SOX17 maintains latent pluripotency of seminoma cells

Since seminomas always show high expression of both SOX17 and OCT4, an essential role of these two transcription factors for maintaining seminoma cell fate was suggested. So far, I could demonstrate that SOX17 binds to somatic (i.e.

neuroectodermal) genes, but also to pluripotency genes in seminoma cells via the compressed (SOX17/OCT4) and the canonical (SOX2/OCT4) binding sites. I have hypothesized that the transactivation of PRDM1 and TFAP2C expression by SOX17 may be essential to suppress the somatic differentiation program in seminoma cells otherwise activated by SOX17. Now, functional analysis needs to demonstrate whether a loss of SOX17 results in suppression of TFAP2C and PRDM1 expression, as well as an overall loss of pluripotency and induction of differentiation.

Therefore, I continued by analysing the effects of SOX17 depletion in seminoma cells.

For this, TCam-2 cells were transfected with two different single guide RNAs (gRNAs) homologous to the second exon of the SOX17 gene locus. CRISPR/Cas9-mediated gene editing using both gRNAs should result in a deletion of approximately 130 bp (Fig. 31). TCam-2 cells contain six copies of chromosome 8 [59], where SOX17 is encoded. Therefore CRISPR/Cas9-mediated gene editing results in a mixture of cells displaying deletions in 0-6 alleles of SOX17.

Figure 31: CRISPR/Cas9 mediated gene editing of SOX17 gene locus

Two gRNAs were designed, directed against the second exon of the human SOX17 gene (yellow arrows). CRISPR/Cas9-mediated gene editing using both gRNAs should result in a final deletion of approximately 130 bp.

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Within 72 hours following transfection of TCam-2 cells with the CRISPR/Cas9 constructs qRT-PCR demonstrated significant reduction of SOX17 expression, suggesting successful gene editing at least in some cells (Fig. 32). Additionally, I was able to demonstrate significant downregulation of the pluripotency markers NANOG, TFAP2C, POU5F1, PRDM14, ALPL and PRDM1 (Fig. 32). All of these genes were shown to be bound by SOX17 according to our ChIP analysis. The fact that downregulation of SOX17 results in downregulation of these genes shows that SOX17 transactivates these genes. Furthermore, these analyses show that downregulation or loss of SOX17 ultimately results in a loss of the latent pluripotent state in seminoma cells, possibly allowing for cellular differentiation. Notably, since differentiated cells lose their capacity to self-renew and divide the derivation of single cell clones was prohibited and the following analyses were performed on the TCam-2 Δ SOX17 bulk population only.

Figure 32: Expression of pluripotency and germ cell markers after depletion of SOX17 in TCam-2 cells

qRT-PCR of ChIP-validated targets of SOX17-mediated transcription (red) in TCam-2 Δ SOX17 bulk population and GFP-transfected TCam-2 as control (72 hours following transfection). Expression is normalized against GAPDH as housekeeping gene.

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While reduction of SOX17 and its downstream target genes NANOG, TFAP2C, POU5F1, PRDM14, ALPL and PRDM1 was clearly evident on mRNA level, protein levels of TFAP2C, OCT4, LIN28A and NANOG in the TCam-2 Δ SOX17 bulk population were not affected (Fig. 33). However, due to the heterogeneity of the TCam-2 Δ SOX17 bulk population and the presence of SOX17 wildtype cells within this population, Western blot analysis of the whole protein lysate may not have been sensitive enough to detect the effects of SOX17 depletion in individual single cells.

Also, mRNA and protein levels can deviate from one another, due to the prolonged half-life of proteins compared to mRNA.

Figure 33: Depletion of SOX17 in TCam-2 cells

Western blot showing levels of SOX17, TFAP2C, OCT4, LIN28A and NANOG protein 72 hours following CRISPR/Cas9 mediated gene editing of SOX17 gene locus in the TCam-2 Δ SOX17 bulk population (Δ SOX17). The wildtype control represents TCam-2 cells that were transiently transfected with a GFP-coding plasmid. ACTIN was used as loading control.

The analysis of TCam-2 cell morphology 10-15 days following gene editing of SOX17, however, revealed signs of cell differentiation, such as the formation of polynucleated cells and an enlarged cytoplasm within these differentiated cell colonies (Fig. 34).

Differentiated areas were negative for the pluripotency marker alkaline phosphatase (AP), while cells resembling TCam-2 wildtype cells stained positive for AP activity (Fig.

35). In comparison, TCam-2 control cells that were transfected with a GFP-coding plasmid remained 100% positive for AP activity (Fig. 35). This confirms that the

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reduction or loss of SOX17 in TCam-2 cells leads to the downregulation of pluripotency resulting in induction of differentiation.

Figure 34: Morphology of TCam-2 Δ SOX17 bulk

Morphology of TCam-2 cells 10 and 15 days following CRISPR/Cas9 mediated gene editing of SOX17 gene locus (right) compared to wildtype TCam-2 control cells (left). Scalebar = 250 µm.

Figure 35: Alkaline phosphatase activity of TCam-2 Δ SOX17 bulk

AP activity of TCam-2 cells following CRISPR/Cas9 mediated gene editing of SOX17 gene locus (right) compared to TCam-2 (GFP-transfected) control cells (left). Scalebar = 250 µm.

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However, since the analysis of the whole protein lysate by Western blot was not sensitive enough to detect loss of TFAP2C and OCT4 on protein level in the TCam-2 Δ SOX17 bulk population, I additionally performed immunofluorescence staining on individual cells that stained either weakly or completely negative for SOX17 protein (Fig. 36). As expected, those cells that showed only weak staining for SOX17 protein also showed reduced levels of OCT4 and TFAP2C protein. This correlates with the ChIP-seq and qRT-PCR data and again confirms TFAP2C and OCT4 as direct targets of SOX17-mediated transcriptional activation in TCam-2 cells and shows that depletion of SOX17 results in loss of pluripotency and germ-cell-identity in TCam-2. Since morphological alterations already suggested induction of differentiation of TCam-2 cells (Fig. 34), I addressed the question whether the cells differentiate into random cell fates or if the induced differentiation is restricted to a specific cell fate.

Due to the resemblance to multinucleated trophoblast giant cells I analysed expression of trophectodermal markers (GATA3, HAND1, αHCG, CDX2, EOMES) (Fig. 37), as well as additional germ-cell related markers (SPRY4, NANOS3) (Fig. 37) in the TCam-2 Δ SOX17 bulk population. Notably, in humans HAND1 is expressed in the trophectoderm layer, where it regulates formation of the amniotic membrane [150].

GATA3 is expressed within the stem cell compartment of the placenta [151]. Human chorionic gonadotropin (αHCG) is a hormone involved in trophoblast differentiation and fusion [152]. qRT-PCR demonstrated a loss in germ-cell related markers and significant induction of GATA3, HAND1, as well as upregulation of αHCG (Fig. 37).

Upregulation of GATA3 was additionally confirmed by immunofluorescence staining (Fig. 37).

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Figure 36: SOX17, OCT4 and TFAP2C protein expression in TCam-2 Δ SOX17 bulk

Immunofluorescence showing expression of SOX17, TFAP2C and OCT4 protein following CRISPR/Cas9-mediated gene editing of SOX17 gene locus. The wildtype control represents TCam-2 cells that were transiently transfected with a GFP-coding plasmid. Scalebar = 250 µm.

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Figure 37: Expression of germ cell markers and trophoblast differentiation markers after depletion of SOX17 in TCam-2 cells

qRT-PCR of germ cell related markers (brown) and markers of extra-embryonic lineages (green) in TCam-2 Δ SOX17 bulk and GFP-transfected TCam-2 as control. Expression is normalized against GAPDH as housekeeping gene.

Altogether this indicates that reduction or loss of SOX17 in TCam-2 cells forces the cells to initiate differentiation to a trophectodermal cell fate. Interestingly, GATA3 protein was only detected in TCam-2 cells that were low, but not completely devoid of SOX17 protein (Fig. 38). Thus, it seems like different levels of SOX17 lead to formation of different cell types, meaning only a reduction but not a complete loss of SOX17 will lead to a GATA3+ cell population. Collectively, this shows that SOX17 is essential to maintain the latent pluripotency of seminoma cells and to prevent cellular differentiation. The analysis of additional markers specific for embryonic (mesoderm, endoderm, ectoderm) lineages, as well as for different extra-embryonic cell types of the placenta may help to fully understand the plasticity and differentiation potential of TCam-2 cells after reduction or complete loss of SOX17.

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Figure 38: SOX17 and GATA3 protein expression in TCam-2 Δ SOX17 bulk population

Immunofluorescence showing expression of SOX17 and GATA3 protein following CRISPR/Cas9 mediated gene editing of SOX17 gene locus. The wildtype control represents TCam-2 cells that were transiently transfected with a GFP-coding plasmid. Scalebar = 250 µm.

5.8. In TGCT cells NANOG is a common downstream target of SOX2 and