Pluripotency Factors Oct4 Research Articles

MicroRNA Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1.

MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes, as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation, a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 overexpression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion, we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate. Stem Cells 2016;34:1284-1296.

Hierarchical Oct4 Binding in Concert with Primed Epigenetic Rearrangements during Somatic Cell Reprogramming.

The core pluripotency factor Oct4 plays key roles in somatic cell reprogramming through transcriptional control. Here, we profile Oct4 occupancy, epigenetic changes, and gene expression in reprogramming. We find that Oct4 binds in a hierarchical manner to target sites with primed epigenetic modifications. Oct4 binding is temporally continuous and seldom switches between bound and unbound. Oct4 occupancy in most of promoters is maintained throughout the entire reprogramming process. In contrast, somatic cell-specific enhancers are silenced in the early and intermediate stages, whereas stem cell-specific enhancers are activated in the late stage in parallel with cell fate transition. Both epigenetic remodeling and Oct4 binding contribute to the hyperdynamic enhancer signature transitions. The hierarchical Oct4 bindings are associated with distinct functional themes at different stages. Collectively, our results provide a comprehensive molecular roadmap of Oct4 binding in concert with epigenetic rearrangements and rich resources for future reprogramming studies.

P73 and IGF1R Regulate Emergence of Aggressive Cancer Stem-like Features via miR-885-5p Control.

Cancer stem-like cells (CSC) have been proposed to promote cancer progression by initiating tumor growth at distant sites, suggesting that stem-like cell features can support metastatic efficiency. Here, we demonstrate that oncogenic DNp73, a dominant-negative variant of the tumor-suppressor p73, confers cancer cells with enhanced stem-like properties. DNp73 overexpression in noninvasive melanoma and lung cancer cells increased anchorage-independent growth and elevated the expression of the pluripotency factors CD133, Nanog, and Oct4. Conversely, DNp73 depletion in metastatic cells downregulated stemness genes, attenuated sphere formation and reduced the tumor-initiating capability of spheroids in tumor xenograft models. Mechanistic investigations indicated that DNp73 acted by attenuating expression of miR-885-5p, a direct regulator of the IGF1 receptor (IGF1R) responsible for stemness marker expression. Modulating this pathway was sufficient to enhance chemosensitivity, overcoming DNp73-mediated drug resistance. Clinically, we established a correlation between low p73 function and high IGF1R/CD133/Nanog/Oct4 levels in melanoma specimens that associated with reduced patient survival. Our work shows how DNp73 promotes cancer stem-like features and provides a mechanistic rationale to target the DNp73-IGF1R cascade as a therapeutic strategy to eradicate CSC.

Expression of the Pluripotency Factor OCT4 in Cutaneous Mast Cell Tumours

Expression of the Pluripotency Factor OCT4 in Cutaneous Mast Cell Tumours

MiR-302 regulates pluripotency, teratoma formation and differentiation in stem cells via an AKT1/OCT4-dependent manner.

Pluripotency makes human pluripotent stem cells (hPSCs) promising for regenerative medicine, but the teratoma formation has been considered to be a major obstacle for their clinical applications. Here, we determined that the downregulation of miR-302 suppresses the teratoma formation, hampers the self-renewal and pluripotency, and promotes hPSC differentiation. The underlying mechanism is that the high endogenous expression of miR-302 suppresses the AKT1 expression by directly targeting its 3'UTR and subsequently maintains the pluripotent factor OCT4 at high level. Our findings reveal that miR-302 regulates OCT4 by suppressing AKT1, which provides hPSCs two characteristics related to their potential for clinical applications: the benefit of pluripotency and the hindrance of teratoma formation. More importantly, we demonstrate that miR-302 upregulation cannot lead OCT4 negative human adult mesenchymal stem cells (hMSCs) to acquire the teratoma formation in vivo. Whether miR-302 upregulation can drive hMSCs to acquire a higher differentiation potential is worthy of deep investigation.

Role of Endothelial Progenitor Cells in Maintaining Stemness and Enhancing Differentiation of Mesenchymal Stem Cells by Indirect Cell-Cell Interaction.

A hot issue in current research regarding stem cells for regenerative medicine is the retainment of the stemness and multipotency of stem cell. Endothelial progenitor cells (EPCs) are characterized by an angiogenic switch that induces angiogenesis and further ameliorates the local microenvironment in ischemic organs. This study investigated whether EPCs could modulate the multipotent and differential abilities of mesenchymal stem cells (MSCs) in vitro and in vivo. We established an EPC/MSC indirect Transwell coculture system and then examined the effects of EPCs on the regulation of MSC biological properties in vitro and bone formation in vivo. The in vitro studies showed that cocultured MSCs (coMSCs) display no overt changes in cell morphology but an enhanced MSC phenotype compared with monocultured MSCs (monoMSCs). Our studies regarding the cellular, molecular, and protein characteristics of coMSCs and monoMSCs demonstrated that EPCs greatly promote the proliferation and differentiation potentials of coMSCs under indirect coculture condition. The expression of the pluripotency factors OCT4, SOX2, Nanog, and Klf4 was also upregulated in coMSCs. Furthermore, coMSCs combined with fibrin glue showed improved bone regeneration when used to repair rat alveolar bone defects compared with monoMSC grafts in vivo. This study is the first to demonstrate that EPCs have dynamic roles in maintaining MSC stemness and regulating MSC differentiation potential.

TGF-βI Regulates Cell Migration through Pluripotent Transcription Factor OCT4 in Endometriosis.

Transforming growth factor (TGF-β)/TGF-β receptor signal is known to promote cell migration. Up-regulation of TGF-β in serum/peritoneal fluid and increased levels of pluripotent transcription factor OCT4 in endometriotic tissues are frequently observed in patients with endometriosis. However, the mechanisms underlying how TGF-β/TGF-β receptor and OCT4 affect endometriotic cell migration still remain largely unknown. Therefore, endometriotic tissue with high cell migratory capacity were collected from patients with adenomyotic myometrium (n = 23) and chocolate cyst (n = 24); and endometrial tissue with low cell migratory capacity in normal endometrium or hyperplastic endometrium (n = 8) were collected as the controls. We found the mRNA levels of TGF-β receptor I (TGF-β RI) and OCT4 were significantly higher in the high-migratory ectopic endometriotic tissues than those of the low-migratory normal or hyperplastic endometrium. Positive correlations between TGF-β RI and OCT4, and either TGF-β RI or OCT4 with migration-related genes (SNAIL, SLUG and TWIST) regarding the mRNA levels were observed in human endometriotic tissues. TGF-βI dose-dependently increased the gene and protein levels of OCT4, SNAIL and N-Cadherin (N-CAD) and silencing of endogenous OCT4 significantly suppressed the TGF-βI-induced expressions of N-CAD and SNAIL in primary human endometriotic stromal cells and human endometrial carcinoma cell lines RL95-2 and HEC1A. Furthermore, TGF-βI significantly increased the migration ability of endometriotic cells and silencing of OCT4 dramatically suppressed the TGF-βI-induced cell migration activity evidenced by wound-closure assay, transwell assay, and confocal image of F-actin cellular distribution. In conclusion, the present findings demonstrate that the niche TGF-β plays a critical role in initiating expressions of pluripotent transcription factor OCT4 which may contribute to the ectopic endometrial growth by stimulating endometrial cell migration. These findings would be useful for developing therapeutic strategies targeting TGF-β-OCT4 signaling to prevent endometriosis in the future.

TALEN-induced disruption of Nanog expression results in reduced proliferation, invasiveness and migration, increased chemosensitivity and reversal of EMT in HepG2 cells.

Accumulating evidence indicates that Nanog plays a central role in modulating the biological behaviors of human hepatocellular carcinoma (HCC). However, the underlying mechanisms remain unclear. In the present study, we employed transcription activator-like effector nucleases (TALEN) to disrupt Nanog expression in HepG2 cells and obtained subcloned cells with diallelic Nanog mutations. Significantly, we found that the expression of pluripotency factors Sox2, Oct4 and Klf4, as well as expression of cancer stem cell (CSC) marker CD133, in the Nanog-targeted HepG2 cells was markedly downregulated. This finding suggests that Nanog may play an important role in maintaining the pluripotency and malignancy of HepG2 cells. We also revealed that Nanog regulated cell proliferation by modulating the expression of cyclin D1/D3/E1 and CDK2, respectively. Additionally, the disruption of Nanog resulted in the downregulation of epithelial-mesenchymal transition (EMT) regulators Snail and Twist, which contributed to the elevated level of epithelial marker E-cadherin, and to the decreased level of mesenchymal markers N-cadherin and vimentin in the HepG2 cells. In addition, the Nanog-targeted HepG2 cells exhibited reduced ability of invasion, migration and chemoresistance in vitro. In conclusion, the disruption of Nanog expression results in less proliferation, invasiveness, migration, more chemosensitivity and reversal of EMT in HepG2 cells, by which Nanog plays crucial roles in influencing the malignant phenotype of HepG2 cells.

Transgene-free human induced pluripotent stem cell line (HS5-SV.hiPS) generated from cesarean scar-derived fibroblasts

Transgene-free human HS5-SV.hiPS line was generated from human cesarean scar-derived fibroblasts using temperature-sensitive Sendai virus vectors carrying Oct4, Sox2, cMyc and Klf4 exogenous transcriptional factors. The viral constructs were eliminated from HS5-SV.hiPS line through heat treatment. Transgene-free HS5-SV.hiPS cells expressed pluripotent associated transcription factors Oct4, Nanog, Sox2, Rex1 and surface markers SSEA-4, TRA-1-60 and OCT4. HS5-SV.hiPS cells formed embryoid bodies and differentiated into three embryonic germ layers in vivo. HS5-SV.hiPS cells maintained their normal karyotype (46, XX) after culture for extended period. HS5-SV.hiPS displayed the similar pattern of DNA fingerprinting to the parenteral scar-derived fibroblasts.

Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States.

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

ATPS-61NEW SMALL MOLECULES THAT KILL HYPOXICALLY TRANSFORMED GLIOMA STEM-LIKE CELLS

High grade gliomas (HGGs) are lethal brain cancers, accounting for 78% of adult onset primary CNS malignancies. HGGs are resistant to current conventional therapies, including radiation therapy (RT), chemotherapy, and treatment with anti-angiogenic agents. Populations of cancer cells surviving these treatments regenerate leading to recurrence in more than 95% of patients. Spontaneous tumor necrosis within avascular tumor domains imparts poor clinical prognosis as glioma cells are reprogrammed by hypoxic transcription factors to adopt ‘stem-cell’ like properties; commonly referred to as glioma ‘stem-like’ cells (GSCs). GSCs possess several properties of normal stem cells, including self-aggregation, expression of stem cell markers, ability to survive in hypovascularized tumor regions, ability to survive detachment, and most importantly, the ability of tumor regrowth. GSCs can stop proliferating in hypoxic and acidic perinecrotic regions, which increases their resistance to RT and conventional anti-mitotic therapies. Hypoxically reprogrammed GSCs represent an important drug target to prevent the recurrence of HGGs. Drug targeting of hypovascularized GSCs is challenging as acidic and hypoxic tumor domains promote cell cycle arrest. Thus, therapeutic agents must be cytotoxic, not cytostatic, and need to kill independently of proliferative status. We developed novel small molecules that triggers ‘mis-trafficking’ of endosomes containing urokinase protein cargo in HGGs, and killing non-proliferating and proliferating glioma cells by an irreversible caspase-independent necrotic pathway. We identified that more than 90% of glioma cells within 22 patient derived xenografts (PDX) express intracellular PAI-1 and uPA and that 40-80% of these cells co-express the pluripotent stem cell transcription factors Oct4 and Nanog. Our lead small molecule crosses the intact rodent BBB and selectively kills glioma cells within intracerebral PDX glioma xenografts (PDX). The anti-glioma cytotoxicities of these small molecules is cell cycle independent, unaffected by hypoxia, and could represent an effective adjunctive therapy to be combined with radiotherapy, anti-mitotic, and/or anti-angiogenic therapies.

Plasma DCLK1 is a marker of hepatocellular carcinoma (HCC): Targeting DCLK1 prevents HCC tumor xenograft growth via a microRNA-dependent mechanism.

Tumor stem cell marker Doublecortin-like kinase1 (DCLK1) is upregulated in several solid tumors. The role of DCLK1 in hepatocellular carcinoma (HCC) is unclear. We immunostained tissues from human livers with HCC, cirrhosis controls (CC), and non-cirrhosis controls (NCC) for DCLK1. Western blot and ELISA analyses for DCLK1 were performed with stored plasma samples. We observed increased immunoreactive DCLK1 in epithelia and stroma in HCC and CCs compared with NCCs, and observed a marked increase in plasma DCLK1 from patients with HCC compared with CC and NCC. Analysis of the Cancer Genome Atlas' HCC dataset revealed that DCLK1 is overexpressed in HCC tumors relative to adjacent normal tissues. High DCLK1-expressing cells had more epithelial-mesenchymal transition (EMT). Various tumor suppressor miRNAs were also downregulated in HCC tumors. We evaluated the effects of DCLK1 knockdown on Huh7.5-derived tumor xenograft growth. This was associated with growth arrest and a marked downregulation of cMYC, and EMT transcription factors ZEB1, ZEB2, SNAIL, and SLUG via let-7a and miR-200 miRNA-dependent mechanisms. Furthermore, upregulation of miR-143/145, a corresponding decrease in pluripotency factors OCT4, NANOG, KLF4, and LIN28, and a reduction of let-7a, miR-143/145, and miR-200-specific luciferase activity was observed. These findings suggest that the detection of elevated plasma DCLK1 may provide a cost-effective, less invasive tool for confirmation of clinical signs of cirrhosis, and a potential companion diagnostic marker for patients with cirrhosis and HCC. Our results support evaluating DCLK1 as a biomarker for detection and as a therapeutic target for eradicating HCC.

Immunohistochemical Expression of the Pluripotency Factor OCT4 in Canine Mast Cell Tumours

Cancer stem cells (CSCs) are related to malignancy and resistance to chemotherapy in several tumours. OCT4 is a 'pluripotency factor' that is expressed by these cells. The aim of the present study was to investigate OCT4 expression in canine cutaneous mast cell tumours (MCTs) by means of immunohistochemistry. Twenty-eight cases were evaluated and showed variable immunolabelling patterns. The dogs were treated by surgery alone and followed up for a minimum of 180 days. No significant difference was found between histopathological grades and similar results were obtained for mortality due to the disease and post-surgical survival. These preliminary results suggest that OCT4 expression is not a precise prognostic indicator for canine MCT.

A Biorepository of Induced Pluripotent Stem Cells from Subjects With Cholangiopathies

Introduction: Regenerative medicine, including the application of induced pluripotent stem cell (iPSC) technology, has the potential to dramatically transform healthcare in the coming years. Chronic biliary disease is the cause of significant morbidity and mortality and end-stage cholangiopathies are essentially untreatable without liver transplantation. As such, the biliary system is an attractive target for regenerative medicine approaches to study and treat the disorders. Methods: Based on emerging cholangiocyte developmental pathways, we pioneered a unique and reproducible system to create iPSC-derived cholangiocytes (iDCs). In order to take full advantage of this novel protocol, we began a project to develop a biorepository of iPSC from subjects with cholangiopathies. To accomplish this, we employed a collaborative effort at Mayo Clinic Rochester, involving the Center for Cell Signaling in Gastroenterology and the Regenerative Medicine Biotrust. Participants were identified and enrolled at three sites (Minnesota, Arizona, and Florida). Subjects were sent to the Regenerative Medicine Consult Clinic for skin biopsy and samples were processed in the Biotrust Laboratory. Fibroblasts were isolated, expanded, and transduced with the Sendai RNA viral reprograming system to force expression of the pluripotency factors KLF4, OCT4, SOX2, and c-MYC. The individualized iPSCs were differentiated in vitro to mature cholangiocytes using a step-wise differentiation strategy via definitive endoderm, hepatic specification, and hepatic progenitors using a process of temporal exposure to key biliary morphogens. Results: We have biobanked numerous fibroblast lines from patients with cholangiopathies (e.g. PSC, PBC, cholangiocarcinoma, polycystic liver disease, Caroli disease, cystic fibrosis, and Alagille syndrome). Several of these lines are being reprogrammed to iPSC and differentiated to cholangiocytes in our Liver Regenerative Medicine Laboratory. Conclusion: Once established, this novel biorepository will represent a limitless source of high-quality, individualized liver cells for future studies that can: 1) be analyzed in vitro as subject-specific models of liver diseases; 2) be treated in vitro to test putative therapeutic compounds for efficacy and toxicity; 3) undergo genetic modification to correct underlying disease-causing defects; and 4) serve as a platform for studying pathophysiology and developing individualized, cell-based, regenerative therapies for the cholangiopathies.

Abstract 4220: Silencing DCLK1 prevents breast cancer cell self-renewal, epithelial mesenchymal transition, circulating tumor cells and metastasis

Abstract Introduction: Solid tumor cancers including breast cancer possess a rare population of cells that display stem cell-like properties with high metastatic potential and relative resistance to conventional therapies. These features render these cells as highly desirable albeit elusive therapeutic targets. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is overexpressed in many cancers including breast cancer. In this study, we establish the scientific rationale for targeting DCLK1 to inhibit breast cancer proliferation, tumor stem cell growth, self-renewal, EMT, circulating tumor cells (CTCa) and metastasis. Methods: Small interfering RNA (siRNA) against DCLK1 was transfected into breast cancer cell lines (MCF7 and MDAMB231 (triple negative breast cancer cell line)). For analyzing circulating tumor cells MDAMB231 cells are stably transfected with RFP (red fluorescence protein. Cell proliferation, colony formation, mammosphere formation assay (self-renewal), cell migration and invasion assays were assessed to evaluate the effect of DCLK1 knockdown on breast cancer cells self-renewal and metastatic potential. Isograft model was used to identify the role of DCLK1 knockdown on breast cancer growth and circulating tumor cells in vivo. The assays were analyzed utilizing experimental protocols for cell culture, clonogenic culture, circulating tumor cells by gradient centrifugation, immunohistochemistry, real-time PCR and Western blotting. Results: Expression of DCLK1 was ∼36% higher in human breast cancer tissues than in normal tissue. DCLK1 siRNA inhibited the expression of DCLK1 mRNA and protein in breast cancer cells. Silencing DCLK1 decreased the proliferation (∼40%), colony formation (∼70%), and self-renewal (∼85%), of the breast cancer cell lines in vitro. In vitro migration and invasion assays demonstrated that knocking down DCLK1 led to significantly less (40-80%) breast cancer cells migrated and invaded. Further, expression of EMT associated factors (Slug, Snail, Twist, Zeb-1, Zeb-2 and Vimentin) and pluripotency factors (c-Myc, Sox2, Nanog and Oct-4) were significantly (p<0.05) reduced between 30 and 50%, after silencing DCLK1. Inhibition of DCLK1 in isograft models resulted in reduced tumor growth (∼81%) and CTCs (∼98%) in vivo. Conclusions: DCLK1 expression is associated with breast cancer cell survival, self-renewal, metastasis and tumorigenesis. Silencing DCLK1 reduced breast cancer cell self-renewal, CTCs, metastasis, tumor growth and survival. DCLK1 inhibition may represent a novel targeted therapeutic approach to reduce the morbidity and mortality associated with breast cancer. Citation Format: PARTHASARATHY CHANDRAKESAN, Nathaniel Weygant, Vivian Taylor, William Berry, Randal May, Dongfeng Qu, Meghna Singh, Sripathi Sureban, Naushad Ali, Michael Bronze, Courtney Houchen. Silencing DCLK1 prevents breast cancer cell self-renewal, epithelial mesenchymal transition, circulating tumor cells and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4220. doi:10.1158/1538-7445.AM2015-4220

Tryptophan derivatives regulate the transcription of Oct4 in stem-like cancer cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to environmental toxicants, is increasingly recognized as a key player in embryogenesis and tumorigenesis. Here we show that a variety of tryptophan derivatives that act as endogenous AhR ligands can affect the transcription level of the master pluripotency factor Oct4. Among them, ITE enhances the binding of the AhR to the promoter of Oct4 and suppresses its transcription. Reduction of endogenous ITE levels in cancer cells by tryptophan deprivation or hypoxia leads to Oct4 elevation, which can be reverted by administration with synthetic ITE. Consequently, synthetic ITE induces the differentiation of stem-like cancer cells and reduces their tumorigenic potential in both subcutaneous and orthotopic xenograft tumour models. Thus, our results reveal a role of tryptophan derivatives and the AhR signalling pathway in regulating cancer cell stemness and open a new therapeutic avenue to target stem-like cancer cells.

Abstract 1: Oct4 is Re-activated in Smooth Muscle Cells through KLF4/Hif1α and Hydroxymethylation Mechanisms and Plays a Critical Role in Atherogenesis

The pluripotency factor Oct4 is a protein that regulates embryonic stem cell (ESC) totipotency and is the only gene thought to be indispensable for production of induced pluripotent stem (iPS) cells. Although there is evidence that Oct4 may be expressed in non-ESC, this is controversial and as yet there is no direct evidence that Oct4 plays a functional role within somatic cells. We demonstrate that Oct4 is markedly induced in cells within atherosclerotic lesions from ApoE-/- mice fed a high-fat diet and within human coronary artery atherosclerotic lesions. We show that smooth muscle cell (SMC)-specific conditional knockout (KO) of Oct4 within Western diet-fed ApoE-/- mice resulted in increased atherosclerotic lesion size and other changes consistent with decreased plaque stability. Moreover, using a novel SMC lineage tracing mouse developed in our lab, we also show that lesions from SMC-specific conditional Oct4 KO mice contained far fewer de-differentiated (Acta2-) SMCs likely as the result of impaired SMC de-differentiation and migration from the media into the intima and not to altered proliferation or apoptosis. Further mechanistic studies using in vitro and in vivo Chromatin Immunoprecipitation assays, glycosylation-coupled methylation sensitive qPCR and hMeDIP show that activation of Oct4 within SMCs and within atherosclerotic lsions was associated with hydroxymethylation of the Oct4 promoter and was Klf4- and Hif1α-dependent. In addition, using in situ hybridization/proximity ligation (ISH-PLA) assay developed in our lab we showed hydroxymethylation of the Oct4 promoter within individual SMC-derived cells in atherosclerotic lesions of our SMC-lineage tracing ApoE-/- mice. These results are the first to show that Oct4 plays a direct functional role in regulating phenotypic transitions in somatic cells, and provide molecular mechanisms required for Oct4 re-activation.

Tex10 Coordinates Epigenetic Control of Super-Enhancer Activity in Pluripotency and Reprogramming

Super-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage-specific transcription factors driving expression of genes that define cell identity. In embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination.

Targeting glioma stem cells using T cells recognizing the pluripotency factor Oct4

Targeting glioma stem cells using T cells recognizing the pluripotency factor Oct4

Viruses from Within

Early human embryos contain virus-like structures in the cytoplasm that appear to originate with an endogenous retrovirus, according to a new study [1]. The viral proteins turn-on an antiviral response and also may regulate the expression of many cellular RNAs. Only recently have technologies emerged to examine endogenous retroviruses closely during early development— and it’s clear that they are up to something. These retoviruses are highly expressed after fertilization, and some of their transcripts exhibit peculiar behavior—such as fusing with cellular RNAs to form RNA chimeras. Some endogenous retroviruses have been implicated in the initiation or maintenance of pluripotency. In the new study, Edward Grow et al. examined data from a previous single-cell analysis of RNA profiles in early human embryos. Similar to previous findings, the researchers found that distinct classes of retroviruses were transcribed at distinct stages. The researchers then focused on the most recently acquired retrovirus—HERVK (HML_2). Nearly 1/3 of its proviruses in the human genome are unique to humans. Unlike most other endogenous retroviruses, HERVK has retained the ability to encode intact retroviral proteins. It is normally transcriptionally silent, but can be activated in germ cell tumors, melanoma, and other pathological conditions. Grow et al. show that HERVK becomes active in eight-cellstage human embryos, and forms virus-like particles. The retrovirus remains active in epiblast cells, and shuts down in cultured epiblast cells as they begin to lose pluripotency. In cell culture, HERVK expression bumped up the cell-surface expression of IFITM1, a protein that combats viral infection, and thwarted infection by a flu virus. The researchers also provided hints about how HERVK is turned on—its expression was induced in human cells by the combination of high levels of the pluripotency factor OCT-4 (POU5F1) and low methylation levels (conditions also present in the early embryo). By turning on an antiviral response, HERVK may serve to protect the embryo from destruction by microbes (like the flu) and from endogenous retroviruses attempting to re-enter a cell. HERVK seems to also have a broader regulatory role: one HERVK protein, Rec, bound numerous cellular RNAs and appeared to regulate their expression. Also, HERVK could possibly re-integrate into the genome, which is a question for future studies.