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OCT3/4 Signaling Pathway

Figure 1. OCT3/4 signaling pathway. Red letters indicate that OCT3/4 promotes the expression of those genes while the green letters indicate the opposite.

An overview of OCT3/4

OCT3/4 (encoded by Pou5f1, also known as OCT3, OCT4) was first identified in mice as an ESC-specific and germline-specific transcription factor. In humans, OCT4 is the product of the OTF3 gene, and has been reported to have three isoforms, OCT4A, OCT4B and OCT4B1. Among these isoforms, only OCT4A – which shares 87% amino acid sequence identity with mouse OCT4 – has been demonstrated to maintain the pluripotency of stem cells; it is referred to as OCT4 in the vast majority of reports.

The Oct4 gene is driven by a TATA-less promoter, a proximal enhancer (PE) and a distal enhancer (DE). Comparative analysis of Oct4 regulatory elements in different species identified four conservative regions (CRs): CR1 (in proximal promoter), CR2 and CR3 (PE), as well as CR4 (DE). The DE/CR4 is essential for regulating Oct4 expression in morula, inner cell mass of blastocysts and primordial germ cells; while the PE/CR2 activates Oct4 in the epiblast stage. Several positive and negative regulators bind to the Oct4 gene to regulate its expression. LRH-1 binds to the PE and activates Oct4 in undifferentiated ES cells through an uncharacterized mechanism. Other positive regulators include SF1 (Steroidogenic Factor-1) and RXR-Beta (Retinoid X Receptor-Beta). As a separate mechanism, retinoic acid induces GCNF (early period) and ARP-1/COUP-TFII and EAR-3/COUP-TFII (late period) contribute to the suppression of the Oct4 gene. Wwp2 (WW domain containing E3 Ubiquitin Protein ligase-2) can directly bind to OCT4 through WW domains. It promotes ubiquitination of OCT4, leading to the degradation of OCT4.

The OCT4 protein comprises three domains: the central POU (Pit-Oct-Unc) domain for DNA binding, the N-terminal transactivation domain and the C-terminal domain, which appears to be cell type-specific transactivation domain. OCT4 belongs to the POU transcription factor family, in which the members control the expression of their target genes through binding to the octameric sequence motif of the AGTCAAAT consensus sequence.

Function of OCT3/4

OCT4 is one of four transcription factors (OCT4, Sox2, Nanog and Lin28), the minimal set, required to induce pluripotency in somatic cells (reprogramming to pluripotency). Currently, the emphasis is placed on investigating the molecular mechanisms by which OCT4 contributes to establishing the gene expression programs that regulate pluripotency and early differentiation processes. It functions by recognizing and binding to DNA regulatory regions alone or in cooperation with other transcription factors as follows:

OCT3/4 with chromatin regulation

Recent experiments indicate that chromatin organization is dynamic and is subject to regulatory mechanisms that enforce the transcriptional potential of the genome during cellular commitment and differentiation. Chromatin is remodeled into transcriptionally permissive or repressive conformations by complexes. Those complexes were proposed to interact with OCT4. Subunits of the NuRD, Polycomb, SWI/SNF (stem cell-specific esBAF), and LSD1 complexes were found in all reported interactomes of OCT4. Many genes implicated in chromatin remodeling were correlated to Oct4 (e.g., Ash2l, Phc1, Rnf134, Bmi1 and Phc3).

OCT3/4 with cell cycle

Cell cycle comprises four different phases: the S phase, the M phase, and two gap phases between S phase and M phase (G1 phase for synthesis of proteins and lipids, and G2 phase for checking DNA integrity).

OCT4 promotes the phosphorylation of hypo-phosphorylated RB (retinoblastoma, a prerequisite for the R-point transition) by downregulating PP1 (protein phosphatase 1) and upregulating CDK4/6-Cyclin D in early and mid G1 phase. At the G2/M phase, CDK1-Cyclin B can enhance the binding of OCT4 to the promoter and suppress the transcription of homeobox protein CDX2, a typical differentiation marker. In contrast, OCT4 can inhibit the activation of CDK1 by cell division cycle phosphorylation, which is independent of its transcriptional activity. Many genes (Ccnf, Nipp1, lgf2bp1, D14Abb1e and Jarid1b) are involved in this process.

Oct3/4 with pluripotency

OCT4, NANOG and SOX2, have been considered to act as a core transcriptional regulatory circuitry in pluripotent stem cells, as they collectively occupy a large number of their target genes. A large number of regulatory elements of the OCT4 target gene contain a composite Oct–Sox element separated by several nucleotides. OCT4 and SOX2 bind the element simultaneously and synergistically activate the expression of these genes, including Oct4, Sox2, Nanog, Utf1 and Zfp206, all of which play important roles in maintaining pluripotency. Consistent with recent reports that TGFβ/ACTIVIN/NODAL signaling is critical for maintaining hESC pluripotency, expression of Nodal ligand, its co-receptor Tdgf1, and the antagonists Lefty1 and Lefty2 are reduced following OCT4 knockdown.

OCT3/4 with apoptosis and DNA damage & repair

The initial phase of apoptosis involves the caspase mediated induction of DNA strand breaks and the recruitment of DNA repair genes that act in concert to halt cell cycle progression and restore genomic stability. But if the damage cannot be repaired, further cutting of the DNA, nuclear foaming and other processes that are elegantly and thoroughly described elsewhere eventually leads to programmed cell death. All apoptosis inducing genes Bin1, Blp1, Serpinb9, Sh3glb1, and Casp6, were found to be negatively correlated to Oct4, and Sh3glb1 and Casp6 were confirmed as targets. Tdrd7, Brca1, and Parp1 are involved in DNA damage and repair as direct Oct4 targets.

Implications in disease

Recently, accumulative evidence suggests that Oct4 is overexpressed in various solid tumors, including breast cancer, bladder cancer and lung cancer. Targeting of Oct4 promotes cell death in breast and lung cancer cells, and makes drug-resistant hepatocellular carcinoma sensitive to chemotherapy. However, the mechanism by which Oct4 influences transcriptional reprogramming that leads to somatic cancer progression remains unclear. The target gene networks of Oct4 in somatic cancer are also undiscovered. Another study found that activation of Oct-4 results in dysplastic growths in epithelial tissues that are dependent on continuous Oct-4 expression. In the intestine, Oct-4 expression causes dysplasia by inhibiting cellular differentiation in a manner similar to that in embryonic cells.

References:

1. Simandi Z; et al. Oct4 acts as an integrator of pluripotency and signal-induced differentiation. Molecular Cell. 2016, 63(4):647.
2. She S; et al. Cell cycle and pluripotency: Convergence on octamer‑binding transcription factor 4 (Review). Molecular Medicine Reports. 2017, 16(5).
3. Shi G; Jin Y. Role of oct4 in maintaining and regaining stem cell pluripotency. Stem Cell Research & Therapy. 2010, 1(5):39.
4. Mullen A C; et al. Oct4 directs TGF-beta signaling in embryonic stem cells. Gastroenterology. 2011, 140(5):S-321-S-321.
5. Vaidya A; Kale V P. TGF-β signaling and its role in the regulation of hematopoietic stem cells. Syst Synth Biol. 2015, 9(1-2):1-10.
6. Yeom YI; et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development. 1996, 122:881–894.
7. Nordhoff V; et al. Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences. Mamm. Genome Official J. Int. Mamm. Genome Soc. 2001, 12:309–317.
8. Freberg CT; et al. Epigenetic reprogramming of OCT4 and NANOG regulatory regions by embryonal carcinoma cell extract. Mol. Biol. Cell. 2007, 18:1543–1553.
9. Babaie, Y.; et al. Analysis of Oct4‐dependent transcriptional networks regulating self‐renewal and pluripotency in human embryonic stem cells. STEM CELLS. 2007, 25:500-510.
10. Tang Y-A; et al. Global Oct4 target gene analysis reveals novel downstream PTEN and TNC genes required for drug-resistance and metastasis in lung cancer. Nucleic Acids Research. 2015, 43(3):1593-1608.
11. Hochedlinger K; et al. Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell. 2005, 121(3):465-477.

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