Oct4 In Embryonic Stem Cells Research Articles

Dichlorodiphenyltrichloroethane metabolites inhibit DNMT1 activity which confers methylation-specific modulation of the sex determination pathway.

Dichlorodiphenyltrichloroethane (DDT) poses a significant health risk to humans which is associated with genomic DNA hypomethylation. However, the mechanism and biological consequences remain poorly understood. Invitro assays confirmed that the DDT metabolites 2,2-bis(p-chlorophenyl)-acetic acid (DDA) and 1-chloro-2,2-bis-(p-chlorophenyl)ethylene (DDMU), but not other DDT metabolites, significantly inhibited DNA methyltransferase 1 (DNMT1) activity, leading to genomic hypomethylation in cell culture assays. DNMT1 as a target for DNA hypomethylation induced by DDT metabolites was also confirmed using cell cultures in which DNMT1 was silenced or highly expressed. DDA and DDMU can modify methylation markers in the promoter regions of sexual development-related genes, and change the expression of Sox9 and Oct4 in embryonic stem cells. Molecular docking indicated that DDA and DDMU bound to DNMT1 with high binding affinity. Molecular dynamic simulation revealed that DDA and DDMU acted as allosteric modulators that reshaped the conformation of the catalytic domain of DNMT1. These findings provide a new insight into DDT-induced abnormalities in sexual development and demonstrate that selective binding to DNMT1 by DDA and DDMU can interfere with human DNMT1 activity and regulate the expression of the Sox9 and Oct4 genes.

Kap1 regulates the self-renewal of embryonic stem cells and cellular reprogramming by modulating Oct4 protein stability.

Oct4 plays a crucial role in the regulation of self-renewal of embryonic stem cells (ESCs) and reprogramming of somatic cells to induced pluripotent stem cells. However, the molecular mechanisms underlying posttranslational regulation and protein stability of Oct4 remain unclear. Using affinity purification and mass spectrometry analysis, we identified Kap1 as an Oct4-binding protein. Silencing of Kap1 reduced the protein levels of Oct4 in ESCs, whereas the overexpression of Kap1 stimulated the levels of Oct4. In addition, Kap1 overexpression stimulated the self-renewal of ESCs and attenuated the spontaneous differentiation of ESCs in response to LIF withdrawal. Kap1 overexpression increased the stability of Oct4 by inhibiting the Itch-mediated ubiquitination of Oct4. Silencing of Kap1 augmented Itch-mediated ubiquitination and inhibited the stability of Oct4. We identified the lysine 133 (K133) residue in Oct4 as a ubiquitination site responsible for the Kap1-Itch-dependent regulation of Oct4 stability. Preventing ubiquitination at the lysine residue by mutation to arginine augmented the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. These results suggest that Kap1 plays a crucial role in the regulation of the pluripotency of ESCs and somatic cell reprogramming by preventing Itch-mediated ubiquitination and the subsequent degradation of Oct4.

The PI3K pathway impacts stem gene expression in a set of glioblastoma cell lines

The PI3K pathway controls diverse cellular processes including growth, survival, metabolism, and apoptosis. Nuclear FOXO factors were observed in cancers that harbor constitutively active PI3K pathway output and stem signatures. FOXO1 and FOXO3 were previously published to induce stem genes such as OCT4 in embryonic stem cells. Here, we investigated FOXO-driven stem gene expression in U87MG glioblastoma cells. PI3K-activated cancer cell lines were investigated for changes in gene expression, signal transduction, and clonogenicity under conditions with FOXO3 disruption or exogenous expression. The impact of PI3K pathway inhibition on stem gene expression was examined in a set of glioblastoma cell lines. We found that CRISPR-Cas9-mediated FOXO3 disruption in U87MG cells caused decreased OCT4 and SOX2 gene expression, STAT3 phosphorylation on tyrosine 705 and clonogenicity. FOXO3 over expression led to increased OCT4 in numerous glioblastoma cancer cell lines. Strikingly, treatment of glioblastoma cells with NVP-BEZ235 (a dual inhibitor of PI3K and mTOR), which activates FOXO factors, led to robust increases OCT4 gene expression. Direct FOXO factor recruitment to the OCT4 promoter was detected by chromatin immunoprecipitation analyses using U87MG extracts. We show for the first time that FOXO transcription factors promote stem gene expression glioblastoma cells. Treatment with PI3K inhibitor NVP-BEZ235 led to dramatic increases in stem genes in a set of glioblastoma cell lines. Given that, PI3K inhibitors are actively investigated as targeted cancer therapies, the FOXO-mediated induction of stem genes observed in this study highlights a potential hazard to PI3K inhibition. Understanding the molecular underpinnings of stem signatures in cancer will allow refinements to therapeutic strategies. Targeting FOXO factors to reduce stem cell characteristics in concert with PI3K inhibition may prove therapeutically efficacious.

Gaining Insights into the Function of Post-Translational Protein Modification Using Genome Engineering and Molecular Cell Biology

Modifications by kinases are a fast and reversible mechanism to diversify the function of the targeted proteins. The OCT4 transcription factor is essential for preimplantation development and pluripotency of embryonic stem cells (ESC), and its activity is tightly regulated by post-transcriptional modifications. Several phosphorylation sites have been identified by systemic approaches and their functions proposed. Here, we combined molecular and cellular biology with CRISPR/Cas9-mediated genome engineering to pinpoint the function of serine 12 of OCT4 in ESCs. Using chemical inhibitors and an antibody specific to OCT4 phosphorylated on S12, we identified cyclin-dependent kinase (CDK) 7 as upstream kinase. Surprisingly, generation of isogenic mESCs that endogenously ablate S12 revealed no effects on pluripotency and self-renewal, potentially due to compensation by other phosphorylation events. Our approach reveals that modification of distinct amino acids by precise genome engineering can help to clarify the functions of post-translational modifications on proteins encoded by essential gene in an endogenous context.

Sox2/Oct4: A delicately balanced partnership in pluripotent stem cells and embryogenesis

Considerable progress has been made in understanding the roles of Sox2 and Oct4 in embryonic stem cells and mammalian embryogenesis. Specifically, significant progress has been made in answering three questions about the functions of Sox2 and Oct4, which are the focus of this review. 1) Are the first or second cell lineage decisions during embryogenesis controlled by Oct4 and/or Sox2? 2) Do the levels of Oct4 and Sox2 need to be maintained within narrow limits to promote normal development and to sustain the self-renewal of pluripotent stem cells? 3) Do Oct4 and Sox2 work closely together or is the primary role of Sox2 in pluripotent cells to ensure the expression of Oct4? Although significant progress has been made in answering these questions, additional studies are needed to resolve several important remaining issues. Nonetheless, the preponderance of the evidence suggests there is considerable crosstalk between Sox2 and Oct4, and further suggests Sox2 and Oct4 function as molecular rheostats and utilize negative feedback loops to carefully balance their expression and other critical genes during embryogenesis. This article is part of a Special Issue entitled: The Oct transcription factor family, edited by Dr. Dean Tantin.

Oct4 was a novel target of Wnt signaling pathway

The specific expression of Oct4 during early mouse development is required for the correct maintenance of pluripotent cells, and the regulatory control of the Oct4 expression is important. Wnt signaling could have multiple and/or complex effects on embryonic stem (ES) cells characteristics. Elucidation of the molecular mechanisms affecting Wnt signaling in ES cells could provide a better understanding of how these effects occur. The purpose of this study was to determine whether Oct4 was regulated by Wnt signaling in undifferentiated ES cells. Here, we report Oct4 as a novel target of β-catenin-mediated transcription. First, we observe that Wnt signaling pathway is activated in undifferentiated mouse ES cells. In 239T cells, Oct4 promoter was regulated by β-catenin. Through promoter mapping and chromatin immuno-precipitation assays, we found that Oct4 is a direct target of β-catenin/TCF-mediated transcription and the binding site at -875/-881 of Oct4 promoter is critical for b-catenin/TCF-dependent expression regulation. We further detect the expression of Oct4 in treatment with glycogen syntheses kinase (GSK)-3-specific inhibitor in mouse ES cells and HepG2 cells. We found that GSK-3-specific inhibitor can maintain the expression of Oct4 in ES cells and can enhance the expression of Oct4 in HepG2 cells. Our results suggest that Oct4 might be a novel target of β-catenin/TCF-mediated downstream gene in Wnt-activated cells.

Master Transcription Factors Determine Cell-Type-Specific Responses to TGF-β Signaling

Transforming growth factor beta (TGF-β) signaling, mediated through the transcription factors Smad2 and Smad3 (Smad2/3), directs different responses in different cell types. Here we report that Smad3 co-occupies the genome with cell-type-specific master transcription factors. Thus, Smad3 occupies the genome with Oct4 in embryonic stem cells (ESCs), Myod1 in myotubes, and PU.1 in pro-B cells. We find that these master transcription factors are required for Smad3 occupancy and that TGF-β signaling largely affects the genes bound by the master transcription factors. Furthermore, we show that induction of Myod1 in nonmuscle cells is sufficient to redirect Smad3 to Myod1 sites. We conclude that cell-type-specific master transcription factors determine the genes bound by Smad2/3 and are thus responsible for orchestrating the cell-type-specific effects of TGF-β signaling.

Targeting of De Novo DNA Methylation Throughout the Oct-4 Gene Regulatory Region in Differentiating Embryonic Stem Cells

Differentiation of embryonic stem (ES) cells is accompanied by silencing of the Oct-4 gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyse the progression of DNA methylation of the ∼2000 base pair regulatory region of Oct-4 in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the Oct-4 promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a. Analysis of the Oct-4 regulatory domain as a whole has allowed us to detect targeted de novo methylation and to refine our understanding the roles of key protein components in this process.

More synergetic cooperation of Yamanaka factors in induced pluripotent stem cells than in embryonic stem cells

The role of Yamanaka factors as the core regulators in the induction of pluripotency during somatic cell reprogramming has been discovered recently. Our previous study found that Yamanaka factors regulate a developmental signaling network in maintaining embryonic stem (ES) cell pluripotency. Here, we established completely reprogrammed induced pluripotent stem (iPS) cells and analyzed the global promoter occupancy of Yamanaka factors in these cells by ChIP-chip assays. We found that promoters of 565 genes were co-bound by four Yamanaka factors in iPS cells, a 10-fold increase when compared with their binding in ES cells. The promoters occupied by a single Yamanaka factor distributed equally in activated and repressed genes in iPS cells, while in ES cells Oct4, Sox2, or Klf4 distributed mostly in repressed genes and c-Myc in activated ones. Pathway analysis of the ChIP-chip data revealed that Yamanaka factors regulated 16 developmental signaling pathways in iPS cells, among which 12 were common and 4 were unique compared to pathways regulated in ES cells. We further analyzed another recently published ChIP-chip dataset in iPS cells and observed similar results, showing the power of ChIP-chip plus pathway analysis for revealing the nature of pluripotency maintenance and regeneration. Next, we experimentally tested one of the repressive signaling pathways and found that its inhibition indeed improved efficiency of cell reprogramming. Taken together, we proposed that there is a core developmental signaling network necessary for pluripotency, with TGF-beta, Hedgehog, Wnt, p53 as repressive (Yin) regulators and Jak-STAT, cell cycle, focal adhesion, adherens junction as active (Yang) ones; and Yamanaka factors synergistically regulate them in a Yin-Yang balanced way to induce pluripotency.

A general mechanism for transcription regulation by Oct1 and Oct4 in response to genotoxic and oxidative stress

Oct1 and Oct4 are homologous transcription factors with similar DNA-binding specificities. Here we show that Oct1 is dynamically phosphorylated in vivo following exposure of cells to oxidative and genotoxic stress. We further show that stress regulates the selectivity of both proteins for specific DNA sequences. Mutation of conserved phosphorylation target DNA-binding domain residues in Oct1, and Oct4 confirms their role in regulating binding selectivity. Using chromatin immunoprecipitation, we show that association of Oct4 and Oct1 with a distinct group of in vivo targets is inducible by stress, and that Oct1 is essential for a normal post-stress transcriptional response. Finally, using an unbiased Oct1 target screen we identify a large number of genes targeted by Oct1 specifically under conditions of stress, and show that several of these inducible Oct1 targets are also inducibly bound by Oct4 in embryonic stem cells following stress exposure.

Ovarian surface epithelium stem cells: oogenesis in vitro?

Background: The dogma that the total number of follicles and oocytes available for reproduction are determined at birth, and that one follicle and one oocyte are recruited from the existing pool to mature in each menstrual cycle still persists. There is an increasing experimental evidence that this might not be true. In the ovarian surface epithelium there might be nondifferentiated stem cells that might differentiate into oocytes also in adult life. In this research we aimed at isolating putative stem cells from the ovarian surface epithelium and at evaluating in vitro oogenesis in the ovarian cell culture in vitro in women without naturally present follicles and oocytes in their ovarian cortex – postmenopausal women and women with premature ovarian failure. Methods: Ovarian surface epithelium was scraped from the ovaries of 20 postmenopausal women and 5 women with premature ovarian failure. We tried to find putative stem cells in ovarian scraping and to confirm them by transcription markers Oct-4, Sox-2, and Nanog, and by surface antigen SSEA-4. Cell culture was set up by scraped cells in DMEM/F-12 medium with phenol red, which shows a weak estrogenic activity. Ovarian cell culture was cultured for 20 days in a CO2-incubator at 37 °C and 5 % CO2. Development of cells in the culture was followed and the presence of oocyte-like cells was evaluated by using different methods – evaluation of morphology, transcription markers, surface antigen markers, oocyte immunohistochemical markers, and flow cytometry after propidium iodide staining. Results: In all postmenopausal women and in 4 out of the 5 women with premature ovarian failure putative stem cells were isolated from the ovarian surface epithelium, which were positive for transcription markers of embryonic stem cells Oct-4, Sox-2, Nanog, and for surface antigen SSEA-4. In all these women ovarian cell culture was successfully established. In all cultures oocyte-like cells developed approximately on day 5 of culture, which were positive for transcription marker Oct-4 and immunohistological germ cell markers c-kit, Oct-4, ZP2, DAZL, and even meiotic marker SCP3. Marker SCP3 was expressed in oocyte-like cells developed in ovarian cell culture of postmenopausal women, but not in patients with premature ovarian failure. Flow cytometry after propidium iodide staining revealed a population of potentially haploid oocyte like cells in postmenopausal women. Stem cells developed also into some somatic cell types, including fibroblasts, neuron-, and mioblast-like cells. In patients with premature ovarian failure oocyte-like cells were developed only at the presence of autologous patient’s serum and mostly at the presence of heterologous follicular fluid serum, whereas they did not develop at the presence of fetal calf serum. Conclusions: First results are promising as they indicate the new insight into the physiology of the adult human ovary. In the ovarian surface epithelium putative stem cells with embryonic character are present, which can develop into the oocyte-like cells and other types of cells in vitro. Further research is required to understand better these new findings.

A negative feedback loop of transcription factors that controls stem cell pluripotency and self‐renewal

Embryonic stem (ES) cells possess the ability to renew themselves while maintaining the capacity to differentiate into virtually all cell types of the body. Current evidence suggests that ES cells maintain their pluripotent state by expressing a battery of transcription factors including Oct4 and Nanog. However, little is known about how ES cells maintain the expression of these pluripotent factors in ES cells. Here we present evidence that Oct4, Nanog, and FoxD3 form a negative feedback loop to maintain their expression in pluripotent ES cells. First, Oct4 maintains Nanog activity by directly activating its promoter at sub-steady-state concentration but repressing it at or above steady-state levels. On the other hand, FoxD3 behaves as a positive activator of Nanog to counter the repressive effect of Oct4. The expression of Oct4 is activated by FoxD3 and Nanog but repressed by Oct4 itself, thus, exerting an important negative feedback loop to limit its own activity. Indeed, overexpression of either FoxD3 or Nanog in ES cells failed to increase the concentration of Oct4 beyond the steady-state concentration, whereas knocking down either FoxD3 or Nanog reduces the expression of Oct4 in ES cells. Finally, overexpression of Oct4 or Nanog failed to compensate the loss of Nanog or Oct4, respectively, suggesting that both are required for ES self-renewal and pluripotency. Our results suggest the FoxD3-Nanog-Oct4 loop anchors an interdependent network of transcription factors that regulate stem cell pluripotency.

Core transcriptional regulatory circuitry in human hepatocytes

We mapped the transcriptional regulatory circuitry for six master regulators in human hepatocytes using chromatin immunoprecipitation and high-resolution promoter microarrays. The results show that these regulators form a highly interconnected core circuitry, and reveal the local regulatory network motifs created by regulator–gene interactions. Autoregulation was a prominent theme among these regulators. We found that hepatocyte master regulators tend to bind promoter regions combinatorially and that the number of transcription factors bound to a promoter corresponds with observed gene expression. Our studies reveal portions of the core circuitry of human hepatocytes.

Identification of Putative Downstream Genes of Oct-4 by Suppression-Subtractive Hybridization

As a step toward understanding how toti/pluripotence is maintained by Oct-4, we have first constructed a cell model with differentially expressed Oct-4 in embryonic stem cells, and then used suppression-subtractive hybridization (SSH) method to identify the downstream genes of Oct-4. Among the 384 clones we screened, 40 clones were detected as differentially expressed genes with colony hybridization, and 13 clones were confirmed as the putative downstream genes of Oct-4 by Northern blot analysis. Sequencing showed 12 different genes, 8 known genes (Oct-4, Rex-1, Sox-2, Creatine kinase B, Makorin 1, Importin β, Histone H2A.Z, Ribosomal protein S7) and 4 new genes. Except Oct-4 and Rex-1, the other genes have not been reported to be regulated by Oct-4. These results showed that SSH provides a very efficient means to identify the downstream genes of transcription factor. Some known genes identified may provide new insight of the function of Oct-4 in stem cells.

Characterization of a negative retinoic acid response element in the murine Oct4 promoter.

Expression of Oct4 in embryonic stem cells is controlled by a distal upstream stem cell-specific enhancer that is deactivated during retinoic acid (RA)-induced differentiation by an indirect mechanism not involving binding of RA receptors (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997-3005, 1991). Here we report that in RA-treated P19 embryonal carcinoma cells the Oct4 promoter is also subject to negative regulation by RA. The minimal Oct4 promoter sequence mediating repression consists of a promoter-proximal sequence containing a GC-rich SP1 consensus-like sequence and several hormone response element half-sites that can be arranged into direct repeats with different spacing. The GC box binds a nuclear factor that is invariably present in undifferentiated and RA-treated differentiated P19 cells. By contrast, the hormone response element-containing sequence binds factors that are induced following RA treatment. Mutational analysis and competition experiments show that the functional entity binding the RA-induced factor is a direct repeat sequence with a spacing of one nucleotide, previously shown to be a binding site for COUP transcription factors (COUP-TFs). Cotransfected orphan receptors COUP-TF1, ARP-1, and EAR-2 were able to repress the activity of Oct4 promoter-driven reporters in P19 EC cells, albeit with different efficiencies. Furthermore, the negative transcriptional effect of COUP-TFs is dominant over the activating effect of the Oct4 embryonic stem cell-specific enhancer. These results show that negative regulation of Oct4 expression during RA-induced differentiation of embryonic stem cells is controlled by two different mechanisms, including deactivation of the embryonic stem cell-specific enhancer and promoter silencing by orphan nuclear hormone receptors.

Characterization of a Negative Retinoic Acid Response Element in the Murine Oct4 Promoter

Expression of Oct4 in embryonic stem cells is controlled by a distal upstream stem cell-specific enhancer that is deactivated during retinoic acid (RA)-induced differentiation by an indirect mechanism not involving binding of RA receptors (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997-3005, 1991). Here we report that in RA-treated P19 embryonal carcinoma cells the Oct4 promoter is also subject to negative regulation by RA. The minimal Oct4 promoter sequence mediating repression consists of a promoter-proximal sequence containing a GC-rich SP1 consensus-like sequence and several hormone response element half-sites that can be arranged into direct repeats with different spacing. The GC box binds a nuclear factor that is invariably present in undifferentiated and RA-treated differentiated P19 cells. By contrast, the hormone response element-containing sequence binds factors that are induced following RA treatment. Mutational analysis and competition experiments show that the functional entity binding the RA-induced factor is a direct repeat sequence with a spacing of one nucleotide, previously shown to be a binding site for COUP transcription factors (COUP-TFs). Cotransfected orphan receptors COUP-TF1, ARP-1, and EAR-2 were able to repress the activity of Oct4 promoter-driven reporters in P19 EC cells, albeit with different efficiencies. Furthermore, the negative transcriptional effect of COUP-TFs is dominant over the activating effect of the Oct4 embryonic stem cell-specific enhancer. These results show that negative regulation of Oct4 expression during RA-induced differentiation of embryonic stem cells is controlled by two different mechanisms, including deactivation of the embryonic stem cell-specific enhancer and promoter silencing by orphan nuclear hormone receptors.