Limits...
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

Levels of DNA methylation and demethylation enzymes and overlap of aberrantly methylated regions with genomic binding sites of reprogramming factors. A. Expression levels of enzymes involved in methylation and demethylation in pluripotent cells measured using qPCR. Data is log2 transformed and normalized to levels of these enzymes in human fibroblasts. Values shown are the mean of the following number of samples: n = 2 (ESCs), n = 3 (Y-iPSCs) and n = 3 (T-iPSCs). B and C. Random sampling simulation to determine the distribution of the expected number of genes that are targets of the indicated reprogramming factor and have aberrant DNA methylation CpGs overlapping the binding site. The histograms and box plots represent the  distribution for the overlap between random sampling DNA methylation aberrations and each transcription factor binding sites. The diamonds represent the experimentally observed number of overlap between Y-iPSCs (B) or T-iPSCs (C) DNA methylation aberrations and each transcription factor binding sites. Red diamonds highlight a number of overlaps that fall completely outside of the  distribution.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Levels of DNA methylation and demethylation enzymes and overlap of aberrantly methylated regions with genomic binding sites of reprogramming factors. A. Expression levels of enzymes involved in methylation and demethylation in pluripotent cells measured using qPCR. Data is log2 transformed and normalized to levels of these enzymes in human fibroblasts. Values shown are the mean of the following number of samples: n = 2 (ESCs), n = 3 (Y-iPSCs) and n = 3 (T-iPSCs). B and C. Random sampling simulation to determine the distribution of the expected number of genes that are targets of the indicated reprogramming factor and have aberrant DNA methylation CpGs overlapping the binding site. The histograms and box plots represent the distribution for the overlap between random sampling DNA methylation aberrations and each transcription factor binding sites. The diamonds represent the experimentally observed number of overlap between Y-iPSCs (B) or T-iPSCs (C) DNA methylation aberrations and each transcription factor binding sites. Red diamonds highlight a number of overlaps that fall completely outside of the distribution.

Mentions: Among other possibilities, differences in the classes of aberrations in Y-iPSCs and T-iPSCs might be due to different levels of DNA methylation and demethylation enzymes during reprogramming or it might be due to different targeting of these enzymes to the genomic regions containing the aberrations. To determine whether the levels and the targeting of DNA methylation machinery may contribute to the observed aberrations, we first tested whether the levels of DNA methylation and demethylation enzymes are aberrant. We found that the level of DNMT3b was lower in T-iPSCs than in ESCs or Y-iPSCs, which may potentially contribute to the de novo methylation defects in Y-iPSCs (Figure 4A). Moreover, TET3 was higher in T-iPSCs than ESCs or Y-iPSCs and may potentially contribute to excessive demethylation in Y-iPSCs (Figure 4A).Figure 4


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)

Levels of DNA methylation and demethylation enzymes and overlap of aberrantly methylated regions with genomic binding sites of reprogramming factors. A. Expression levels of enzymes involved in methylation and demethylation in pluripotent cells measured using qPCR. Data is log2 transformed and normalized to levels of these enzymes in human fibroblasts. Values shown are the mean of the following number of samples: n = 2 (ESCs), n = 3 (Y-iPSCs) and n = 3 (T-iPSCs). B and C. Random sampling simulation to determine the distribution of the expected number of genes that are targets of the indicated reprogramming factor and have aberrant DNA methylation CpGs overlapping the binding site. The histograms and box plots represent the  distribution for the overlap between random sampling DNA methylation aberrations and each transcription factor binding sites. The diamonds represent the experimentally observed number of overlap between Y-iPSCs (B) or T-iPSCs (C) DNA methylation aberrations and each transcription factor binding sites. Red diamonds highlight a number of overlaps that fall completely outside of the  distribution.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Levels of DNA methylation and demethylation enzymes and overlap of aberrantly methylated regions with genomic binding sites of reprogramming factors. A. Expression levels of enzymes involved in methylation and demethylation in pluripotent cells measured using qPCR. Data is log2 transformed and normalized to levels of these enzymes in human fibroblasts. Values shown are the mean of the following number of samples: n = 2 (ESCs), n = 3 (Y-iPSCs) and n = 3 (T-iPSCs). B and C. Random sampling simulation to determine the distribution of the expected number of genes that are targets of the indicated reprogramming factor and have aberrant DNA methylation CpGs overlapping the binding site. The histograms and box plots represent the distribution for the overlap between random sampling DNA methylation aberrations and each transcription factor binding sites. The diamonds represent the experimentally observed number of overlap between Y-iPSCs (B) or T-iPSCs (C) DNA methylation aberrations and each transcription factor binding sites. Red diamonds highlight a number of overlaps that fall completely outside of the distribution.
Mentions: Among other possibilities, differences in the classes of aberrations in Y-iPSCs and T-iPSCs might be due to different levels of DNA methylation and demethylation enzymes during reprogramming or it might be due to different targeting of these enzymes to the genomic regions containing the aberrations. To determine whether the levels and the targeting of DNA methylation machinery may contribute to the observed aberrations, we first tested whether the levels of DNA methylation and demethylation enzymes are aberrant. We found that the level of DNMT3b was lower in T-iPSCs than in ESCs or Y-iPSCs, which may potentially contribute to the de novo methylation defects in Y-iPSCs (Figure 4A). Moreover, TET3 was higher in T-iPSCs than ESCs or Y-iPSCs and may potentially contribute to excessive demethylation in Y-iPSCs (Figure 4A).Figure 4

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