Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal

Abstract

In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.

Figure 1.

Sox17 is expressed in the preimplantation embryo. (A) Microarray analysis of Sox17, Dab2, Laminin, and Sparc expression at defined stages of mouse preimplantation development (n = 3). (B–G) Optical sections of immunostained mouse preimplantation embryos. (B) Optical sections and reconstructed projections of Oct4 and Sox17 expression. (C) Schematic representation of the Sox17∷Tomato reporter gene. Sox17∷Tomato heterozygous mice were intercrossed, and the resulting embryos were immunostained with Tomato, Oct4, and Sox17 antibodies. Optical sections of mouse embryo immunofluorescently stained with Sox17 and Gata4 (D), Gata6 (E), Laminin (F), and Nanog (G) antibodies.

Figure 2.

Sox17 is required for XEN cell derivation and regulates ExEn gene expression. (A) Sox17, Gata4, Laminin, Dab2, Sparc, and Nanog immunostaining in XEN and ES cell cultures. (B) Forty-one embryos resulting from a Sox17^+/− heterozygous intercross were used to derive XEN cell lines. (C,D) Analysis of day 6 EBs generated from Sox17^+/+, Sox17^+/−, and Sox17^−/− ES cells. (C) Epifluorescence images of Tomato expression and localization in Sox17^+/−∷Tomato and Sox17^−/−∷Tomato EBs. (D) Quantitative RT–PCR analysis of Afp, Amn, Nr2a1, Sox7, Sparc, Pem, Foxa2, Nr2f1, and Sall4 transcript levels, with values adjusted to Gapdh and relative expression reflected as a percent of the expression observed in wild-type Sox17^+/+ EBs (n = 4).

Figure 3.

Cells expressing Sox17 are committed to differentiate. (A) Epifluorescent image of Sox17∷GFP expression under standard mouse ES cell culture conditions. (B) Epifluorescent image of Sox17∷GFP ES cell following lentiviral transduction with Ubiquitin∷Tomato. (C) Manually picked Sox17∷GFP-high or Sox17∷GFP-low cells expressing Ubiquitin∷Tomato prior to clonal culture or blastocyst injection. (D,E) Single Sox17∷GFP-low cells (D) and single Sox17∷GFP-high subclones (E) plated under standard mouse ES culture conditions. (F, G) One hour post-injection of a single Sox17∷GFP-low (F) or Sox17∷GFP-high (G) cell into wild-type blastocysts. (H, I) Reconstructed projections of E6–E6.5 representative embryos that had incorporated a single or multiple Sox17∷GFP-low (H) or Sox17∷GFP-high (I) cell(s) prior to the establishment of the definitive endoderm (Hoechst nuclear overlay). (H) Representative dissected embryo from the transfer of a blastocyst injected with eight to 10 Ubiquitin∷Tomato-positive/Sox17∷GFP-low cells. (I) Representative dissected embryo from the transfer of a blastocyst injected with a single Ubiquitin∷Tomato-positive/Sox17∷GFP-high cell.

Figure 4.

Sox17 binds to target genes required for extraembryonic differentiation. (A) Examples of DNA sequences bound by Sox17. The unprocessed ChIP enrichment ratio was plotted with the associated chromosome and gene location, exon (box), intron (horizontal line), transcription direction, and start site (arrow) for each Sox17-bound region. (B) ChIP followed by quantitative PCR using primers spanning the Sox17-bound regions. Fold enrichment was normalized to Ct value of Ppil4 and Rosa26 loci (n = 3). (C) The Sox17-binding consensus motif of ATTGT was identified using de novo motif analysis. The gray box indicates similarities in the genomic consensus motif at Sox17-enriched sites. (D) Sox17 occupies the promoter sequence of Nanog and Sox2 in Sox17-induced mouse ES cells (black and blue) but not in XEN cells (red).

Figure 5.

Sox17 expression is sufficient to activate ExEn target genes. (A) Schematic of the inducible Sox17 construct. (SA) Splice acceptor; (pA) polyadenylation signal; (TetOP) tetracycline/doxycycline-responsive element; (3XFLAG) Flag epitope. (B) Immunostaining of Sox17-inducible ES cells in the absence or presence of doxycycline. Quantitative RT–PCR of Sox17 (C); Col4a1, Col4a2, Lama1, Gata4, Gata6, and Sall4 (D); and Oct4, Nanog, and Sox2 (E) in Sox17-inducible ES cells without or with doxycycline treatment (48 h). The relative expression was reflected as a percent of Gapdh expression (n = 4).

Figure 6.

Epistatic relationship between Sox17 and other transcriptional regulators. (A) Wild-type, Sox17^+/−, Sox17^−/−, Nanog-overexpressing (OE), Gata4^−/−, Gata6^−/−, and Gata4^−/− and Gata6^−/− compound mutant day 6 EBs were examined by immunostaining for Oct4, Nanog, Sox2, Sox17, Laminin, Dab2, Gata4, and Gata6. Quantitative RT–PCR analysis of Nanog, Oct4, and Sox2 (B); Sox17, Gata6, and Gata4 (C); and Laminin and Dab2 (D), with relative expression reflected as a percent of Gapdh expression (n = 4).

Figure 7.

Sox17 inhibits ES cell self-renewal by displacing Nanog. (A) Control ES (KH2), Sox17-inducible, Sox17 mutant, and Nanog-overexpressing cells were plated at the same initial cell density in the presence or absence of doxycycline (n = 3). (*) P < 0.01; (+) P < 0.05. (B) ChIP followed by quantitative PCR analysis of Nanog enrichment at the Oct4, Sox2, Lama1, Ppm1b, Vitrin, and Col4a1 regulatory regions was compared in Sox17-uninduced and Sox17-induced cells following 48 h of doxycycline induction (n = 3). (*) P < 0.01. (C) Sox17 ChIP–chip targets were compared with the regulatory regions bound by Sox2, Nanog, and Oct4 (as published previously in Chen et al. 2008; Kim et al. 2008). Representative example of the target gene, Lama1. Unprocessed ChIP enrichment ratios were plotted for each probe, together with the associated chromosome and genomic location, exon (box), intron (horizontal line), transcription direction, and start site (arrow). (D) A model for Sox17 gene regulation in ExEn differentiation. The Oct4, Sox2, and Nanog transcription factor network is a feed-forward loop maintaining mouse ES cell pluripotency (Boyer et al. 2006), while inhibiting genes involved in differentiation. Gata6 lies upstream of Gata4 in the differentiation cascade (depicted as dashed lines, indicating no evidence to date that these interactions are direct) (Chazaud et al. 2006). Sox17 lies downstream from Gata6 and directly regulates the expression of Gata6 and Gata4 (solid lines). Sox17 binds directly to and activates the transcription of genes known to function in ExEn differentiation. (E) Nanog and Sox17 were bound reciprocally to shared Nanog/Sox17 genomic sites, represented here by the region upstream of the Lama1 start site (arrow). This suggests a mechanism for initiation of differentiation in which Sox17 displaces repressive Nanog complexes from shared binding sites and, in turn, activates gene expression.