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

Embryonic Expression

OCT4

In humans, the POUF5F1 alternative splicing gives rise to two Oct4 isoforms, Oct4-IA and Oct4-IB, that differs by the N-terminal region. Oct4-IA is required to self-renewal maintenance of stem cells and Oct4-IB is not related to stemness ^{[7] [8]}. Oct4 is present in all stages of embryo development ^{[9] [10] [11]}. The Oct4 expression pattern differs between the blastomeres in the same development stage by the protein cytoplasmic localization^[12]. From the unfertilized oocyte to the solid morula no Oct4 protein is observed in the nucleus ^[12]. During compaction, Oct4 expression becomes ubiquitous and abundant in the nucleus of all morula blastomeres ^[12]. In the blastocyst, the Oct4 mRNA and protein are present in the inner cell mass ^[11].

Maternal murine 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 ^{[13] [14] [15] [16]}. 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 ^{[13] [14] [15] [16]}. During gastrulation, Oct4 is down-regulated and, after day 8 of gestation, it is confined to primordial germ cells ^{[13] [15] [17] [18]}.

SOX2

Sox2 is persistently expressed during embryonic development and it is first expressed in the morula stage. Later it becomes specifically located in the inner cell mass of blastocyst and epiblast ^[19]. After gastrulation it is predominantly expressed in the central nervous system ^[20]. It is known that zygotic deletion of Sox2 is lethal due to the failure to form pluripotent epiblast whilst the absence of Sox2 has little effect on the trophectoderm formation ^[19]. The depletion of Sox2 compromised the stemness of both mouse and human embryonic stem cells, changing their morphology and pluripotent marker expression and they differentiate primarily into trophectoderm ^{[21] [22]}.

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)^[23] and both of them act in the DNA removal from the core histones ^[24]. 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) ^{[23] [24] [16]}. The OCT4-POUS and SOX2-HMG DBDs engage major and minor grooves, respectively ^[24]. The DNA remains attached and straightened around the OCT4 site but is detached around the SOX2 motif ^[24].

OCT4 recognizes a partial motif, engaging DNA with its POUS domain, whereas the POUHD is not engaged ^[23]. On free DNA, both POU domains engage the major groove over 8bp on opposite sides of the DNA ^[24]. SOX2 competes with histones for DNA binding and kinks DNA by ~90° at SHL-6.5 away from the histones ^[16]. This is accomplished by intercalation of the SOX2 Phe48 and Met49 ‘wedge’ at the TT base step ^[16]. SOX2 kinks the DNA and synergistically with OCT4 releases the DNA from the core histones Movie1^[23].

Figure 1 - Extracted from the article Mechanisms of OCT4-SOX2 motif readout on nucleosomes (Alicia K. Michael et al., 2020)^[23]. (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)^[23]. (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.

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 ^[25]. Oct4 keeps the undifferentiated cells from becoming trophoblast or endoderm ^[25] and Sox2 is critical in the formation of pluripotent epiblast cells ^[26]. 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 ^{[21] [22]}. 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 ^[26]. They activate the transcription of genes involved in the self renewal of embryonic stem cells and besides, they bind themselves to the promoters of their own genes activating them ^[25].

iPSC/Yamanaka factors

In 2006, Yamanaka and Takahashi first demonstrated the factors necessary for the induced Pluripotent Stem Cells (iPSC) generation. The Yamanaka factors, including Oct3/4 and Sox2 (also Klf4 and c-Myc), represent an important milestone in life sciences and have been widely used in the research and medical fields ^[27].

Related Diseases

Tumorigenicity

References

1. ↑ Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell. 2005 Sep 23;122(6):947-56. doi: 10.1016/j.cell.2005.08.020. PMID:16153702 doi:http://dx.doi.org/10.1016/j.cell.2005.08.020
2. ↑ Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J, Loh YH, Yeo HC, Yeo ZX, Narang V, Govindarajan KR, Leong B, Shahab A, Ruan Y, Bourque G, Sung WK, Clarke ND, Wei CL, Ng HH. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell. 2008 Jun 13;133(6):1106-17. doi: 10.1016/j.cell.2008.04.043. PMID:18555785 doi:http://dx.doi.org/10.1016/j.cell.2008.04.043
3. ↑ Schepers GE, Teasdale RD, Koopman P. Twenty pairs of sox: extent, homology, and nomenclature of the mouse and human sox transcription factor gene families. Dev Cell. 2002 Aug;3(2):167-70. doi: 10.1016/s1534-5807(02)00223-x. PMID:12194848 doi:http://dx.doi.org/10.1016/s1534-5807(02)00223-x
4. ↑ Bowles J, Schepers G, Koopman P. Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Dev Biol. 2000 Nov 15;227(2):239-55. doi: 10.1006/dbio.2000.9883. PMID:11071752 doi:http://dx.doi.org/10.1006/dbio.2000.9883
5. ↑ Schaefer T, Lengerke C. SOX2 protein biochemistry in stemness, reprogramming, and cancer: the PI3K/AKT/SOX2 axis and beyond. Oncogene. 2020 Jan;39(2):278-292. doi: 10.1038/s41388-019-0997-x. Epub 2019 Sep, 2. PMID:31477842 doi:http://dx.doi.org/10.1038/s41388-019-0997-x
6. ↑ Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 2008 Jan;26(1):101-6. doi: 10.1038/nbt1374. Epub 2007 Nov 30. PMID:18059259 doi:http://dx.doi.org/10.1038/nbt1374
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11. ↑ ^{11.0 11.1} Fujii T, Sakurai N, Osaki T, Iwagami G, Hirayama H, Minamihashi A, Hashizume T, Sawai K. Changes in the expression patterns of the genes involved in the segregation and function of inner cell mass and trophectoderm lineages during porcine preimplantation development. J Reprod Dev. 2013;59(2):151-8. doi: 10.1262/jrd.2012-122. Epub 2012 Dec 20. PMID:23257836 doi:http://dx.doi.org/10.1262/jrd.2012-122
12. ↑ ^{12.0 12.1 12.2} Cauffman G, Van de Velde H, Liebaers I, Van Steirteghem A. Oct-4 mRNA and protein expression during human preimplantation development. Mol Hum Reprod. 2005 Mar;11(3):173-81. doi: 10.1093/molehr/gah155. Epub 2005 Feb , 4. PMID:15695770 doi:http://dx.doi.org/10.1093/molehr/gah155
13. ↑ ^{13.0 13.1 13.2} Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell. 1998 Oct 30;95(3):379-91. doi: 10.1016/s0092-8674(00)81769-9. PMID:9814708 doi:http://dx.doi.org/10.1016/s0092-8674(00)81769-9
14. ↑ ^{14.0 14.1} Palmieri SL, Peter W, Hess H, Scholer HR. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol. 1994 Nov;166(1):259-67. doi: 10.1006/dbio.1994.1312. PMID:7958450 doi:http://dx.doi.org/10.1006/dbio.1994.1312
15. ↑ ^{15.0 15.1 15.2} Pesce M, Scholer HR. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells. 2001;19(4):271-8. doi: 10.1634/stemcells.19-4-271. PMID:11463946 doi:http://dx.doi.org/10.1634/stemcells.19-4-271
16. ↑ ^{16.0 16.1 16.2 16.3 16.4} Jerabek S, Merino F, Scholer HR, Cojocaru V. OCT4: dynamic DNA binding pioneers stem cell pluripotency. Biochim Biophys Acta. 2014 Mar;1839(3):138-54. doi: 10.1016/j.bbagrm.2013.10.001., Epub 2013 Oct 18. PMID:24145198 doi:http://dx.doi.org/10.1016/j.bbagrm.2013.10.001
17. ↑ Pan GJ, Chang ZY, Scholer HR, Pei D. Stem cell pluripotency and transcription factor Oct4. Cell Res. 2002 Dec;12(5-6):321-9. doi: 10.1038/sj.cr.7290134. PMID:12528890 doi:http://dx.doi.org/10.1038/sj.cr.7290134
18. ↑ Kehler J, Tolkunova E, Koschorz B, Pesce M, Gentile L, Boiani M, Lomeli H, Nagy A, McLaughlin KJ, Scholer HR, Tomilin A. Oct4 is required for primordial germ cell survival. EMBO Rep. 2004 Nov;5(11):1078-83. doi: 10.1038/sj.embor.7400279. PMID:15486564 doi:http://dx.doi.org/10.1038/sj.embor.7400279
19. ↑ ^{19.0 19.1} Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 2003 Jan 1;17(1):126-40. doi: 10.1101/gad.224503. PMID:12514105 doi:http://dx.doi.org/10.1101/gad.224503
20. ↑ Wegner M, Stolt CC. From stem cells to neurons and glia: a Soxist's view of neural development. Trends Neurosci. 2005 Nov;28(11):583-8. doi: 10.1016/j.tins.2005.08.008. Epub, 2005 Aug 31. PMID:16139372 doi:http://dx.doi.org/10.1016/j.tins.2005.08.008
21. ↑ ^{21.0 21.1} Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K, Okochi H, Okuda A, Matoba R, Sharov AA, Ko MS, Niwa H. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol. 2007 Jun;9(6):625-35. doi: 10.1038/ncb1589. Epub 2007 May 21. PMID:17515932 doi:http://dx.doi.org/10.1038/ncb1589
22. ↑ ^{22.0 22.1} Fong H, Hohenstein KA, Donovan PJ. Regulation of self-renewal and pluripotency by Sox2 in human embryonic stem cells. Stem Cells. 2008 Aug;26(8):1931-8. doi: 10.1634/stemcells.2007-1002. Epub 2008, Apr 3. PMID:18388306 doi:http://dx.doi.org/10.1634/stemcells.2007-1002
23. ↑ ^{23.0 23.1 23.2 23.3 23.4 23.5} Michael AK, Grand RS, Isbel L, Cavadini S, Kozicka Z, Kempf G, Bunker RD, Schenk AD, Graff-Meyer A, Pathare GR, Weiss J, Matsumoto S, Burger L, Schubeler D, Thoma NH. Mechanisms of OCT4-SOX2 motif readout on nucleosomes. Science. 2020 Apr 23. pii: science.abb0074. doi: 10.1126/science.abb0074. PMID:32327602 doi:http://dx.doi.org/10.1126/science.abb0074
24. ↑ ^{24.0 24.1 24.2 24.3 24.4} Soufi A, Garcia MF, Jaroszewicz A, Osman N, Pellegrini M, Zaret KS. Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming. Cell. 2015 Apr 23;161(3):555-568. doi: 10.1016/j.cell.2015.03.017. Epub 2015 Apr , 16. PMID:25892221 doi:http://dx.doi.org/10.1016/j.cell.2015.03.017
25. ↑ ^{25.0 25.1 25.2} Zeineddine D, Hammoud AA, Mortada M, Boeuf H. The Oct4 protein: more than a magic stemness marker. Am J Stem Cells. 2014 Sep 5;3(2):74-82. eCollection 2014. PMID:25232507
26. ↑ ^{26.0 26.1} Zhang S, Cui W. Sox2, a key factor in the regulation of pluripotency and neural differentiation. World J Stem Cells. 2014 Jul 26;6(3):305-11. doi: 10.4252/wjsc.v6.i3.305. PMID:25126380 doi:http://dx.doi.org/10.4252/wjsc.v6.i3.305
27. ↑ Yamanaka S, Takahashi K. [Induction of pluripotent stem cells from mouse fibroblast cultures]. Tanpakushitsu Kakusan Koso. 2006 Dec;51(15):2346-51. PMID:17154061

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