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SOX17 is a critical specifier of human primordial germ cell fate.

Irie N, Weinberger L, Tang WW, Kobayashi T, Viukov S, Manor YS, Dietmann S, Hanna JH, Surani MA - Cell (2014)

Bottom Line: Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells.Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs.We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge CB2 3EG, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 3EG, UK.

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Immunofluorescence for Epigenetic Modifications and Modifiers in Embryoids, Related to Figure 3(A–C) Immunofluorescence analysis was carried out for (A) 5-methylcytosine (5mC); (B) DNMT3A; (C) UHRF1 on day 4 embryoids. TFAP2C, OCT4 or BLIMP1 were used to counterstain for hPGCLCs. hPGCLCs were highlighted by white dashed lines. White arrowheads showed examples of KI-67-positive PGCLCs while yellow arrows showed the KI-67-negative PGCLCs (C). Scale bars = 50 μm.(D) Expression level of methylation related epigenetic modifiers from RNA-Seq dataset. Mean normalized read counts from two biological replicates were shown.(E) Immunofluoresence of PRMT5 on cryosections of embryoids collected at day 1, 2, 4 and 8 post-hPGCLC induction. hPGCLCs were counterstained with OCT4 as highlighted. Scale bar = 70 μm.
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figs3: Immunofluorescence for Epigenetic Modifications and Modifiers in Embryoids, Related to Figure 3(A–C) Immunofluorescence analysis was carried out for (A) 5-methylcytosine (5mC); (B) DNMT3A; (C) UHRF1 on day 4 embryoids. TFAP2C, OCT4 or BLIMP1 were used to counterstain for hPGCLCs. hPGCLCs were highlighted by white dashed lines. White arrowheads showed examples of KI-67-positive PGCLCs while yellow arrows showed the KI-67-negative PGCLCs (C). Scale bars = 50 μm.(D) Expression level of methylation related epigenetic modifiers from RNA-Seq dataset. Mean normalized read counts from two biological replicates were shown.(E) Immunofluoresence of PRMT5 on cryosections of embryoids collected at day 1, 2, 4 and 8 post-hPGCLC induction. hPGCLCs were counterstained with OCT4 as highlighted. Scale bar = 70 μm.

Mentions: The RNA-seq of hPGCLC also revealed gene expression changes that indicate initiation of the epigenetic program with downregulation of UHRF1, DNMT3A, and DNMT3B and upregulation of TET1 and TET2 (Figure S3D). Notably, we found a significant increase in 5-hydroxymethylacytosine (5hmC) in hPGCLCs, which is consistent with an increase in the expression of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5hmC (Figures 3E–3G), together with a small but significant decline in 5mC (Figures 3G and S3A). This indicates that, as in the mouse PGCs, loss of 5mC might be coupled with the conversion of 5mC to 5hmC (Hackett et al., 2013). At the same time, we detected a decline in the expression of de novo DNA methyltransferase 3A (DNMT3A) and UHRF1 in hPGCLCs compared to the neighboring somatic cells in the embryoids (Figures 3G, S3B, and S3C). UHRF1 targets DNMT1 to replication foci to confer maintenance of DNA methylation (Liu et al., 2013). The repression of UHRF1 in proliferating (KI-67-positive) hPGCLCs would allow DNA-replication-coupled loss of 5mC, which is analogous to the observations on the early mouse germline.


SOX17 is a critical specifier of human primordial germ cell fate.

Irie N, Weinberger L, Tang WW, Kobayashi T, Viukov S, Manor YS, Dietmann S, Hanna JH, Surani MA - Cell (2014)

Immunofluorescence for Epigenetic Modifications and Modifiers in Embryoids, Related to Figure 3(A–C) Immunofluorescence analysis was carried out for (A) 5-methylcytosine (5mC); (B) DNMT3A; (C) UHRF1 on day 4 embryoids. TFAP2C, OCT4 or BLIMP1 were used to counterstain for hPGCLCs. hPGCLCs were highlighted by white dashed lines. White arrowheads showed examples of KI-67-positive PGCLCs while yellow arrows showed the KI-67-negative PGCLCs (C). Scale bars = 50 μm.(D) Expression level of methylation related epigenetic modifiers from RNA-Seq dataset. Mean normalized read counts from two biological replicates were shown.(E) Immunofluoresence of PRMT5 on cryosections of embryoids collected at day 1, 2, 4 and 8 post-hPGCLC induction. hPGCLCs were counterstained with OCT4 as highlighted. Scale bar = 70 μm.
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figs3: Immunofluorescence for Epigenetic Modifications and Modifiers in Embryoids, Related to Figure 3(A–C) Immunofluorescence analysis was carried out for (A) 5-methylcytosine (5mC); (B) DNMT3A; (C) UHRF1 on day 4 embryoids. TFAP2C, OCT4 or BLIMP1 were used to counterstain for hPGCLCs. hPGCLCs were highlighted by white dashed lines. White arrowheads showed examples of KI-67-positive PGCLCs while yellow arrows showed the KI-67-negative PGCLCs (C). Scale bars = 50 μm.(D) Expression level of methylation related epigenetic modifiers from RNA-Seq dataset. Mean normalized read counts from two biological replicates were shown.(E) Immunofluoresence of PRMT5 on cryosections of embryoids collected at day 1, 2, 4 and 8 post-hPGCLC induction. hPGCLCs were counterstained with OCT4 as highlighted. Scale bar = 70 μm.
Mentions: The RNA-seq of hPGCLC also revealed gene expression changes that indicate initiation of the epigenetic program with downregulation of UHRF1, DNMT3A, and DNMT3B and upregulation of TET1 and TET2 (Figure S3D). Notably, we found a significant increase in 5-hydroxymethylacytosine (5hmC) in hPGCLCs, which is consistent with an increase in the expression of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5hmC (Figures 3E–3G), together with a small but significant decline in 5mC (Figures 3G and S3A). This indicates that, as in the mouse PGCs, loss of 5mC might be coupled with the conversion of 5mC to 5hmC (Hackett et al., 2013). At the same time, we detected a decline in the expression of de novo DNA methyltransferase 3A (DNMT3A) and UHRF1 in hPGCLCs compared to the neighboring somatic cells in the embryoids (Figures 3G, S3B, and S3C). UHRF1 targets DNMT1 to replication foci to confer maintenance of DNA methylation (Liu et al., 2013). The repression of UHRF1 in proliferating (KI-67-positive) hPGCLCs would allow DNA-replication-coupled loss of 5mC, which is analogous to the observations on the early mouse germline.

Bottom Line: Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells.Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs.We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge CB2 3EG, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 3EG, UK.

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Related in: MedlinePlus