Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells.

The transcription factor Oct4 can rightfully be considered a pivotal element in maintaining pluripotency. In addition, its ability to function as a pioneer factor enables the reprogramming of somatic cells back into a pluripotent state. To better understand the regulation of the Oct4-encoding gene (Pou5f1), the main genetic elements that regulate its expression in different states of pluripotency ought to be identified. While some elements have been well characterized for their ability to drive Pou5f1 expression, others have yet to be determined. In this work, we show that translocation of the Pou5f1 gene fragment purported to span all essential cis-elements, including the well-known distal and proximal enhancers (DE and PE), into the Rosa26 locus impairs the self-renewal of mouse embryonic stem cells (ESCs) in the naïve pluripotency state, as well as their further advancement through the formative and primed pluripotency states, inducing overall differentiation failure. These results suggest that regulatory elements located outside the previously determined Pou5f1 boundaries are critical for the proper spatiotemporal regulation of this gene during development, indicating the need for their better characterization.

The functional diversity of the POUV-class proteins across vertebrates.

POUV is a relatively newly emerged class of POU transcription factors present in jawed vertebrates (Gnathostomata). The function of POUV-class proteins is inextricably linked to zygotic genome activation (ZGA). A large body of evidence now extends the role of these proteins to subsequent developmental stages. While some functions resemble those of other POU-class proteins and are related to neuroectoderm development, others have emerged de novo. The most notable of the latter functions is pluripotency control by Oct4 in mammals. In this review, we focus on these de novo functions in the best-studied species harbouring POUV proteins-zebrafish, Xenopus (anamniotes) and mammals (amniotes). Despite the broad diversity of their biological functions in vertebrates, POUV proteins exert a common feature related to their role in safeguarding the undifferentiated state of cells. Here we summarize numerous pieces of evidence for these specific functions of the POUV-class proteins and recap available loss-of-function data.

Key features of the POU transcription factor Oct4 from an evolutionary perspective.

Oct4, a class V POU-domain protein that is encoded by the Pou5f1 gene, is thought to be a key transcription factor in the early development of mammals. This transcription factor plays indispensable roles in pluripotent stem cells as well as in the acquisition of pluripotency during somatic cell reprogramming. Oct4 has also been shown to play a role as a pioneer transcription factor during zygotic genome activation (ZGA) from zebrafish to human. However, during the past decade, several studies have brought these conclusions into question. It was clearly shown that the first steps in mouse development are not affected by the loss of Oct4. Subsequently, the role of Oct4 as a genome activator was brought into doubt. It was also found that the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) could proceed without Oct4. In this review, we summarize recent findings, reassess the role of Oct4 in reprogramming and ZGA, and point to structural features that may underlie this role. We speculate that pluripotent stem cells resemble neural stem cells more closely than previously thought. Oct4 orthologs within the POUV class hold key roles in genome activation during early development of species with late ZGA. However, in Placentalia, eutherian-specific proteins such as Dux overtake Oct4 in ZGA and endow them with the formation of an evolutionary new tissue-the placenta.

hnRNP-K Targets Open Chromatin in Mouse Embryonic Stem Cells in Concert with Multiple Regulators.

The transcription factor Oct4 plays a key regulatory role in the induction and maintenance of cellular pluripotency. In this article, we show that ubiquitous and multifunctional poly(C) DNA/RNA-binding protein hnRNP-K occupies Oct4 (Pou5f1) enhancers in embryonic stem cells (ESCs) but is dispensable for the initiation, maintenance, and downregulation of Oct4 gene expression. Nevertheless, hnRNP-K has an essential cell-autonomous function in ESCs to maintain their proliferation and viability. To better understand mechanisms of hnRNP-K action in ESCs, we have performed ChIP-seq analysis of genome-wide binding of hnRNP-K and identified several thousands of hnRNP-K target sites that are frequently co-occupied by pluripotency-related and common factors (Oct4, TATA-box binding protein, Sox2, Nanog, Otx2, etc.), as well as active histone marks. Furthermore, hnRNP-K localizes exclusively within open chromatin, implying its role in the onset and/or maintenance of this chromatin state. Stem Cells 2019;37:1018-1029.