotent stem cells (iPSCs) differs from that of embryonic stem cells (ESCs). Whether different sets of reprogramming factors influence the type and extent of aberrant DNA methylation in iPSCs differently remains unknown. In order to help resolve this critical question, we generated human iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and cMYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28), and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identified Yamanaka-iPSC (Y-iPSC)-specific and Thomson-iPSC (T-iPSC)-specific recurrent aberrations. Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs. Differences in the level of transcripts encoding DNMT3b and TET3 between Y-iPSCs and T-iPSCs may contribute partially to the distinct types of aberrations. Finally, de novo aberrantly methylated genes in Y-iPSCs were enriched for NANOG targets that are also aberrantly methylated in some cancers. Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs. These findings may provide clues into how to produce human iPSCs with fewer DNA methylation abnormalities.

No MeSH data available.


Related in: MedlinePlus

DNA methylation aberrations that are found only in Y- or T-iPSCs. A. Principal Component Analysis showing that methylomes of Y-iPSCs, T-iPSCs and ESCs segregate into separate groups. B. Volcano plots of all CpG sites analyzed. The beta value difference in DNA methylation between Y-iPSCs and T-iPSCs is plotted on the x-axis, and the p-value for a FDR-corrected Wilcoxon signed-rank test of differences between Y-iPSCs and T-iPSCs (shown on − log10 scale) is plotted on the y-axis. CpGs that are significantly different between the 2 subtypes are shown on the upper left corner (significantly hypermethylated in T-iPSCs) and upper right corner (significantly hypermethylated in Y-iPSCs). C. CpGs that are hypomethylated in fibroblasts but are aberrantly methylated in iPSCs. D. CpGs that are hypomethylated in fibroblasts but fail to acquire methylation in iPSCs. E. CpGs that are hypermethylated in fibroblasts but aberrantly demethylated. F. CpGs that hypermethylated in fibroblasts but aberrantly gets demethylated in iPSCs. G. Summary of the classes of DNA methylation aberrations found only in Y-iPSCs (left) or T-iPSCs (right) but not both.
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Fig3: DNA methylation aberrations that are found only in Y- or T-iPSCs. A. Principal Component Analysis showing that methylomes of Y-iPSCs, T-iPSCs and ESCs segregate into separate groups. B. Volcano plots of all CpG sites analyzed. The beta value difference in DNA methylation between Y-iPSCs and T-iPSCs is plotted on the x-axis, and the p-value for a FDR-corrected Wilcoxon signed-rank test of differences between Y-iPSCs and T-iPSCs (shown on − log10 scale) is plotted on the y-axis. CpGs that are significantly different between the 2 subtypes are shown on the upper left corner (significantly hypermethylated in T-iPSCs) and upper right corner (significantly hypermethylated in Y-iPSCs). C. CpGs that are hypomethylated in fibroblasts but are aberrantly methylated in iPSCs. D. CpGs that are hypomethylated in fibroblasts but fail to acquire methylation in iPSCs. E. CpGs that are hypermethylated in fibroblasts but aberrantly demethylated. F. CpGs that hypermethylated in fibroblasts but aberrantly gets demethylated in iPSCs. G. Summary of the classes of DNA methylation aberrations found only in Y-iPSCs (left) or T-iPSCs (right) but not both.

Mentions: Though previous studies have identified recurrent DNA methylation alterations in iPSCs when compared to ESCs, no systematic study has been done to determine how the extent and type of DNA methylation aberrations depend on the factors used for reprogramming. Our study was specifically designed to identify these differences. We performed a Principal Component Analysis of global DNA methylation values, and observed that though all the iPSCs are closer to ESCs than fibroblasts, they separated in two distinct groups according to the reprogramming factors used to generate them (Figure 3A). We identified 6,011 CpGs that were differentially methylated between T-iPSCs and Y-iPSCs (1,722 CpGs were hypermethylated in T-iPSCs and 4,289 CpGs were hypermethylated in Y-iPSCs) (Figure 3B; Additional file 2: Table S1). Note that these are not a subset of aberrantly methylated CpGs found in all iPSCs (Figure 2). Among these 6,011 CpGs we observed that in both T-iPSCs and Y-iPSCs they corresponded to all 4 classes of DNA methylation aberrations described above (Figure 3C to G). Interestingly, both T-iPSCs and Y-iPSCs had fewer de novo aberrations (Class I and IV) than expected by chance (p < 0.001) and more failure aberrations (Class II and III) than expected by chance (p < 0.001) (Additional file 1: Figure S4). Moreover, T-iPSCs had substantially more Class II and fewer Class III aberrations than Y-iPSCs (Figure 3G and Additional file 1: Figure S4). Thus, Y-iPSCs mainly suffer from demethylation failure while T-iPSCs mainly suffer from methylation failure. We also observed that de novo aberrations (both methylation and demethylation) preferentially present at non-CpG island regions in both Y-iPSCs and T-iPSCs, while failure aberrations (Class II and Class III) are enriched at CpG islands or shores (Additional file 1: Figure S5). Note that approximately 57% (3446 out of 6011 CpGs identified in Figure 3B) of the CpGs that showed recurrent differences between Y-iPSCs and T-iPSCs did not fall into Class I or IV. Interestingly, aberrantly methylated CpGs (Class I) in Y-iPSCs and T-iPSCs were enriched at transcription start sites (TSS) (Additional file 1: Figure S6).Figure 3


Aberrant DNA methylation reprogramming during induced pluripotent stem cell generation is dependent on the choice of reprogramming factors.

Planello AC, Ji J, Sharma V, Singhania R, Mbabaali F, Müller F, Alfaro JA, Bock C, De Carvalho DD, Batada NN - Cell Regen (Lond) (2014)

DNA methylation aberrations that are found only in Y- or T-iPSCs. A. Principal Component Analysis showing that methylomes of Y-iPSCs, T-iPSCs and ESCs segregate into separate groups. B. Volcano plots of all CpG sites analyzed. The beta value difference in DNA methylation between Y-iPSCs and T-iPSCs is plotted on the x-axis, and the p-value for a FDR-corrected Wilcoxon signed-rank test of differences between Y-iPSCs and T-iPSCs (shown on − log10 scale) is plotted on the y-axis. CpGs that are significantly different between the 2 subtypes are shown on the upper left corner (significantly hypermethylated in T-iPSCs) and upper right corner (significantly hypermethylated in Y-iPSCs). C. CpGs that are hypomethylated in fibroblasts but are aberrantly methylated in iPSCs. D. CpGs that are hypomethylated in fibroblasts but fail to acquire methylation in iPSCs. E. CpGs that are hypermethylated in fibroblasts but aberrantly demethylated. F. CpGs that hypermethylated in fibroblasts but aberrantly gets demethylated in iPSCs. G. Summary of the classes of DNA methylation aberrations found only in Y-iPSCs (left) or T-iPSCs (right) but not both.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: DNA methylation aberrations that are found only in Y- or T-iPSCs. A. Principal Component Analysis showing that methylomes of Y-iPSCs, T-iPSCs and ESCs segregate into separate groups. B. Volcano plots of all CpG sites analyzed. The beta value difference in DNA methylation between Y-iPSCs and T-iPSCs is plotted on the x-axis, and the p-value for a FDR-corrected Wilcoxon signed-rank test of differences between Y-iPSCs and T-iPSCs (shown on − log10 scale) is plotted on the y-axis. CpGs that are significantly different between the 2 subtypes are shown on the upper left corner (significantly hypermethylated in T-iPSCs) and upper right corner (significantly hypermethylated in Y-iPSCs). C. CpGs that are hypomethylated in fibroblasts but are aberrantly methylated in iPSCs. D. CpGs that are hypomethylated in fibroblasts but fail to acquire methylation in iPSCs. E. CpGs that are hypermethylated in fibroblasts but aberrantly demethylated. F. CpGs that hypermethylated in fibroblasts but aberrantly gets demethylated in iPSCs. G. Summary of the classes of DNA methylation aberrations found only in Y-iPSCs (left) or T-iPSCs (right) but not both.
Mentions: Though previous studies have identified recurrent DNA methylation alterations in iPSCs when compared to ESCs, no systematic study has been done to determine how the extent and type of DNA methylation aberrations depend on the factors used for reprogramming. Our study was specifically designed to identify these differences. We performed a Principal Component Analysis of global DNA methylation values, and observed that though all the iPSCs are closer to ESCs than fibroblasts, they separated in two distinct groups according to the reprogramming factors used to generate them (Figure 3A). We identified 6,011 CpGs that were differentially methylated between T-iPSCs and Y-iPSCs (1,722 CpGs were hypermethylated in T-iPSCs and 4,289 CpGs were hypermethylated in Y-iPSCs) (Figure 3B; Additional file 2: Table S1). Note that these are not a subset of aberrantly methylated CpGs found in all iPSCs (Figure 2). Among these 6,011 CpGs we observed that in both T-iPSCs and Y-iPSCs they corresponded to all 4 classes of DNA methylation aberrations described above (Figure 3C to G). Interestingly, both T-iPSCs and Y-iPSCs had fewer de novo aberrations (Class I and IV) than expected by chance (p < 0.001) and more failure aberrations (Class II and III) than expected by chance (p < 0.001) (Additional file 1: Figure S4). Moreover, T-iPSCs had substantially more Class II and fewer Class III aberrations than Y-iPSCs (Figure 3G and Additional file 1: Figure S4). Thus, Y-iPSCs mainly suffer from demethylation failure while T-iPSCs mainly suffer from methylation failure. We also observed that de novo aberrations (both methylation and demethylation) preferentially present at non-CpG island regions in both Y-iPSCs and T-iPSCs, while failure aberrations (Class II and Class III) are enriched at CpG islands or shores (Additional file 1: Figure S5). Note that approximately 57% (3446 out of 6011 CpGs identified in Figure 3B) of the CpGs that showed recurrent differences between Y-iPSCs and T-iPSCs did not fall into Class I or IV. Interestingly, aberrantly methylated CpGs (Class I) in Y-iPSCs and T-iPSCs were enriched at transcription start sites (TSS) (Additional file 1: Figure S6).Figure 3

Bottom Line: Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs.Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs.These findings may provide clues into how to produce human iPSCs with fewer DNA methylation abnormalities.

View Article: PubMed Central - PubMed

Affiliation: Campbell Family Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2 M9 Canada ; Department of Morphology, Piracicaba Dental School, University of Campinas, Piracicaba, SP Brazil.

ABSTRACT
The conversion of somatic cells into pluripotent stem cells via overexpression of reprogramming factors involves epigenetic remodeling. DNA methylation at a significant proportion of CpG sites in induced pluripotent stem cells (iPSCs) differs from that of embryonic stem cells (ESCs). Whether different sets of reprogramming factors influence the type and extent of aberrant DNA methylation in iPSCs differently remains unknown. In order to help resolve this critical question, we generated human iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and cMYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28), and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identified Yamanaka-iPSC (Y-iPSC)-specific and Thomson-iPSC (T-iPSC)-specific recurrent aberrations. Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs. Differences in the level of transcripts encoding DNMT3b and TET3 between Y-iPSCs and T-iPSCs may contribute partially to the distinct types of aberrations. Finally, de novo aberrantly methylated genes in Y-iPSCs were enriched for NANOG targets that are also aberrantly methylated in some cancers. Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs. These findings may provide clues into how to produce human iPSCs with fewer DNA methylation abnormalities.

No MeSH data available.


Related in: MedlinePlus