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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 common in Y- and T-iPSCs. A. Hypomethylated CpGs in fibroblasts that undergo de novo methylation. B. Hypomethylated CpGs that fail to be methylated. C. Hypermethylated CpGs in fibroblasts that fail to be demethylated. D. Hypermethylated CpGs in fibroblasts that get aberrantly methylated. E. Summary of the classes of DNA methylation aberrations found in all the iPSCs. F. DNA methylation of CpGs at the transcription start site of 9 genes reported to be aberrantly methylated in all iPSCs [15].
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Fig2: DNA methylation aberrations that are common in Y- and T-iPSCs. A. Hypomethylated CpGs in fibroblasts that undergo de novo methylation. B. Hypomethylated CpGs that fail to be methylated. C. Hypermethylated CpGs in fibroblasts that fail to be demethylated. D. Hypermethylated CpGs in fibroblasts that get aberrantly methylated. E. Summary of the classes of DNA methylation aberrations found in all the iPSCs. F. DNA methylation of CpGs at the transcription start site of 9 genes reported to be aberrantly methylated in all iPSCs [15].

Mentions: In order to identify the CpGs that are differentially methylated between iPSCs and ESCs, we used a DNA methylation difference of more than 0.2 or less than −0.2 and a False Discovery Rate (FDR)-corrected p-value lower than 0.05 as a threshold (Additional file 1: Figure S3). DNA methylation aberrations shared by iPSCs were partitioned into 4 classes: Class I represented de novo methylation (defined as methylated regions in iPSCs which are unmethylated in ESCs and in parental cells), Class II represented failed methylation (defined as unmethylated regions in iPSCs which are unmethylated in parental cells but methylated in ESCs), Class III represented failed demethylation (defined as methylated regions in iPSCs which are methylated in parental cells but unmethylated in ESCs); and Class IV represented de novo demethylation (defined as unmethylated sites in iPSCs that are methylated in both parental cells and ESCs). Consistent with previous findings, all the iPSCs had DNA methylation aberrations (Figure 2A-E). About 62% of the aberrations were Class II (failed methylation) (Figure 2B) and about 32% of the aberrations were Class III (failed demethylation) (Figure 2D). These results suggest that inadequate change of parental DNA methylation state (also referred to as tissue of origin memory), rather than de novo changes in DNA methylation, is the type of aberration shared by all iPSCs regardless of whether the Yamanaka or the Thomson reprogramming factors were used.Figure 2


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 common in Y- and T-iPSCs. A. Hypomethylated CpGs in fibroblasts that undergo de novo methylation. B. Hypomethylated CpGs that fail to be methylated. C. Hypermethylated CpGs in fibroblasts that fail to be demethylated. D. Hypermethylated CpGs in fibroblasts that get aberrantly methylated. E. Summary of the classes of DNA methylation aberrations found in all the iPSCs. F. DNA methylation of CpGs at the transcription start site of 9 genes reported to be aberrantly methylated in all iPSCs [15].
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230737&req=5

Fig2: DNA methylation aberrations that are common in Y- and T-iPSCs. A. Hypomethylated CpGs in fibroblasts that undergo de novo methylation. B. Hypomethylated CpGs that fail to be methylated. C. Hypermethylated CpGs in fibroblasts that fail to be demethylated. D. Hypermethylated CpGs in fibroblasts that get aberrantly methylated. E. Summary of the classes of DNA methylation aberrations found in all the iPSCs. F. DNA methylation of CpGs at the transcription start site of 9 genes reported to be aberrantly methylated in all iPSCs [15].
Mentions: In order to identify the CpGs that are differentially methylated between iPSCs and ESCs, we used a DNA methylation difference of more than 0.2 or less than −0.2 and a False Discovery Rate (FDR)-corrected p-value lower than 0.05 as a threshold (Additional file 1: Figure S3). DNA methylation aberrations shared by iPSCs were partitioned into 4 classes: Class I represented de novo methylation (defined as methylated regions in iPSCs which are unmethylated in ESCs and in parental cells), Class II represented failed methylation (defined as unmethylated regions in iPSCs which are unmethylated in parental cells but methylated in ESCs), Class III represented failed demethylation (defined as methylated regions in iPSCs which are methylated in parental cells but unmethylated in ESCs); and Class IV represented de novo demethylation (defined as unmethylated sites in iPSCs that are methylated in both parental cells and ESCs). Consistent with previous findings, all the iPSCs had DNA methylation aberrations (Figure 2A-E). About 62% of the aberrations were Class II (failed methylation) (Figure 2B) and about 32% of the aberrations were Class III (failed demethylation) (Figure 2D). These results suggest that inadequate change of parental DNA methylation state (also referred to as tissue of origin memory), rather than de novo changes in DNA methylation, is the type of aberration shared by all iPSCs regardless of whether the Yamanaka or the Thomson reprogramming factors were used.Figure 2

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