Scientists claim the forkhead box O (FOXO) transcription factor FOXO1 is an essential component of the cellular control mechanism that maintains pluripotency in human embryonic stem cells (hESCs). Studies by a Mount Sinai School of Medicine-led team suggest the protein may directly control OCT4 and SOX2 gene expression by activating the genes’ promoters.
Knocking out the human FOXO1 gene in cells resulted in loss of pluripotency markers and eventually evidence of differentiation. Saghi Ghaffari, Ph.D., and colleagues published their results in Nature Cell Biology in a paper titled “FOXO1 is an essential regulator of pluripotency in human embryonic stem cells.”
Research to date has shown that hESC pluripotency is maintained by OCT4 (octamer-binding transcription factor 4), NANOG, and SOX2 (SRY-box containing protein). These proteins form a feedback circuit that positively regulates their own genes and activates other genes encoding critical components of pluripotency, while at the same time repressing those involved in developmental processes, the Mount Sinai team reports.
FOXO proteins, meanwhile, are known tumor suppressors, which studies suggest also play key roles in primary stem and progenitor cells. To address whether FOXO proteins play a role in human development, Dr. Ghaffari’s team analyzed their expression in hESCs.
They found FOXO1 was the most abundant FOXO at the messenger RNA level in undifferentiated pluripotent H1 hESCs and was at least seven times more abundant than FOXO3A and FOXO4. Conversely, the expression of FOXO1 was markedly downregulated during embryoid body formation and cell commitment to mesoderm and hematopoietic differentiation. In two undifferentiated self-renewing hESC lines most FOXO1 was located in the nucleus. These observations hinted that FOXO1 may also be involved in regulating ESC fate, the authors note.
To investigate a potential role for FOXO1 in hESC pluripotency, the team generated inducible FOXO1-specific knockdown systems based on two different short hairpin RNAs. They found that inhibition of FOXO1 mRNA in H1 cells using either of the distinct shRNAs resulted in >90% depletion of FOXO1 protein expression within 72 hours, which was accompanied by rapid downregulation of OCT4, NANOG, and SOX2 expression. In parallel to the changes in gene expression the cells lost surface markers of pluripotency.
Indeed, FOXO1 knockdown resulted in the spontaneous differentiation of hESCs maintained under pluripotent self-renewal conditions, as shown by the induction of mesoderm and endoderm lineage markers, none of which had previously been reported to be directly directed by FOXO proteins.
“These results also indicate that FOXO1 regulation of pluripotency is due in part to its participation in suppressing the mesoderm and endoderm lineage commitment,” the researchers note. Interestingly, the effect of FOXO1 knockdown on hESC pluripotency genes was reversible: reinstating FOXO1 expression in the knockdown within a few days resulted in recovery of OCT4, NANOG, and SOX2.
The researchers moved on to evaluate the effects of forced FOXO1 expression on hESC pluripotency. Lentiviral-mediated ectopic FOXO1 expression induced a significant upregulation of OCT4, NANOG, and SOX2 in two different cell lines, whereas ectopic expression of FoxO3A had no effect on the expression of the three pluripotency genes. Of particular interest, the team adds, was the observation that overexpression of FOXO1 in hESCs also resulted in upregulation of KLF4 and REST, which are genes associated with mouse embryonic stem cell potency.
Conversely, knockdown of FOXO1 in hESCs undergoing a number of passages in self-renewing conditions resulted in the cells’ morphology changing to a more flattened, epithelial appearance, which was accompanied by a nearly complete loss of alkaline phosphatase activity, cell surface markers of pluripotency, and transcripts for OCT4, NANOG, and SOX2. These changes were accompanied by the full acquisition of differentiation markers and were not reversible after a few passages, “indicating that FOXO1 is critical for the maintenance of pluripotency in hESCs over time,” the authors state.
Adding more evidence to the notion that FOXO1 is critical for pluripotency, the researchers further found that the mouse orthologue of FOXO1 was also critical for the regulation of pluripotency and differentiation of mouse embryonic stem cells.
hESCs form teratoma-like masses when introduced into immunodeficient mice, and this is a generally accepted approach to demonstrate the developmental potential of pluripotent hESCs in vivo, the researchers continue. “We reasoned that if FOXO1 is critical for hESC pluripotency, loss of its activity should prevent hESCs from forming teratomas in vivo.”
This was, indeed, the outcome of knocking out FOXO1 in hESCs transplanted into SCID mice: Whereas all the animals injected with control cells formed cystic tumors containing elements of all three embryhonic germ layers, only two of nine mice administered with the FOXO1 knockouts formed tumors, and these were relatively small.
The next stage was to investigate the mechanism by which FOXO1 regulates pluripotency. Genetic analysis identified highly conserved regions, upstream of the pluripotency genes OCT4 and SOX2 (although not NANOG), which contained putative FOXO binding sites. Chromatin immunoprecipitation studies further confirmed that in pluripotent self-renewing hESCs, endogenous FOXO1 bound specifically to sequences within regulatory regions upstream of the OCT4 and SOX2 genes. Reporter assays supported these findings and demonstrated that FOXO1 activates OCT4 and SOX2 transcription through the regulation of specific gene regions.
“The present study demonstrates an essential function for FOXO1 in the regulation of hESC fate,” the authors conclude. “Thus, the longevity FOXO proteins emerge as critical nonredundant regulators of both somatic and embryonic stem cell activity. These findings indicate that FOXO1 is a component of the circuitry of hESC pluripotency and support the notion that activation of FOXO1 may be used for improving somatic cell reprogramming."