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Tet-mediated imprinting erasure in H19 locus following reprogramming of spermatogonial stem cells to induced pluripotent stem cells.

Bermejo-Álvarez P, Ramos-Ibeas P, Park KE, Powell AP, Vansandt L, Derek B, Ramirez MA, Gutiérrez-Adán A, Telugu BP - Sci Rep (2015)

Bottom Line: RNA-seq revealed extensive reprogramming at the transcriptional level with 8124 genes differentially expressed between SSC and SSCiPSC and only 490 between SSCiPSC and fiPSC.A closer look at H19 ICR revealed complete erasure in SSCiPSC in contrast to fiPSC.These results suggest that imprinting erasure during reprogramming depends on the epigenetic landscape of the precursor cell and is mediated by TETs at the H19 locus.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal and Avian Sciences, University of Maryland, MD, USA.

ABSTRACT
Selective methylation of CpG islands at imprinting control regions (ICR) determines the monoparental expression of a subset of genes. Currently, it is unclear whether artificial reprogramming induced by the expression of Yamanaka factors disrupts these marks and whether cell type of origin affects the dynamics of reprogramming. In this study, spermatogonial stem cells (SSC) that harbor paternalized imprinting marks, and fibroblasts were reprogrammed to iPSC (SSCiPSC and fiPSC). The SSCiPSC were able to form teratomas and generated chimeras with a higher skin chimerism than those derived from fiPSC. RNA-seq revealed extensive reprogramming at the transcriptional level with 8124 genes differentially expressed between SSC and SSCiPSC and only 490 between SSCiPSC and fiPSC. Likewise, reprogramming of SSC affected 26 of 41 imprinting gene clusters known in the mouse genome. A closer look at H19 ICR revealed complete erasure in SSCiPSC in contrast to fiPSC. Imprinting erasure in SSCiPSC was maintained even after in vivo differentiation into teratomas. Reprogramming of SSC from Tet1 and Tet2 double knockout mice however lacked demethylation of H19 ICR. These results suggest that imprinting erasure during reprogramming depends on the epigenetic landscape of the precursor cell and is mediated by TETs at the H19 locus.

No MeSH data available.


Related in: MedlinePlus

CpG methylation of H19 Imprinted Controlled region following reprogramming and differentiation.Bisulfite sequencing of H19 ICR revealed that CpG methylation was almost completely erased following reprogramming of (A) SSCiPSC (68% in SSC vs 0–2% in SSCiPSC) in comparison to (B) fiPSC (48% in fibroblast vs 20–39.3% in fiPSC). (C) The loss of imprinting erasure in SSCiPSC is maintained even following differentiation.
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f3: CpG methylation of H19 Imprinted Controlled region following reprogramming and differentiation.Bisulfite sequencing of H19 ICR revealed that CpG methylation was almost completely erased following reprogramming of (A) SSCiPSC (68% in SSC vs 0–2% in SSCiPSC) in comparison to (B) fiPSC (48% in fibroblast vs 20–39.3% in fiPSC). (C) The loss of imprinting erasure in SSCiPSC is maintained even following differentiation.

Mentions: To investigate the extent and mechanism of imprinting erasure, the methylation profiles at H19 ICR in the original cells (Gfra1+ SSC, Gfra1-spermatogonia, and fetal fibroblast) and in 3 iPSC cell lines derived from each resultant cell type were analyzed (Fig. 3). As expected, H19 was highly, but not totally methylated in Gfra1+ cells, in agreement with previous findings in immature spermatogonia1021, whereas fibroblast showed the typical pattern of methylation with a mix of methylated paternal alleles and unmethylated maternal alleles. The methylation profile of the differentiated spermatogonia (Gfra1-) did not differ from that of the SSC (Gfra1+). Following reprogramming of SSC to SSC-iPSC, methylation at H19 ICR was erased almost completely in all three representative SSC-iPSC cell lines (Fig. 3A). In contrast, the reprogramming of fibroblast to the fiPSC did not significantly alter the methylation of H19 ICR based on ANOVA (p < 0.05) (Fig. 3B). Next, we questioned whether the imprinting marks could be restored in SSC-iPSC following in vivo differentiation, by analyzing the methylation status of H19 ICR in teratomas. In vivo differentiation of iPSC did not affect the methylation levels at H19 ICR, as teratomas produced either by intramuscular or subcutaneous injections revealed similar methylation patterns as the SSCiPSC line from which they were derived (Fig. 3C).


Tet-mediated imprinting erasure in H19 locus following reprogramming of spermatogonial stem cells to induced pluripotent stem cells.

Bermejo-Álvarez P, Ramos-Ibeas P, Park KE, Powell AP, Vansandt L, Derek B, Ramirez MA, Gutiérrez-Adán A, Telugu BP - Sci Rep (2015)

CpG methylation of H19 Imprinted Controlled region following reprogramming and differentiation.Bisulfite sequencing of H19 ICR revealed that CpG methylation was almost completely erased following reprogramming of (A) SSCiPSC (68% in SSC vs 0–2% in SSCiPSC) in comparison to (B) fiPSC (48% in fibroblast vs 20–39.3% in fiPSC). (C) The loss of imprinting erasure in SSCiPSC is maintained even following differentiation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556992&req=5

f3: CpG methylation of H19 Imprinted Controlled region following reprogramming and differentiation.Bisulfite sequencing of H19 ICR revealed that CpG methylation was almost completely erased following reprogramming of (A) SSCiPSC (68% in SSC vs 0–2% in SSCiPSC) in comparison to (B) fiPSC (48% in fibroblast vs 20–39.3% in fiPSC). (C) The loss of imprinting erasure in SSCiPSC is maintained even following differentiation.
Mentions: To investigate the extent and mechanism of imprinting erasure, the methylation profiles at H19 ICR in the original cells (Gfra1+ SSC, Gfra1-spermatogonia, and fetal fibroblast) and in 3 iPSC cell lines derived from each resultant cell type were analyzed (Fig. 3). As expected, H19 was highly, but not totally methylated in Gfra1+ cells, in agreement with previous findings in immature spermatogonia1021, whereas fibroblast showed the typical pattern of methylation with a mix of methylated paternal alleles and unmethylated maternal alleles. The methylation profile of the differentiated spermatogonia (Gfra1-) did not differ from that of the SSC (Gfra1+). Following reprogramming of SSC to SSC-iPSC, methylation at H19 ICR was erased almost completely in all three representative SSC-iPSC cell lines (Fig. 3A). In contrast, the reprogramming of fibroblast to the fiPSC did not significantly alter the methylation of H19 ICR based on ANOVA (p < 0.05) (Fig. 3B). Next, we questioned whether the imprinting marks could be restored in SSC-iPSC following in vivo differentiation, by analyzing the methylation status of H19 ICR in teratomas. In vivo differentiation of iPSC did not affect the methylation levels at H19 ICR, as teratomas produced either by intramuscular or subcutaneous injections revealed similar methylation patterns as the SSCiPSC line from which they were derived (Fig. 3C).

Bottom Line: RNA-seq revealed extensive reprogramming at the transcriptional level with 8124 genes differentially expressed between SSC and SSCiPSC and only 490 between SSCiPSC and fiPSC.A closer look at H19 ICR revealed complete erasure in SSCiPSC in contrast to fiPSC.These results suggest that imprinting erasure during reprogramming depends on the epigenetic landscape of the precursor cell and is mediated by TETs at the H19 locus.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal and Avian Sciences, University of Maryland, MD, USA.

ABSTRACT
Selective methylation of CpG islands at imprinting control regions (ICR) determines the monoparental expression of a subset of genes. Currently, it is unclear whether artificial reprogramming induced by the expression of Yamanaka factors disrupts these marks and whether cell type of origin affects the dynamics of reprogramming. In this study, spermatogonial stem cells (SSC) that harbor paternalized imprinting marks, and fibroblasts were reprogrammed to iPSC (SSCiPSC and fiPSC). The SSCiPSC were able to form teratomas and generated chimeras with a higher skin chimerism than those derived from fiPSC. RNA-seq revealed extensive reprogramming at the transcriptional level with 8124 genes differentially expressed between SSC and SSCiPSC and only 490 between SSCiPSC and fiPSC. Likewise, reprogramming of SSC affected 26 of 41 imprinting gene clusters known in the mouse genome. A closer look at H19 ICR revealed complete erasure in SSCiPSC in contrast to fiPSC. Imprinting erasure in SSCiPSC was maintained even after in vivo differentiation into teratomas. Reprogramming of SSC from Tet1 and Tet2 double knockout mice however lacked demethylation of H19 ICR. These results suggest that imprinting erasure during reprogramming depends on the epigenetic landscape of the precursor cell and is mediated by TETs at the H19 locus.

No MeSH data available.


Related in: MedlinePlus