OCT4 and SOX2 transcription factors
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Introduction
Oct4 and Sox2 are two transcription factors (TFs) involved in various roles in murine and primate cells, mainly related to the maintenance of pluripotency and self-renewal properties in embryonic stem cells. These two factors, encoded by the POU5F1 (POU Class 5 Homeobox 1) and SOX2 (SRY-Box Transcription Factor 2) genes, respectively, serve as repprogramming TFs and occupy the same target genes in vivo [1][2], forming the complex OCT4-SOX2, which is the main way in which they act, although they are not obligate heterodimers in solution.
OCT4
The OCT4 transcription factor (octamer-binding transcription factor 4), also known as OCT-3, OCT3 / 4, OTF3 or NF-A3, was discovered almost three decades ago, where its use relationship with pluripotent CTE in primate and rodent species (Zeineddine et al., 2014). This protein is encoded by the POU5F1 gene, which is located on chromosome 6 in humans and 17 in rats, and belongs to the POU family (Pit, October, Unc) of DNA-binding proteins, which regulate the expression of target genes (Zeineddine et al., 2014; Malakootian et al., 2017). In humans, through alternative splicing, POU5F1 generates less than eight distinct RNA transcripts, these being OCT4A, OCT4B-190, OCT4B-265, OCT4B-164, OCT4B1 and more recently reported as OCT4C, OCT4C1 and OCT4B4 variants [16; 17] In addition to the generated isoforms, many studies have been carried out mainly with respect to the functions of the OCT4A isoform. Studies targeting the OCT4B isoforms (190, 265 and 164) that are not able to support an automatic restoration of the CTE, but they can respond to cellular stress, whereas the functions of OCT4B1, OCT4C and OCT4C1 have not yet been clarified (Wang and Dai, 2010). OCT4A is normally expressed in the early stages of embryonic development and represents one of the main regulatory factors for pluripotency and self-review of embryonic stem cells, being considered a marker of pluripotency (Da SILVA et al., 2017). A further differentiation of CTE into cells used for different tissues depends on rapid and rapid expression of OCT4A, and these cells are differentiated remain with the OCT4A factor silenced (Villodre et al., 2016; Atlasi et al, 2008; Hatefi et al., 2012). However, we have already documented an open expression of transcription factors such as OCT4, SOX2 and NANOG, together or controlled, lead to tumors, metastases and the greatest recurrence after use, in different types of cancer (Zeineddine et al., 2014).
SOX2
The SOX/Sox (SRY homology box) family of proteins comprises 20 individual members in man and mouse [3], which SOX2 is the most explored. SOX proteins are principally defined by a conserved DNA-binding element, the so-called high mobility group (HMG) that relates to a transcriptional master regulator of virility (i.e., SEX determining factor Y, SRY) and thus functionally qualifies SOX/Sox proteins as DNA-binders [4][5]. While Sox proteins contribute to various cellular functionalities, reprogramming capacity is largely confined to members of the SoxB1 group (i.e., Sox1, Sox2, and Sox3)[6]. SOX2 significantly often imposes transcription modulatory in conjunction with co-factors, such as Oct3/4. OCT3/4 in ES cells
Murine Expression
Maternal Oct4 mRNA and protein are deposited in the oocyte and they are necessary for the development until the stage of 4 cells. Proteins are present at low levels at these early stages of murine embryogenesis. Transcription of zygotic Pou5f1 gene is activated at the 4 to 8-cell stage [rev oct 4, 6-8].
With the blastocyst is formation, the expression of Oct4 remains high in the inner cell mass and it is not observed in the trophectoderm. After the murine embryo implantation, the transient upregulation of Oct4 in a subset of cells from the inner cell mass, triggers their differentiation into hypoblast (primitive endoderm). After that, the Oct4 expression decreases in these cells [rev oct 4, 6-8]. During gastrulation, Oct4 is down-regulated and, after day 8 of gestation, it is confined to primordial germ cells [rev oct 4 - 7, 9, 10].
Human Expression
Gatekeeper for Embryonic Stem Cell Pluripotency
The pluripotent identity is ruled by transcriptional factor such as Oct4 and Sox2, that act as key pluripotency regulators among the mammals [rev oct4]. Oct4 keeps the undifferentiated cells from becoming trophoblast or endoderm [rev oct4] and Sox2 is critical in the formation of pluripotent epiblast cells [artg sox2]. The forced expression of Oct4 in Sox2-null mouse embryonic stem cells can rescue the pluripotency, indicating that the role of Sox2 in maintaining the pluripotent state of embryonic stem cells is primarily to sustain a sufficient level of Oct4 expression [ artigo sox2 - 2,13]. Oct4 and Sox2 cooperate to keep the pluripotency of embryonic stem cells by co-occupying a large number of enhancers and/or promoters and regulating the expression levels of their target genes [ref artigo sox2]. They activate the transcription of genes involved in the self renewal of embryonic stem cells [ref revisao oct4]. Besides, they bind themselves to the promoters of their own genes activating them [revisao oct4].
Intersection between Transcriptional Core and Lif Signalling
The OCT4-SOX2 mechanism in the nucleosome
The nucleosome is the chromatin basic unit, composed of a 147 pb DNA segment wrapped around 8 histone proteins. It is a convention that the sites in which a DNA major groove is pointed to the nucleosome core are called "superhelix location" (SHL). The SHL are enumerated from 0 to ±7, having 0 as the nucleosome main axis, known as "dyad". The OCT4-SOX2 binds in the SHL-6 site (Fig 1) and both of them act in the DNA removal from the core histones [ref1]. OCT4 has a bipartite DNA binding domain (DBD) comprised of a POU-specific (POUS) and POU-homeo-domain (POUHD) separated by 17-residues (Fig 2) and SOX2 has a high-mobility group (HMG) domain (Fig 2) [refs 1, 8]. The OCT4-POUS and SOX2-HMG DBDs engage major and minor grooves, respectively [ref1]. The DNA remains attached and straightened around the OCT4 site but is detached around the SOX2 motif [ref1].
OCT4 recognizes a partial motif, engaging DNA with its POUS domain, whereas the POUHD is not engaged. On free DNA, both POU domains engage the major groove over 8bp on opposite sides of the DNA [ref1]. SOX2 competes with histones for DNA binding and kinks DNA by ~90° at SHL-6.5 away from the histones [ref. 8]. This is accomplished by intercalation of the SOX2 Phe48 and Met49 ‘wedge’ at the TT base step [ref 8]. SOX2 kinks the DNA and synergistically with OCT4 releases the DNA from the core histones [movie1].
Figure 1 - Extracted from the article Mechanisms of OCT4-SOX2 motif readout on nucleosomes (Alicia K. Michael et al., 2020). (B) OCT4-SOX2-NCPSHL+6 model. (C) Details of SOX2-induced DNA kink.
Figure 2 - Extracted from the article Mechanisms of OCT4-SOX2 motif readout on nucleosomes (Alicia K. Michael et al., 2020). (A) Domain schematic of OCT4 and SOX2 constructs.
Movie 1 - Extracted from the Supplementary Materials for the article Mechanisms of OCT4-SOX2 motif readout on nucleosomes (Alicia K. Michael et al., 2020). OCT4-SOX2 binding at SHL-6 removes DNA from the histone core. A morph video modelling the structural change induced in the nucleosome upon OCT4-SOX2 binding at SHL-6. Morph is between the DNA of the NCP-SHL-6 and OCT4-SOX2-NCP-SHL-6 models.
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