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40 results on '"NANOG"'

1. Cryo-electron microscopy studies of pluripotency factor Nanog

Author: Guild, Abbie, Chambers, Ian, and Spagnolo, Laura
Subjects: Cryo-electron microscopy studies, pluripotency factor Nanog, Embryonic stem (ES) cells, Culture of ES cells, network of pluripotency, Nanog, homeodomain protein, cytokine-independent self-renewal, undifferentiated ES cells, tryptophan repeat (WR) region, oligomerisation of Nanog
Abstract: Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst in the early mammalian embryo. These cells are characterised by the ability to self-renew and to give rise to cell types from all lineages, known as pluripotency. Culture of ES cells in vitro requires addition of signalling molecules such as LIF to block differentiation signals and maintain self-renewal. At the transcriptional level, self-renewal is governed by the 'network of pluripotency'; a group of transcription factors which includes Nanog, Sox2 and Oct4. Nanog is a homeodomain protein which, when over-expressed, can drive cytokine-independent self-renewal of undifferentiated ES cells. Dimerisation of Nanog through the tryptophan repeat (WR) region, a low complexity domain including 10 tryptophan residues, has been shown to be vital for this function. Although Nanog is well characterised from a biological perspective, detailed structural information has previously been limited to the homeodomain only. To provide a structural basis for how oligomerisation of Nanog drives self-renewal, cryo-electron microscopy and single particle analysis were utilised to determine a 3D structure of Nanog. By performing size exclusion chromatography and analytical ultracentrifugation, it was discovered that Nanog forms large oligomers of 860 kDa which corresponds to a complex of 24 Nanog protomers. Negative stain electron microscopy showed that the oligomers are globular with a size of 220 x 200 Å. Cryo-electron microscopy and single particle analysis approaches produced a model of the Nanog oligomers showing a triangular shape, however, inherent flexibility within the assembly limited the resolution to approximately 10 Å. Attempts to limit this flexibility for further structural studies included binding of Nanog to DNA and to partner protein Sox2. Negative stain microscopy of Nanog bound to a DNA element derived from the Tcf3 promoter showed a strikingly different, larger complex compared to Nanog alone. Preliminary biophysical and negative stain studies suggested that the Nanog-Sox2 complex may exist in a variety of oligomeric species. Altogether, this thesis provides insight on how Nanog behaves structurally and biophysically. Further work will be required to limit flexibility within these Nanog homo-oligomers to allow determination of a high resolution structure.
Published: 2023
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2. Functional analysis of the NANOG tryptophan repeat

Author: Vojtek, Matúš, Chambers, Ian, and Mullin, Nicholas
Subjects: 572, Nanog, embryonic stem cells
Abstract: The homeodomain transcription factor NANOG promotes self-renewal of embryonic stem cells (ESCs) and specification of primordial germ cells (PGCs). NANOG binds thousands of sites within the ESC genome together with OCT4, SOX2 and RNA polymerase II (RNAPII) regulating transcription of its target genes. Replacing tryptophans within the NANOG tryptophan repeat (WR) region for alanines (W10A) causes NANOG to lose its capacity to homodimerise and to interact with other proteins including RNAPII, SOX2 and RESF1. NANOGW10A mutant is also unable to sustain LIF-independent self-renewal. However, the requirements of the WR to interact with other proteins for NANOG function remains unclear. To characterise the cellular localisation of RESF1, endogenously tagged Resf1 mouse embryonic stem cell lines were produced. RESF1 is a widely expressed nuclear protein which interacts with NANOG in nuclei of ESCs. To quantify the requirements of Resf1 for ESC self-renewal and NANOG function, Resf1-/- ES cell lines were derived. In serum/LIF culture, Resf1 deletion promotes cell differentiation and decreases expression of Pou5f1, Nanog and Esrrb. Episomal expression of NANOG sustains LIF-independent self-renewal independently of Resf1. Resf1-/- ESCs are also capable to differentiate into epiblast stem cells. To investigate the role of Resf1 in PGC specification, Resf1-/- ESCs were differentiated into PGC-like cells (PGCLCs). Deletion of Resf1decreasedPGCLCspecification4-foldandpreventedefficientactivation of Blimp1, Prdm14 and Ap2γ. The effect of NANOG on OCT4, SOX2 and RNAPII chromatin binding was evaluated by inducing NANOG in Nanog-/- mouse ESCs. OCT4, SOX2 and RNAPII occupancy before and shortly after NANOG induction was measuredbyanalysingchromatinimmunoprecipitationfollowedbysequencing data sets. The k-mean clustering of Nanog binding sites revealed seven clusters with different response patterns of OCT4, SOX2 and RNAPII to NANOG. The sites where NANOG increases OCT4, SOX2 and RNAPII chromatin binding are mostly associate with the genes activated by NANOG, whereas sites where RNAPII binding decreases after NANOG induction associate with genes repressed by NANOG. To assess the requirements of the NANOG WR and the NANOG DNA binding for directing OCT4, SOX2 and RNAPII chromatin binding, NANOG variants with debilitating mutations in the WR or the homeodomain were induced in Nanog-/- ESCs and chromatin binding of NANOG, OCT4, SOX2 and RNAPII was assessed. The NANOGW10A has reduced chromatin binding at most of the NANOG binding sites. A reduced DNA binding capacity of recombinant NANOGW10A was also suggested by electrophoretic mobility shift assay. Moreover, NANOGW10A is unable to increase OCT4, SOX2 and RNAPII binding at the sites associated with gene activation. At the sites associated with gene repression, NANOGW10A induction leads to increased OCT4 binding. NANOG variant incapable to bind DNA (NANOGN51A) is unable to direct OCT4 and RNAPII binding. However, SOX2 binding temporarily decreases after NANOGN51A induction. In summary, Resf1 is not essential for ESC self-renewal and NANOG function but it is important for efficient specification of PGCLCs. NANOG directs the core transcription complex regulating self-renewal via the WR. This thesis contributes to understanding of NANOG function in ESC self-renewal and describes the function of previously uncharacterised RESF1 in ESC self-renewal and germ cell specification.
Published: 2020
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3. Nanog-Tcf15 axis during exit from naïve pluripotency

Author: Tatar, Tülin, Chambers, Ian, and Lowell, Sally
Subjects: 571.8, embryonic stem cells, pluripotency, Nanog, Tcf15, transcription factors, differentiating cells
Abstract: Pluripotent cells have the dual abilities to self-renewal and to differentiate into all three germ layers. Pluripotent cells can be isolated from two different stages of mouse embryogenesis. Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of the pre-implantation embryo and are considered to be in a naïve state. On the other hand, cells isolated from epiblast of the post-implantation embryo are referred as epiblast stem cells (EpiSC) and are representative of primed pluripotency. ESCs and EpiSCs are distinct from each other in terms of the morphology, the gene regulatory network and the signalling pathways regulating self-renewal. Under certain conditions, ESCs and EpiSCs can be transitioned into each other. However, the mechanism that regulates this transition from naïve to primed pluripotent state remains to be solved. Nanog, Oct4 and Sox2 form the core gene regulatory network of pluripotency. Additionally, the Id protein family is also important in the maintenance of pluripotency in ESCs. Id proteins function by inhibiting the activity of pro-differentiation factors. Tcf15 is identified as one of the targets of Id inhibition in ESCs. Moreover, Tcf15 has been identified as a repression target of Nanog. In this study, to understand the function of Tcf15, the expression of Tcf15 was characterized in differentiating ESCs. The transient upregulation of Tcf15 mRNA and protein was detected at early stages of differentiation before lineage commitment. Furthermore, Tcf15 protein was heterogeneously expressed in differentiating cells. Mutually exclusive expression of Nanog and Tcf15 proteins were demonstrated in both self-renewing and differentiating ESCs. Further characterization of the effect of Nanog on Tcf15 transcription showed that Tcf15 pre-mRNA was downregulated within 20 minute of Nanog induction. A Nanog binding site was identified at +32kb relative to the Tcf15 transcription start site (TSS). Initially, Nanog binding at this region was confirmed by performing ChIP-PCR experiments. Then, this Nanog binding region was further analysed for its enhancer activity related to the Tcf15 gene. Deletion of the Nanog binding region using CRISPR-Cas9 confirmed that this region acts as Tcf15 enhancer; it was shown that this region was required for the activation of Tcf15 transcription during differentiation. Tcf15 induction experiments were performed in order to the check whether Tcf15 affects Nanog transcription. The results indicate that Nanog is not a direct target of Tcf15, but Tcf15 contributes indirectly to the repression of Nanog. Additional analysis with the Tcf15 enhancer deletion cells showed that Tcf15 is not required for efficient downregulation of naïve markers and the upregulation of primed markers. However, the genes related to the regulation of adhesion properties of cells such as Zyc, Itga3 were induced with lower efficiency in the absence of Tcf15 compared to the wild type cells. In summary, I investigated the reciprocal regulation of Tcf15 and Nanog and the role of Tcf15 for the differentiation. My results suggest that Tcf15 is expressed in the cells that have initiated differentiation but are not lineage-committed. Additionally, Tcf15 can contribute to the regulation of adhesion related genes in order to help the epithelisation of the cells required during the differentiation from naïve to the primed pluripotent state. As a conclusion, Nanog is proposed to help to prevent certain aspects of ESCs differentiation by repressing Tcf15.
Published: 2018

4. Functional analysis of the role of the Nanog tryptophan repeat in ES cells

Author: Zhang, Jingchao, Chambers, Ian, and Lowell, Sally
Subjects: 616.02, ES cells, Nanog, Otx2, tryptophan repeats, pluripotency
Abstract: Nanog is a transcription factor that plays a central part in the gene regulatory network that maintains and induces pluripotency of embryonic stem cells (ESCs). However, the molecular basis by which Nanog achieves its functions is not fully understood. At the centre of C-terminal domain of Nanog a tryptophan repeat (WR) is located, comprising 10 penta-peptide repeats each starting with a tryptophan. A mutant form of Nanog (Nanog-W10A) in which all 10 tryptophan residues have been substituted by alanine has an impaired capacity to drive LIF-independent self-renewal and a reduced efficiency in reprogramming primed epiblast stem cells to naïve pluripotency. To understand how the WR contributes to Nanog function, Nanog-W10A-ERT2 was introduced into Nanog null cells. Upon hydroxytamoxifen addition, the Nanog-ERT2 fusion proteins were detected on chromatin within 1 hour, allowing a comparison of genome-wide transcriptional responses to Nanog and Nanog-W10A by microarray. When treated with LIF, Nanog-W10A can activate most of Nanog targets as efficiently as Nanog. In contrast, Nanog-W10A did not efficiently repress most Nanog targets, including Otx2 and Tcf15 that were previously suggested to prime ESCs for differentiation. The microarray experiments performed in the absence of LIF signalling showed that Nanog and LIF co-regulate an extensive list of targets, including Klf4 and Mras. When LIF is absent, wildtype Nanog can still activate pro-self-renewal factors, including Esrrb and repress differentiation-priming factor, such as Tcf15 and Otx2. In contrast, in the absence of LIF, the activation of pro-self-renewal factors Klf4 and Mras is reduced. In addition, activation of Esrrb by Nanog-W10A induction delays but does not prevent differentiation. These effects allow the de-repression of Otx2 and Tcf15 by Nanog-W10A to dominate. Therefore, the function of Nanog is not only mediated by the activation of pro-self-renewal genes, but also repression of pro-differentiation signals. The functional significance of the repression of Nanog targets was further exemplified by the robust capacity of Otx2 to dominate over the self-renewal signals and to drive differentiation. The Otx2 protein is a direct interacting partner of Nanog that binds the Nanog WR tryptophan residues. The previously identified Otx2 “tail domain” comprises two imperfectly aligned repeats and aromatic residues of each repeat align with aromatic residues of the Sox2 “SXS/TY” motif previously identified to mediate the interaction between Sox2 and Nanog. Aromatic residues of Otx2 were demonstrated to directly interact with both Nanog and Sox2. The interactions between Otx2, Nanog and Sox2 are essential for Otx2 functions in driving ESCs differentiation, as Otx2 mutants with alanine substitutions of the aromatic residues in both or either of the repeats have reduced efficiency to drive differentiation. As Nanog and Sox2 may co-occupy many loci important in maintaining ESC self-renewal, Otx2 may be able to “read” the Nanog/Sox2 co-binding sites to dissolve the pluripotent networks. In summary, the repression function of Nanog is located within the Nanog WR region and represents an important module of Nanog in fine-tuning the balance between self-renewal and differentiation. This module involving Nanog WR can also be recognised by differentiation-priming factor Otx2 and may represent an initial step during the exit of differentiation.
Published: 2016

5. Protein interactions underpinning pluripotency

Author: Gagliardi, Alessia, Chambers, Ian, and Mullin, Nicholas
Subjects: 572, embryonic stem cells, Nanog, Sox2, protein interaction, pluripotency
Abstract: Embryonic stem (ES) cells are maintained in an undifferentiated state by a gene regulatory network centred on the triumvirate of transcription factors Nanog, Oct4 and Sox2. Genome-wide chromatin immunoprecipitation studies indicate that in many cases target genes contain closely localised binding sites for each of these proteins, as well as additional members of the extended pluripotency transcription factor network. However, the biochemical basis of the interactions between these proteins is largely unknown, as are the mechanisms by which these interactions control ES cell identity. By purifying Nanog from ES cells and identifying co-purified proteins, we determined a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members and RNA processing factors. Validation of interactions was obtained by co-immunoprecipitation of Nanog with putative partners. Sox2 was identified as a robust interacting partner of Nanog and the interaction was investigated further. We show that the interaction is independent of DNA binding and that a region of Nanog known as tryptophan repeat, in which tryptophan is present every 5th residue is necessary and sufficient for the binding of Sox2. Furthermore, mutation of tryptophan residues within the Nanog tryptophan repeat (WR) abolishes the interaction with Sox2. A region of Sox2 known as serine rich region, a triple-repeat motif (S X T/S Y) within a stretch of 21 residues is required for the interaction with Nanog. Mutation of tyrosines to alanine within the three motifs (S X T/S Y) abrogates the Nanog–Sox2 interaction. The disruption of the Nanog-Sox2 interaction results in the alteration of expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines of the motif with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal. Together these data shed light on the extent of the interactions of Nanog with protein partners as well as the biochemical nature of the interaction between Nanog and one of the most important partners Sox2, an interaction crucial for maintaining optimal mouse ES cell self-renewal efficiency.
Published: 2014

6. Esrrb is a prominent target of Nanog that substitutes for Nanog function in ES cell self-renewal, reprogramming and germline development

Author: Festuccia, Nicola, Chambers, Ian, Lowell, Sally, and Tomlinson, Simon
Subjects: 572, transcriptional profiling, embryonic stem, Nanog, Estrogen-related receptor b, Esrrb
Abstract: Embryonic stem (ES) cell pluripotency is sustained by a network of transcription factors centred on Oct4, Sox2 and Nanog. Whilst Oct4 and Sox2 expression is relatively uniform, ES cells fluctuate between states of high Nanog expression possessing high self-renewal efficiency, and low Nanog expression exhibiting increased differentiation propensity. Moreover, modulation in the level of Nanog expression determines the efficiency of ES cell self-renewal. To identify genes regulated by Nanog, genome-wide transcriptional profiling was performed on ES cells expressing different Nanog levels and Nanog-null ES cells expressing a Nanog-ERT2 fusion protein in which nuclear Nanog activity can be regulated by tamoxifen. Surprisingly, only a minor fraction of the genes to which Nanog binds showed significant changes in response to Nanog induction. Prominent amongst Nanog-responsive genes is Estrogen-related receptor b (Esrrb). Nanog binds directly to Esrrb, enhances binding and pause-release of RNAPolII from the Esrrb promoter and stimulates Esrrb transcription. Consistent with these findings, elevation of Nanog produces a cell population that expresses uniformly high Esrrb levels. Moreover, double fluorescent reporter lines show that Esrrb and Nanog levels are strongly correlated in individual cells. Loss of Nanog is required for downregulation of Esrrb, which coincides with commitment to differentiate. Esrrb overexpression results in LIF independent self-renewal, and blocks neural differentiation, even in the absence of Nanog. Cell fusion experiments between ES and neural stem (NS) cells show that elevated Esrrb levels allow the reprogramming of the NS cell genome in the absence of Nanog. Esrrb can rescue stalled reprogramming during the derivation of Nanog-/- induced pluripotent stem (iPS) cells. Moreover, targeted knock-in of Esrrb at the Nanog locus rescues the ability of Nanog null ES cells to maintain germ cell development beyond E12. Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent selfrenewal to ES cells. Together these data identify Esrrb as a critical downstream mediator of Nanog function.
Published: 2013

7. Post-translational regulation of Nanog and Nanog-interacting proteins in mouse embryonic stem cells

Author: Roy, Marcia Michelle, Tyers, Mike, and Chambers, Ian
Subjects: 616.02, stem cells, LC-MS/MS, Nanog, small molecules, cell cycle, CRLs
Abstract: Pluripotent embryonic stem cells (ESCs) possess an unlimited capacity for self-renewal. This property of ES cells is both defining and unique. Harnessing this potential of ESCs would provide tremendous opportunity in the field of regenerative medicine and its attempts to combat degenerative diseases such as Parkinson’s, muscular dystrophy, etc. In 2006, Shinya Yamanaka was able to demonstrate that the ectopic expression of four proteins could reverse the process of differentiation and provide somatic cells with the characteristics ESCs. One year later, James Thompson’s group proved the same feat could be accomplished in human somatic cells using a different set of four proteins, including Nanog. The prospect of converting one’s own cells into a stem cell which could subsequently differentiate and repopulate an area of the body afflicted by gross degeneration was revolutionary. In the years following Yamanaka’s and Thompson’s discoveries, however, there has been little insight gained into how these proteins are regulated post-translationally. In this study, four proteins which had previously been identified by Yamanaka as being ‘pluripotency factors’ were used as baits in order to ascertain a protein-protein interaction network. This network was subsequently interrogated using various chemical compounds and small molecules in order to dissect the signal transduction pathways feeding into pluripotency, as well as, post-translational modifications regulating the factors themselves. In this way, the chemical inhibitor H89 was found to decrease the presence of Nanog phosphorylation and possibly its dimerization resulting in the Nanog protein being destabilized and targeted for degradation. Inversely, the pan-cullin inhibitor MLN4924 was identified to increase the abundance of both phosphorylated Nanog and total Nanog protein. In an attempt to identify the Cullin Ring Ligase (CRL) responsible for the degradation of Nanog protein in ESCs, each cullin identified in the protein interaction network was inhibited using specific shRNAs. Quantitative fluorescence microscopy was performed and identified that inhibition of CUL3 increases Nanog protein levels, suggesting that a CUL3-based CRL may be responsible for the post-translation regulation of Nanog. Additionally, the quantitation of Sox2 protein levels in CUL4B shRNA cell line demonstrates that Sox2 protein levels may be regulated by a CUL4B-based CRL. Further studies will reveal whether or not CUL4A depletion also results in elevated Sox2 protein levels. If not, this would include the pluripotency factor Sox2 among the recently identified CUL4B-isoform-specific substrates for degradation and possibly provide the basis for a hypothesis of developmentally regulated substrate specificity. In addition to MLN4924, several other small molecules were identified as being able to increase phospho-Nanog protein levels in this study. Among them were the cell permeable peptides Ht-31 and PKI (14-22) amide. These peptides were found to both stabilize phospho-Nanog and produce ES cell colonies that uniformly express the Nanog protein. The development of a growth medium containing these peptides in order to maintain homogeneous pluripotent ES cells is currently in progress and received backing for a patent application by the University of Edinburgh on February 23, 2012.
Published: 2012

8. Experimental approaches to establish rat embryonic stem cells

Author: Meek, Stephen Earl, Smith, Andrew., Chambers, Ian., and Buehr, Mia
Subjects: 571.6, ES cells, 2i, rat, Oct4, Nanog
Abstract: The rat has been an established experimental animal model within many areas of biological investigation for over one hundred years due to its size, breeding characteristics, and knowledge of its physiology and behaviour. In recent years its status as a leading biomedical model has been somewhat surpassed by the mouse. This is largely the result of the isolation and application of mouse embryonic stem (ES) cells. Mouse ES cells have the capacity for unlimited self-renew in vitro whilst maintaining pluripotency and germline competence, and most importantly are amenable to sophisticated reverse genetics strategies such as gene targeting, which have provided a route to germ line modification. Thus far, the derivation of rat ES cells has proved elusive. The generation of rat ES cells would therefore facilitate equivalent applications to rat genetics and significantly strengthen the rat as an experimental model system. Previous attempts to derive rat ES cells led to the isolation of rat ES-like cells. However, whilst these cells exhibit extensive self-renew in vitro, it was known that they fail to maintain significant levels of the key functional ES cell marker Oct4 and do not contribute to chimeras. Rather, these cells express the trophectoderm markers Cdx2 and CyclinD3, and have been termed ExS cells due to their probable extra-embryonic nature. In the work described in this thesis, further investigation of ExS cells revealed the absence of expression of the key pluripotency gene Nanog, although the expression pattern of Nanog in the rat embryo was shown to be similar to that of mouse. It was hypothesised that expression of exogenous Oct4 and Nanog or Sox2 genes could facilitate reprogramming of ExS cells into a 'true' ES cell state. Initial work described in this thesis demonstrated that it was possible to introduce transgenes into rat ExS cells and obtain stable transformants with long term transgene expression. On this basis Oct 4, Nanog and Sox2 transgene expression vectors were constructed and stably integrated into ExS cells, and transgene expression verified. However, no reactivation of an endogenous gene expression profile, characteristic of a true ES cell-like state, was observed in any of the transgenic lines produced. Concurrent with work on ExS cells, investigations by others using chemically defined, serum-free medium containing small molecule inhibitors of MEK and GSK3 (called 3i/2i medium) had demonstrated that it was possible to readily isolate mouse ES cells, even from strains known to be refractory to ES cell isolation. Therefore, the ability of this culture system to facilitate rat ES cell derivation was investigated. Rat 3i/2i cell lines were established from ICM outgrowths of Fischer, DA and Sprague Dawley E4.5 rat embryos. These cells maintained expression of Oct4 and Nanog and could generate complex teratomas consisting of all three germ layers. They were distinct from epiblast stem cells (EpiSC) in that they expressed Klf4, Rex1 and Stella and most importantly, they could contribute to the formation of adult chimaeras and demonstrated germline competency. Isolation of these authentic rat ES cells paves the way for gene targeting in the rat, a development that should greatly facilitate new biomedical discoveries.
Published: 2011

9. Dynamic control of Nanog expression in embryonic stem cells

Author: Karwacki-Neisius, Violetta Anna, Chambers, Ian, and Mee, Patrick
Subjects: 616.02, Nanog, stem cells
Abstract: Embryonic stem cells are defined by two key characteristics; apparently symmetrical self-renewing cell division and the ability to differentiate into cells of all three germ layers. Self-renewal depends on several extrinsic and intrinsic cues including a gene regulatory network centered around Oct4, Sox2 and Nanog that has been hypothesized to be reinforced by positive reciprocal interactions. Studies measuring Nanog expression by fluorescent reporters and immunoflourescence have shown that some undifferentiated Oct4 positive cells do not express Nanog (Chambers et al., 2007). However, the mechanisms responsible for generating this heterogeneity in Nanog expression are unknown. Here I show that Oct4 heterozygote ES cells lack Nanog-negative cells. Consistent with a model in which ES cell differentiation proceeds effectively through Nanog-negative cells, these Oct4 heterozygotes are retarded in their differentiation kinetics. Importantly, restoring Oct4 levels towards wild type reestablished both heterogenous Nanog expression and rapid differentiation. Analysis of ES cells carrying a mutation in the Oct4 binding site in the proximal Nanog promoter showed that Oct4 acts as a positive activator on the endogenous Nanog. Finally, comparison of gene expression in Nanog expressing and Nanog non-expressing ES cells has identified candidate genes that may be responsible for the switch in Nanog expression.
Published: 2011

10. Signal transduction mechanisms for stem cell differentation into cardiomyocytes

Author: Humphrey, Peter Saah
Subjects: 612.1, Akt, Protein Kinase B, Bisindolylmaleimide I, 8-Bromo-cGMP, Caffeic Acid Phenethyl Ester, CAPE, Cardiomyocytes, Differentiation, Embryonic Stem Cells, H9c2 (2-1), Ly294002, Myosin Chain Light Chain 1, MLC, Myosin Heavy Chain, MHC, Nanog, Nitric Oxide, Nkx2.5, NOC-18, NOC-5, Nuclear Factor-Kappa B, NF-kB, P19 Embryonal Carcinoma Cell, P19 Stem Cells, P38 MAPK, P38 Mitogen-Activated Protein Kinase, Phosphoinositide 3-Kinase, PI3K, Protein Kinase C, PKC, SB203580, Signal Transduction, Signalling, SIN-1, SNAP, Sox2, Transcription Factors, Troponin I, Wnt Proteins, GATA, Bone Morphogenetic Proteins, Leukaemia Inhibitory Factor, Oct-4, Octamer-Binding Protein
Abstract: Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.
Published: 2009

11. Caracterización de marcadores de células madre tumorales en cáncer escamocelular de cavidad oral

Author: Campuzano Castellanos, Marisol, García Flórez, Manuel, Campuzano Castellanos, Marisol, and García Flórez, Manuel
Abstract: Introduction: Squamous cell carcinoma of the oral cavity is a pathology with poor survival rates. When it is not adequately treated, it is a tumor with high recurrence and resistance to treatment. According to new hypotheses, progenitor tumor cells, due to their properties of self-renewal, tumor initiation, migration, and metastasis, could be responsible for the maintenance and renewal of this tumor. However, there is still no consensus on their true participation, subsequent to difficult in their identification and characterization. Materials and methods: In this research, 32 samples provided from patients diagnosis with squamous cell carcinoma of the oral cavity were used. To detect specific markers progenitor tumor cells were used immunofluorescence microscopy. Results: The cells markers OCT4, SSEA4, NANOG and TRA-1-60 were identified in the different stages of the tumor samples, all these findings suggest the role of tumor progenitor cells in the evolution of this pathology. Conclusions: The establishment and correct identification of the progenitor tumor cells provide new therapeutic options for the approach of this tumor seeking to improve the prognosis, survival rate and quality of life of the patient., Introducción: el cáncer escamocelular de cavidad oral es una patología con bajas tasas de sobrevivencia. Cuando no es tratado adecuadamente es un tumor de alta recurrencia y resistente al tratamiento. Nuevas hipótesis plantean que las células tumorales progenitoras por sus propiedades de auto renovación, iniciación tumoral, migración y metástasis pueden ser responsables de la manutención y renovación de este tumor. Sin embargo, aún no existe un consenso sobre la verdadera participación de ellas, debido a que su identificación y caracterización es aún un reto experimental. Objetivo: en este trabajo se busca detectar células con expresión de marcadores de células tumorales Progenitoras en muestras cáncer escamocelular de cavidad oral y relacionarlo con los estadios de diferenciación del tumor. Metodología: en esta investigación se tomaron 32 muestras de pacientes con carcinoma escamocelular de cavidad oral. Se logró detectar in situ, mediante la técnica de inmunofluorescencia, cuatroreconocidos marcadores de células tumorales progenitoras. Resultados: se identificaron los marcadores OCT4, SSEA4, NANOG yTRA-1-60 en los diferentes estadios de diferenciación tumoral, lo que sugiere la participación de las células progenitoras tumorales en la evolución de esta patología. Conclusiones: el establecimiento y correcta identificación de las células tumorales progenitoras abre nuevas vías terapéuticas para el abordaje de este tumor, en busca de mejorar el pronóstico, tasa de sobrevivencia y calidad de vida del paciente.
Published: 2023

12. Maternal Subclinical and Clinical Hypothyroidism Effects on Rat Offspring: A Story of the Skin and its Derivatives

Author: Luzajic Bozinovski, Tijana, Danilović Luković, Jelena, Nikolić, Anja, Radovanović, Anita, Marković, Danica, Kovačević Filipović, Milica, Vasić, Mirjana, Milošević, Ivan, Luzajic Bozinovski, Tijana, Danilović Luković, Jelena, Nikolić, Anja, Radovanović, Anita, Marković, Danica, Kovačević Filipović, Milica, Vasić, Mirjana, and Milošević, Ivan
Abstract: Epidermis stem cells have a crucial role through the processes of proliferation and differentiation, to replace cells that are constantly lost during tissue turnover or following injury. On the other hand, thyroid hormones regulate the proliferation and differentiation of epidermal cells and thus significantly influence the homeostasis of the skin. It is well known that maternal hypothyroidism during pregnancy leads to impaired development of many organ systems in their offspring. However, there is a lack of data about the influence of maternal subclinical hypothyroidism during pregnancy and lactation on the development of the skin and its derivatives in the litter. The aim of this study was to investigate the effects of maternal thyroid dysfunction on the development of the skin and its derivatives in their offspring in the early postnatal period. Antithyroid substance 6-n-propyl-2-thiouracil was added into the drinking water to female Albino Oxfords rats from the beginning of pregnancy and during lactation, with the aim to induce subclinical and overt form of hypothyroidism. Skin samples were taken from male pups within twenty-four hours and seven days after birth. The main findings of this investigation were that both forms of maternal hypothyroidism lead to serious damage of the epidermis in pups in terms of pronounced hyperkeratosis and reduction of the germinal layer along with a reduced number of hair follicles and their delayed morphogenesis. Epidermal impairments were more pronounced in pups with the overt form of hypothyroidism while offspring with the subclinical form had impairments that were less pronounced and delayed in occurrence.
Published: 2023

13. Epigenetic Regulation of Driver Genes in Testicular Tumorigenesis

Author: von Eyben, Finn E., Kristiansen, Karsten, Kapp, Daniel S., Hu, Rong, Preda, Ovidiu, Nogales, Francisco F., von Eyben, Finn E., Kristiansen, Karsten, Kapp, Daniel S., Hu, Rong, Preda, Ovidiu, and Nogales, Francisco F.
Abstract: In testicular germ cell tumor type II (TGCT), a seminoma subtype expresses an induced pluripotent stem cell (iPSC) panel with four upregulated genes, OCT4/POU5F1, SOX17, KLF4, and MYC, and embryonal carcinoma (EC) has four upregulated genes, OCT4/POU5F1, SOX2, LIN28, and NANOG. The EC panel can reprogram cells into iPSC, and both iPSC and EC can differentiate into teratoma. This review summarizes the literature on epigenetic regulation of the genes. Epigenetic mechanisms, such as methylations of cytosines on the DNA string and methylations and acetylations of histone 3 lysines, regulate expression of these driver genes between the TGCT subtypes. In TGCT, the driver genes contribute to well-known clinical characteristics and the driver genes are also important for aggressive subtypes of many other malignancies. In conclusion, epigenetic regulation of the driver genes are important for TGCT and for oncology in general., In testicular germ cell tumor type II (TGCT), a seminoma subtype expresses an induced pluripotent stem cell (iPSC) panel with four upregulated genes, OCT4/POU5F1, SOX17, KLF4, and MYC, and embryonal carcinoma (EC) has four upregulated genes, OCT4/POU5F1, SOX2, LIN28, and NANOG. The EC panel can reprogram cells into iPSC, and both iPSC and EC can differentiate into teratoma. This review summarizes the literature on epigenetic regulation of the genes. Epigenetic mechanisms, such as methylations of cytosines on the DNA string and methylations and acetylations of histone 3 lysines, regulate expression of these driver genes between the TGCT subtypes. In TGCT, the driver genes contribute to well-known clinical characteristics and the driver genes are also important for aggressive subtypes of many other malignancies. In conclusion, epigenetic regulation of the driver genes are important for TGCT and for oncology in general.
Published: 2023

14. Investigation of the mechanisms underlying triple-negative breast cancer radioresistance

Author: Li, Yong, Clinical School St George Hospital, Medicine & Health, UNSW, Ni, Jie, Clinical School St George Hospital, Medicine & Health, UNSW, Beretov, Julia, Clinical School St George Hospital, Medicine & Health, UNSW, Graham, Peter, Clinical School St George Hospital, Medicine & Health, UNSW, Zhu, Ying, Clinical School St George Hospital, Medicine & Health, UNSW, Bai, Xupeng, Clinical School St George Hospital, Medicine & Health, UNSW, Li, Yong, Clinical School St George Hospital, Medicine & Health, UNSW, Ni, Jie, Clinical School St George Hospital, Medicine & Health, UNSW, Beretov, Julia, Clinical School St George Hospital, Medicine & Health, UNSW, Graham, Peter, Clinical School St George Hospital, Medicine & Health, UNSW, Zhu, Ying, Clinical School St George Hospital, Medicine & Health, UNSW, and Bai, Xupeng, Clinical School St George Hospital, Medicine & Health, UNSW
Abstract: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and tends to develop a radioresistant phenotype. The underlying mechanism is not fully understood. In this study, three radioresistant TNBC cell lines were developed and the differentially expressed proteins between radioresistant and their parental cell lines were analysed using label-free quantitative proteomics. The data from canonical pathway core analysis showed that the integrated stress response (ISR) was the most activated signalling in radioresistant TNBC cells. Further investigation indicated that the constitutive phosphorylation of eIF2α in radioresistant TNBC cells promoted the activation of ATF4 and elicited the transcription of genes implicated in glutathione (GSH) biosynthesis, including glutamate-cysteine ligase catalytic subunit (GCLC), solute carrier family 7 member 11 (SLC7A11), and cystathionine γ-lyase (CTH), which increased the intracellular level of reduced GSH and the scavenging of reactive oxygen species after irradiation (IR), leading to a radioresistant phenotype. The cascade was significantly up-regulated in TNBC tissues and associated with unfavourable survival in TNBC patients. Dephosphorylation of eIF2α increased IR-induced cell apoptosis in radioresistant TNBC cells by disrupting ATF4-mediated GSH biosynthesis and sensitized radioresistant TNBC tumours to IR in vivo. On the other hand, cancer stem cells (CSCs) were found enriched in radioresistant TNBC cells, and THOC2 and THOC5 that are critical components of the THO complex in regulating embryogenesis were up-regulated in these cells and associated with a poor prognosis in TNBC patients. Further investigation revealed that THOC2 promoted the stem-like properties and radioresistance of TNBC in a THOC5-dependent manner by facilitating the releasing of SRY (sex-determining region Y)-box transcription factor 2 (SOX2) and NANOG homeobox transcription factor (NANOG) transcripts from the nuclei. Silencing
Published: 2021

15. An improved protocol for stable and efficient culturing of chicken primordial germ cells using small-molecule inhibitors

Author: Ezaki, Ryo, Hirose, Fumiya, Furusawa, Shuichi, Horiuchi, Hiroyuki, Ezaki, Ryo, Hirose, Fumiya, Furusawa, Shuichi, and Horiuchi, Hiroyuki
Abstract: At present, the most reliable method for creating genetically modified chickens is the modification of the DNA sequence of primordial germ cells (PGCs). However, during embryogenesis, only a small number of chicken PGCs can be obtained. Therefore, in vitro PGC culturing is necessary to obtain sufficient cells for further genetic engineering. Previously reported PGC culturing methods lack versatility. We report here a new protocol for stable and efficient culturing of chicken PGCs using small-molecule inhibitors. The growth rate of PGCs was investigated following the addition of three small-molecule inhibitors, including blebbistatin, into the culture medium. Chicken PGC survival and proliferation rates increased after the addition of small-molecule inhibitors, compared with the untreated control. Blebbistatin was shown to be the most effective inducer of PGC growth. Long-term culturing of PGCs with blebbistatin maintained the morphology of typical PGCs, and these cells expressed marker proteins such as chicken vasa homolog (CVH) and NANOG. Additionally, PGCs transfected with a fluorescent protein gene were shown to migrate into the gonads of the recipient embryo, and progeny derived from PGCs cultured by this method were efficiently obtained. These results demonstrate that small-molecule inhibitors represent a useful tool for stable and efficient chicken PGC culturing., This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant Numbers 19H03107, and 19K22286.
Published: 2020

16. Alu retrotransposons modulate Nanog expression through dynamic changes in regional chromatin conformation via aryl hydrocarbon receptor

Author: Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Junta de Extremadura, Red Temática de Investigación Cooperativa en Cáncer (España), Instituto de Salud Carlos III, González-Rico, Francisco J., Vicente-García, Cristina, Fernández, Almudena, Muñoz-Santos, Diego, Montoliu, Lluís, Morales-Hernández, Antonio, Merino, Jaime M., Román, Angel-Carlos, Fernández-Salguero, Pedro M., Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Junta de Extremadura, Red Temática de Investigación Cooperativa en Cáncer (España), Instituto de Salud Carlos III, González-Rico, Francisco J., Vicente-García, Cristina, Fernández, Almudena, Muñoz-Santos, Diego, Montoliu, Lluís, Morales-Hernández, Antonio, Merino, Jaime M., Román, Angel-Carlos, and Fernández-Salguero, Pedro M.
Abstract: Transcriptional repression of Nanog is an important hallmark of stem cell differentiation. Chromatin modifications have been linked to the epigenetic profile of the Nanog gene, but whether chromatin organization actually plays a causal role in Nanog regulation is still unclear. Here, we report that the formation of a chromatin loop in the Nanog locus is concomitant to its transcriptional downregulation during human NTERA-2 cell differentiation. We found that two Alu elements flanking the Nanog gene were bound by the aryl hydrocarbon receptor (AhR) and the insulator protein CTCF during cell differentiation. Such binding altered the profile of repressive histone modifications near Nanog likely leading to gene insulation through the formation of a chromatin loop between the two Alu elements. Using a dCAS9-guided proteomic screening, we found that interaction of the histone methyltransferase PRMT1 and the chromatin assembly factor CHAF1B with the Alu elements flanking Nanog was required for chromatin loop formation and Nanog repression. Therefore, our results uncover a chromatin-driven, retrotransposon-regulated mechanism for the control of Nanog expression during cell differentiation.
Published: 2020

17. Stem cell markers in oral and oropharyngeal squamous cell carcinomas in relation to the site of origin and HPV infection: Clinical implications

Author: Rizzo, Daniela, Graziani, C., Gallus, R., Zannoni, Gian Franco, Lucchetti, Donatella, Parrilla, Claudio, Boninsegna, A., Galli, Jacopo, Paludetti, Gaetano, Bussu, Francesco, Sgambato, Alessandro, Rizzo D. (ORCID:0000-0003-1809-5901), Zannoni G. F. (ORCID:0000-0003-1809-129X), Lucchetti D. (ORCID:0000-0001-8147-0079), Parrilla C., Galli J. (ORCID:0000-0001-6353-6249), Paludetti G. (ORCID:0000-0003-2480-1243), Bussu F. (ORCID:0000-0001-6261-2772), Sgambato A. (ORCID:0000-0002-9487-4563), Rizzo, Daniela, Graziani, C., Gallus, R., Zannoni, Gian Franco, Lucchetti, Donatella, Parrilla, Claudio, Boninsegna, A., Galli, Jacopo, Paludetti, Gaetano, Bussu, Francesco, Sgambato, Alessandro, Rizzo D. (ORCID:0000-0003-1809-5901), Zannoni G. F. (ORCID:0000-0003-1809-129X), Lucchetti D. (ORCID:0000-0001-8147-0079), Parrilla C., Galli J. (ORCID:0000-0001-6353-6249), Paludetti G. (ORCID:0000-0003-2480-1243), Bussu F. (ORCID:0000-0001-6261-2772), and Sgambato A. (ORCID:0000-0002-9487-4563)
Abstract: The expression of potential stem cell markers in HNSCCs was investigated to assess their potential clinical role. 69 primary, previously untreated oral (OSCC) and oropharyngeal squamous cell carcinomas (OPSCC) were enrolled; personal, clinical and follow-up data were collected. HPV infection and expression of 5 potential stem cell markers (CD44, CD133, Oct-4, Nanog, and Sox-2) were evaluated. HPV+ OPSCC showed lower expression of Nanog. The cytoplasmic expression of Nanog was associated with significantly worse prognosis in OPSCC, but not in OSCC. Sox-2 staining was more intense among OPSCCs. Sox-2 nuclear staining was associated with worse prognosis. Nanog expression was associated with HPV-OPSCC and may have a role as a surrogate diagnostic marker. In general, the expression profile of some stem cell markers in HNSCC seems to vary according to the site of origin and HPV infection. Nanog and Sox-2 may also have prognostic value.
Published: 2020

18. Rapid translocation of pluripotency-related transcription factors by external uniaxial forces.

Author: Topal, Tuğba, Topal, Tuğba, Kim, Byoung Choul, Villa-Diaz, Luis G, Deng, Cheri X, Takayama, Shuichi, Krebsbach, Paul H, Topal, Tuğba, Topal, Tuğba, Kim, Byoung Choul, Villa-Diaz, Luis G, Deng, Cheri X, Takayama, Shuichi, and Krebsbach, Paul H
Abstract: Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransduction-mediated bioengineering approaches to influencing stem cell fate determination.
Published: 2019

19. Nanog regulates Pou3f1 expression at the exit from pluripotency during gastrulation

Author: Medical Research Council (UK), MRC Cambridge Stem Cell Institute, Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), Centro Nacional de Investigaciones Cardiovasculares (España), Barral, Antonio, Rollan, isabel, Sánchez-Iranzo, Héctor, Jawaid, Wajid, Badia-Careaga, Claudio, Menchero, Sergio, Gómez, Manuel J., Torroja, Carlos, Sánchez-Cabo, Fátima, Göttgens, Berthold, Manzanares, Miguel, Sainz de Aja, Julio, Medical Research Council (UK), MRC Cambridge Stem Cell Institute, Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), Centro Nacional de Investigaciones Cardiovasculares (España), Barral, Antonio, Rollan, isabel, Sánchez-Iranzo, Héctor, Jawaid, Wajid, Badia-Careaga, Claudio, Menchero, Sergio, Gómez, Manuel J., Torroja, Carlos, Sánchez-Cabo, Fátima, Göttgens, Berthold, Manzanares, Miguel, and Sainz de Aja, Julio
Abstract: Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog. Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation.
Published: 2019

20. Nanog induced intermediate state in regulating stem cell differentiation and reprogramming.

Author: Yu, Peijia, Yu, Peijia, Nie, Qing, Tang, Chao, Zhang, Lei, Yu, Peijia, Yu, Peijia, Nie, Qing, Tang, Chao, and Zhang, Lei
Abstract: BackgroundHeterogeneous gene expressions of cells are widely observed in self-renewing pluripotent stem cells, suggesting possible coexistence of multiple cellular states with distinct characteristics. Though the elements regulating cellular states have been identified, the underlying dynamic mechanisms and the significance of such cellular heterogeneity remain elusive.ResultsWe present a gene regulatory network model to investigate the bimodal Nanog distribution in stem cells. Our model reveals a novel role of dynamic conversion between the cellular states of high and low Nanog levels. Model simulations demonstrate that the low-Nanog state benefits cell differentiation through serving as an intermediate state to reduce the barrier of transition. Interestingly, the existence of low-Nanog state dynamically slows down the reprogramming process, and additional Nanog activation is found to be essential to quickly attaining the fully reprogrammed cell state.ConclusionsNanog has been recognized as a critical pluripotency gene in stem cell regulation. Our modeling results quantitatively show a dual role of Nanog during stem cell differentiation and reprogramming, and the importance of the intermediate state during cell state transitions. Our approach offers a general method for analyzing key regulatory factors controlling cell differentiation and reprogramming.
Published: 2018

21. Functional Dissection of the Enhancer Repertoire in Human Embryonic Stem Cells

Author: Barakat, T.S. (Tahsin Stefan), Halbritter, F. (Florian), Zhang, M. (Man), Rendeiro, A.F. (André F.), Perenthaler, E. (Elena), Bock, G.H. (Geertruida) de, Chambers, I. (Ian), Barakat, T.S. (Tahsin Stefan), Halbritter, F. (Florian), Zhang, M. (Man), Rendeiro, A.F. (André F.), Perenthaler, E. (Elena), Bock, G.H. (Geertruida) de, and Chambers, I. (Ian)
Abstract: Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications functionally define active enhancers remains unclear. Here, we combine chromatin immunoprecipitation with a massively parallel reporter assay (ChIP-STARR-seq) to identify functional enhancers in human embryonic stem cells (ESCs) genome-wide in a quantitative unbiased manner. Although active enhancers associate with TFs, only a minority of regions marked by NANOG, OCT4, H3K27ac, and H3K4me1 function as enhancers, with activity markedly changing under naive versus primed culture conditions. We identify an enhancer set associated with functions extending to non-ESC-specific processes. Moreover, although transposable elements associate with putative enhancers, only some exhibit activity. Similarly, within super-enhancers, large tracts are non-functional, with activity restricted to small sub-domains. This catalog of validated enhancers provides a valuable resource for further functional dissection of the regulatory genome.Barakat et al. use a combination of chromatin immunoprecipitation and a massively parallel reporter assay to identify functional enhancers in primed and naive human embryonic stem cells. This genome-wide catalog of validated enhancers provides a valuable resource for the further dissection of the regulatory genome.
Published: 2018
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22. Thyroid hormones affect the proliferation and/or mobilization of bulge stem cell population

Author: Lužajić, Tijana, Milošević, Ivan, Danilović Luković, Jelena, Marković, Danica, Milošević, Sandra, Kovačević Filipović, Milica, Radovanovic, Anita, Lužajić, Tijana, Milošević, Ivan, Danilović Luković, Jelena, Marković, Danica, Milošević, Sandra, Kovačević Filipović, Milica, and Radovanovic, Anita
Abstract: Thyroid hormones (TH) play a pivotal role in the development of mammalian skin, and are necessary for both the initiation and maintenance of hair growth. Adult hypothyroid rats showed impaired epidermal proliferation, hair growth and wound healing. Stem cells (SCs) located at the bulges of the hair follicles are responsible for hair cycling, and contribute to the regeneration of the new epidermis after wounding. Therefore a reduction in the number or function of bulge stem cells could be a cause of disturbed skin reepithelization and hair follicle maintenance. We hypothesized that a decrease in TH during prenatal development would affect the bulge stem cell population. Subclinical hypothyroidism was induced with propylthiouracil through drinking water in a dose of 1.5 mg/l in pregnant Albino Oxford rats, from the first day of gravidity and during lactation. The control group was untreated. Five, seven-day-old pups were euthanized and skin samples were taken from the dorsal part of their bodies. The number of hair follicles was estimated on an area of 1mm2 of dermis. For the immunohistochemical study, proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology) and NANOG (Thermo Scientific) antibodies were used. The number of PCNA and NANOG positive cells was estimated on the area of 1mm2 of hair follicle. The number of hair follicles was reduced and the expression of PCNA was decreased in the cells of the inner and outer sheath of hair follicles in the hypothyroid pups compared to the controls. They also had an increased number of NANOG positive bulge cells which we presume are multipotent SCs. Decreased expression of PCNA demonstrates low proliferation and increased expression of NANOG, which may be explained in two ways: 1) the inhibited differentiation of putative SCs and/or 2) the reduction of mobilization of these cells to the epidermis or sebaceous gland. Our results confirm that the lack of TH influences the formation of hair follicles in early infant
Published: 2017

23. Histone modifications on the promoters of human OCT4 and NANOG genes at the onset of neural differentiation of NT2/D1 cells

Author: Topalović, Vladanka, Schwirtlich, Marija, Stevanović, Milena, Mojsin, Marija, Topalović, Vladanka, Schwirtlich, Marija, Stevanović, Milena, and Mojsin, Marija
Abstract: Transcription factors OCT4 and NANOG are main constituents of a functional network that controls proliferation and pluripotency maintenance of stem cells as well as early lineage decisions. We investigated expression profiles of OCT4 and NANOG during the early phases of neural differentiation using NT2/D1 cells induced by retinoic acid as an in vitro model system of human neurogenesis. We demonstrated decrease in OCT4 and NANOG mRNA and protein levels following exposure to retinoic acid. Next, by employing chromatin immunoprecipitation, we investigated profiles of selected H3 and H2B histone marks deposited on the promoters of the OCT4 and NANOG genes. We found decline in H3K4me3, H2BK5ac, and H2BK120ac on both promoters, which paralleled the decrease in OCT4 and NANOG expression. Moreover, we found that the H2BK16ac mark is differentially enriched on these two promoters, pointing to differences in epigenetic regulation of OCT4 and NANOG gene expression. Finally, based on our data, we suggest that the early response of pluripotency genes OCT4 and NANOG to the differentiation-inducing stimuli is mediated by dynamic changes in chromatin marks, while DNA methylation is acquired in the later stages of neurogenesis.
Published: 2017

24. Yin Yang 1 is associated with cancer stem cell transcription factors (SOX2, OCT4, BMI1) and clinical implication.

Author: Kaufhold, Samantha, Kaufhold, Samantha, Garbán, Hermes, Bonavida, Benjamin, Kaufhold, Samantha, Kaufhold, Samantha, Garbán, Hermes, and Bonavida, Benjamin
Abstract: The transcription factor Yin Yang 1 (YY1) is frequently overexpressed in cancerous tissues compared to normal tissues and has regulatory roles in cell proliferation, cell viability, epithelial-mesenchymal transition, metastasis and drug/immune resistance. YY1 shares many properties with cancer stem cells (CSCs) that drive tumorigenesis, metastasis and drug resistance and are regulated by overexpression of certain transcription factors, including SOX2, OCT4 (POU5F1), BMI1 and NANOG. Based on these similarities, it was expected that YY1 expression would be associated with SOX2, OCT4, BMI1, and NANOG's expressions and activities. Data mining from the proteomic tissue-based datasets from the Human Protein Atlas were used for protein expression patterns of YY1 and the four CSC markers in 17 types of cancer, including both solid and hematological malignancies. A close association was revealed between the frequency of expressions of YY1 and SOX2 as well as SOX2 and OCT4 in all cancers analyzed. Two types of dynamics were identified based on the nature of their association, namely, inverse or direct, between YY1 and SOX2. These two dynamics define distinctive patterns of BMI1 and OCT4 expressions. The relationship between YY1 and SOX2 expressions as well as the expressions of BMI1 and OCT4 resulted in the classification of four groups of cancers with distinct molecular signatures: (1) Prostate, lung, cervical, endometrial, ovarian and glioma cancers (YY1(lo)SOX2(hi)BMI1(hi)OCT4(hi)) (2) Skin, testis and breast cancers (YY1(hi)SOX2(lo)BMI1(hi)OCT4(hi)) (3) Liver, stomach, renal, pancreatic and urothelial cancers (YY1(lo)SOX2(lo)BMI1(hi)OCT4(hi)) and (4) Colorectal cancer, lymphoma and melanoma (YY1(hi)SOX2(hi)BMI1(lo)OCT4(hi)). A regulatory loop is proposed consisting of the cross-talk between the NF-kB/PI3K/AKT pathways and the downstream inter-regulation of target gene products YY1, OCT4, SOX2 and BMI1.
Published: 2016

25. Inmunodetección de la proteina nanog en embriones preimplantacionales producidos in vitro por fiv y partenogénesis en pequeños rumiantes

Author: Vicente Antón, José Salvador, Paramio Nieto, Maria Teresa, Universitat Politècnica de València. Departamento de Ciencia Animal - Departament de Ciència Animal, Martinez María, Patricia, Vicente Antón, José Salvador, Paramio Nieto, Maria Teresa, Universitat Politècnica de València. Departamento de Ciencia Animal - Departament de Ciència Animal, and Martinez María, Patricia
Abstract: [EN] Since the development of techniques for in vitro embryo production there is an important demand of methods to assess the quality of the generated embryos. One of the main criteria to assess the quality of produced blastocysts is the ratio between the number of inner cell mass (ICM) and trophectoderm (TE). Nanog and Cdx2 proteins are founded in the ICM cells and TE cells respectively in mouse embryos. The aim of this work was tune up, in our laboratory, the immunodetection analysis of Nanog protein in goat and sheep embryos produced by in vitro fertilization (IVF) and parthenogenesis. We have produced 640 ovine embryos by IVF and 1083 by parthenogenetic activation (PA). In caprine we produced 205 embryos by IVF and 66 by PA. All produced embryos are grouped in three different groups: 2 to 8 cells embryos, morulae, and blastocysts. We used the routine techniques of laboratory to carry out IVF and PA. For the Nanog protein analysis we used 286 embryos and different protocols were performed to optimize the suitable one for sheep and goats. By using the original immunodetection protocol (mouse), it was observed that Nanog protein needed an optimization by changing the primary antibody from the original (polyclonal rabbit anti-‐Nanog) to the human one (rabbit anti-‐Human Nanog). We found expression of Nanog protein in each cell in: 2-‐8 cells embryos, morulae and blastocysts. It was contrary to our expectations considering that the mouse Nanog protein is localized, in blastocysts, only in the ICM cells and it is not found in the previous embryonic stages. Our produced blastocysts shows Nanog signal in 66’7% and 89’6% en sheep, and 83’6% and 60’5% in goat of total cells, which indicates that this signal was also localized in the TE cells and not only on the ICM, as it was expected. The Cdx2 was located in 68’97% of cells in the blastocyst stage, indicating that the labeling was correct only in the TE. In conclusion, our data do not show significant differences betw, [ES] Desde que se desarrollaron las técnicas de producción de embriones in vitro existe una demanda de métodos que permiten evaluar la calidad de los embriones generados. Uno de los criterios de la calidad de los blastocistos producidos es la relación entre el número de células del Masa Celular Interna (MCI) y del Trofoectodermo (TE). La proteína Nanog y la Cdx2, son proteínas que se encuentras en la MCI y TE, respectivamente en embriones de ratón. El objetivo de este trabajo fue poner a punto en nuestro laboratorio en embriones caprinos y ovinos producidos por fecundación in vitro (FIV) y partenogénesis, el análisis de inmunodetección de la proteína Nanog. Producimos 640 embriones ovinos mediante FIV y 1083 embriones por activación partenogenética (AP). En caprino producimos 205 embriones por FIV y 66 por AP. Los embriones producidos son agrupados en tres grupos: embriones de 2 a 8 células, mórulas y blastocistos. Las técnicas utilizadas para la FIV y AP son las rutinarias del laboratorio. Para el análisis de la proteína Nanog utilizamos 286 embriones con diferentes protocolos hasta optimizar el adecuado para ovino y caprino. Utilizando el protocolo de inmunodetección original (de ratón), se observó que la proteína Nanog necesitó una optimización para los embriones de oveja y cabra, cambiando el anticuerpo primario original rabbit polyclonal anti-‐Nanog por rabbit anti-‐Human Nanog. Encontramos expresión de la proteína Nanog en cada célula de embriones de 2-‐8 células, mórulas y blastocistos, contrariamente a lo esperado ya que la proteína Nanog de ratón solo se localiza en las células MCI y no en los anteriores estadios embrionarios. En nuestros blastocistos producidos se observa que el número de células con señal Nanog es del 66’7% y 89’6% en ovino, y 83’6% y 60’5% en caprino del total de células, lo que indica que esta señal estuvo localizada también en las células del TE y no solo en las del MCI, como era lo esperado. El Cdx2 se localizó en el 68’97% de las
Published: 2016

26. Genome Binding and Gene Regulation by Stem Cell Transcription Factors

Author: Brandsma, J.H. (Johan) and Brandsma, J.H. (Johan)
Abstract: Nearly all cells of an individual organism contain the same genome. However, each cell type transcribes a different set of genes due to the presence of different sets of cell type-specific transcription factors. Such transcription factors bind to regulatory regions such as promoters and enhancers and regulate their activity in gene transcription. Transcription factors interact with each other and form tissue-specific transcription factor networks. Identification of genome binding and gene regulation by transcription factors will enhance our understanding of how transcription factors specify cell types. Chapter 1 serves as a general introduction into eukaryotic transcription regulation and describes the role of enhancers and transcription factors. Chapters 2 – 4 describe the experimental work of this thesis. Chapter
Published: 2016

27. A class of genes in the HER2 regulon that is poised for transcription in breast cancer cell lines and expressed in human breast tumors.

Author: Rahmatpanah, Farah B, Rahmatpanah, Farah B, Jia, Zhenyu, Chen, Xin, Char, Jessica E, Men, Bozhao, Franke, Anna-Clara, Jones, Frank E, McClelland, Michael, Mercola, Dan, Rahmatpanah, Farah B, Rahmatpanah, Farah B, Jia, Zhenyu, Chen, Xin, Char, Jessica E, Men, Bozhao, Franke, Anna-Clara, Jones, Frank E, McClelland, Michael, and Mercola, Dan
Abstract: HER2-positive breast cancer accounts for 25% of all cases and has a poor prognosis. Although progress has been made in understanding signal transduction, little is known of how HER2 achieves gene regulation. We performed whole genome expression analysis on a HER2⁺ and HER2⁻ breast cancer cell lines and compared these results to expression in 812 primary tumors stratified by their HER2 expression level. Chip-on-chip with anti-RNA polymerase II was compared among breast cancer cell lines to identify genes that are potentially activated by HER2. The expression levels of these HER2-dependent POL II binding genes were determined for the 812 HER2+/- breast cancer tissues. Genes differentially expressed between HER2+/- cell lines were generally regulated in the same direction as in breast cancer tissues. We identified genes that had POLII binding in HER2⁺ cell lines, but without significant gene expression. Of 737 such genes "poised" for expression in cell lines, 113 genes were significantly differentially expressed in breast tumors in a HER2-dependent manner. Pathway analysis of these 113 genes revealed that a large group of genes were associated with stem cell and progenitor cell control as indicated by networks centered on NANOG, SOX2, OCT3/4. HER2 directs POL II binding to a large number of genes in breast cancer cells. A "poised" class of genes in HER2⁺ cell lines with POLII binding and low RNA expression but is differentially expressed in primary tumors, strongly suggests a role of the microenvironment and further suggests a role for stem cells proliferation in HER2-regulated breast cancer tissue.
Published: 2015

28. Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells.

Author: Hayashi, Yohei, Hayashi, Yohei, Caboni, Laura, Das, Debanu, Yumoto, Fumiaki, Clayton, Thomas, Deller, Marc C, Nguyen, Phuong, Farr, Carol L, Chiu, Hsiu-Ju, Miller, Mitchell D, Elsliger, Marc-André, Deacon, Ashley M, Godzik, Adam, Lesley, Scott A, Tomoda, Kiichiro, Conklin, Bruce R, Wilson, Ian A, Yamanaka, Shinya, Fletterick, Robert J, Hayashi, Yohei, Hayashi, Yohei, Caboni, Laura, Das, Debanu, Yumoto, Fumiaki, Clayton, Thomas, Deller, Marc C, Nguyen, Phuong, Farr, Carol L, Chiu, Hsiu-Ju, Miller, Mitchell D, Elsliger, Marc-André, Deacon, Ashley M, Godzik, Adam, Lesley, Scott A, Tomoda, Kiichiro, Conklin, Bruce R, Wilson, Ian A, Yamanaka, Shinya, and Fletterick, Robert J
Abstract: NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein-DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.
Published: 2015

29. Klinički tok karcinoma jajnika u zavisnosti od patohistoloških i imunohistohemijskih karakteristika tumora

Author: Stanojević, Zorica, Vrbić, Svetislav, Janković-Veličković, Ljubinka, Jovanović, Darjana, Krstić, Miljan, Conić, Irena, Stanojević, Zorica, Vrbić, Svetislav, Janković-Veličković, Ljubinka, Jovanović, Darjana, Krstić, Miljan, and Conić, Irena
Abstract: Introduction: According to the frequency of malignant tumours in the world, ovarian carcinoma takes the eighth place; it is also the fifth most frequent tumour in women and the fourth cause of cancer death in women. In early discovering and treatment of ovarian carcinoma, before it expends out of the primary localisation, the five-year relative survival rate is 92%. Unfortunately, only 15% of all ovarian carcinomas are discovered in the early stage. It has been noticed that the survival rate is higher in women under the age of 65 than in the older ones and it varies depending on the morphological type of ovarian carcinoma, patient’s over-all health condition and the stage of the disease diffusion at the time of setting the diagnosis. The exact cause of ovarian carcinoma occurrence is still unknown, but many risk factors are related to the occurrence of this malignoma. The aim of this research was to analyze the clinical and pathological characteristics of ovarian carcinoma, as well as to determine Nanog, Sox2, Oct3/4, Sox4, CD44. CD117, Ezh2 and Stat3 expressions in paraffin sections of ovarian carcinoma and to compare the Nanog, Sox2, Oct3/4, Sox4, CD44, Ezh2 and Stat3 expressions with the clinical and pathological characteristics of ovarian carcinoma. Methods: During the research, the immunohistochemical method was applied. Paraffin sections of the tissue of ovarian carcinoma 5μm thick were used. Conclusion: Unfavourable clinical course of ovarian carcinoma was related to the positive Nanog, Sox2 and Oct3/4 expression, as well as with the absence or low expression of Sox4, CD117 and CD44, while the loss of Ezh2 and Stat3 expressions in ovarian carcinoma, indicated significantly longer survival.
Published: 2014

30. Hepatitis C virus core protein regulates NANOG expression via the stat3 pathway

Author: Zhou, Jia-Jia, Chen, Ru-Fu, Deng, Xiao-Geng, Zhou, Yu, Ye, Xiao, Yu, Min, Tang, Jing, He, Xiao-Yu, Cheng, Di, Zeng, Bing, Zhou, Quan-bo, Li, Zhi-hua, Zhou, Jia-Jia, Chen, Ru-Fu, Deng, Xiao-Geng, Zhou, Yu, Ye, Xiao, Yu, Min, Tang, Jing, He, Xiao-Yu, Cheng, Di, Zeng, Bing, Zhou, Quan-bo, and Li, Zhi-hua
Abstract: HCV Core plays a role in the development of hepatocellular carcinoma. Aberrant expression of NANOG has been observed in many types of human malignancies. However, relationship between Core and NANOG has not been clarified. In this study, we found that Core is capable of up-regulating NANOG expression. Core-induced NANOG expression was accompanied by enforced expression of phosphorylated stat3 protein and was attenuated by inhibition of stat3 phosphorylation. ChIP showed that phosphorylated stat3 directly binds to the NANOG promoter. Core-induced NANOG expression resulted in enhanced cell growth and cell cycle progression. Knockdown of NANOG blocked the cell cycle at the G0/G1 phases and inhibited the cyclin D1 expression. Our findings provide a new insight into the mechanism of hepatocarcinogenesis by HCV infection. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Published: 2014

31. TET2 deficiency inhibits mesoderm and hematopoietic differentiation in human embryonic stem cells.

Author: Langlois, Thierry, Da Costa Reis Monte-Mor, Barbara, Lenglet, Gaëlle, Droin, Nathalie, Marty, Caroline, Le Couédic, Jean Pierre, Almire, Carole, Auger, Nathalie, Mercher, Thomas, Delhommeau, François, Christensen, Jesper, Helin, Kristian, Debili, Najet, Fuks, François, Bernard, Olivier A, Solary, Eric, Vainchenker, William, Plo, Isabelle, Langlois, Thierry, Da Costa Reis Monte-Mor, Barbara, Lenglet, Gaëlle, Droin, Nathalie, Marty, Caroline, Le Couédic, Jean Pierre, Almire, Carole, Auger, Nathalie, Mercher, Thomas, Delhommeau, François, Christensen, Jesper, Helin, Kristian, Debili, Najet, Fuks, François, Bernard, Olivier A, Solary, Eric, Vainchenker, William, and Plo, Isabelle
Abstract: Ten-eleven-translocation 2 (TET2) belongs to the TET protein family that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and plays a central role in normal and malignant adult hematopoiesis. Yet the role of TET2 in human hematopoietic development remains largely unknown. Here, we show that TET2 expression is low in human embryonic stem cell (ESC) lines and increases during hematopoietic differentiation. shRNA-mediated TET2 knockdown had no effect on the pluripotency of various ESCs. However, it skewed their differentiation into neuroectoderm at the expense of endoderm and mesoderm both in vitro and in vivo. These effects were rescued by reintroducing the targeted TET2 protein. Moreover, TET2-driven differentiation was dependent on NANOG transcriptional factor. Indeed, TET2 bound to NANOG promoter and in TET2-deficient cells the methylation of the NANOG promoter correlated with a decreased in NANOG expression. The altered differentiation resulting from TET2 knockdown in ESCs led to a decrease in both the number and the cloning capacities of hematopoietic progenitors. These defects were due to an increased apoptosis and an altered gene expression profile, including abnormal expression of neuronal genes. Intriguingly, when TET2 was knockdown in hematopoietic cells, it increased hematopoietic development. In conclusion, our work suggests that TET2 is involved in different stages of human embryonic development, including induction of the mesoderm and hematopoietic differentiation., info:eu-repo/semantics/published
Published: 2014

32. Stem cell marker (Nanog) and Stat-3 signaling promote MicroRNA-21 expression and chemoresistance in hyaluronan/CD44-activated head and neck squamous cell carcinoma cells.

Author: Bourguignon, LYW, Bourguignon, LYW, Earle, C, Wong, G, Spevak, CC, Krueger, K, Bourguignon, LYW, Bourguignon, LYW, Earle, C, Wong, G, Spevak, CC, and Krueger, K
Abstract: MicroRNAs are often associated with the pathogenesis of many cancers, including head and neck squamous cell carcinoma (HNSCC). In particular, microRNA-21 (miR-21) appears to have a critical role in tumor cell survival, chemoresistance and HNSCC progression. In this study, we investigated matrix hyaluronan (HA)-induced CD44 (a primary HA receptor) interaction with the stem cell markers, Nanog and Stat-3, in HNSCC cells (HSC-3 cells). Our results indicate that HA binding to CD44 promotes Nanog-Stat-3 (also tyrosine phosphorylated Stat-3) complex formation, nuclear translocation and transcriptional activation. Further analyses reveal that miR-21 is controlled by an upstream promoter containing Stat-3 binding site(s), while chromatin immunoprecipitation assays demonstrate that stimulation of miR-21 expression by HA/CD44 signaling is Nanog/Stat-3-dependent in HNSCC cells. This process results in a decrease of a tumor suppressor protein (PDCD4), and an upregulation of i nhibitors of the apoptosis family of proteins (IAPs) as well as chemoresistance in HSC-3 cells. Treatment of HSC-3 cells with Nanog- and/or Stat-3-specific small interfering RNAs effectively blocks HA-mediated Nanog-Stat-3 signaling events, abrogates miR-21 production and increases PDCD4 expression. Subsequently, this Nanog-Stat-3 signaling inhibition causes downregulation of survival protein (IAP) expression and enhancement of chemosensitivity. To further evaluate the role of miR-21 in tumor cell-specific functions, HSC-3 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and block its target functions. Our results demonstrate that anti-miR-21 inhibitor not only upregulates PDCD4 expression but also decreases IAP expression and enhances chemosensitivity in HA-treated HNSCC cells. Together, these findings indicate that the HA-induced CD44 interaction with Nanog and Stat-3 has a pivotal role in miR-21 production leading to PDCD4 reduction, IAP upregulatio
Published: 2012

33. Induction of NANOG expression by targeting promoter sequence with small activating RNA antagonizes retinoic acid-induced differentiation.

Author: Wang, Xiaoling, Wang, Xiaoling, Wang, Ji, Huang, Vera, Place, Robert F, Li, Long-Cheng, Wang, Xiaoling, Wang, Xiaoling, Wang, Ji, Huang, Vera, Place, Robert F, and Li, Long-Cheng
Abstract: RNAa (RNA activation) is a mechanism by which small dsRNA (double-stranded RNA), termed saRNA (small activating RNA), target promoter sequences to induce gene expression. This technique represents a novel approach to gene overexpression without the use of exogenous DNA. In the present study, we investigated whether RNAa can modulate expression of the development-related gene NANOG and manipulate cell fate. Using a lentivirus-based reporter system as a screening tool, we identified synthetic saRNAs that stimulate NANOG expression in human NCCIT embryonic carcinoma cells. Mismatch mutations to saRNA duplexes define sequence requirement for gene activation. Functional analysis of NANOG induction reveals saRNA treatment predictably modulates the expression of several known downstream target genes, including FOXH1 (forkhead box H1), REST (RE1-silencing transcription factor), OCT4 (octamer-binding protein 4) and REX1 (reduced expression protein 1). Treatment with RA (retinoic acid) triggers NCCIT cell differentiation, reducing NANOG and OCT4 expression and up-regulating several neural markers [i.e. ASCL1 (achaete-scute complex homologue 1), NEUROD1 (neuronal differentiation 1) and PAX6 (paired box 6)]. However, co-treatment with saRNA antagonizes NANOG down-regulation and RA-induced differentiation. Ectopic overexpression of NANOG via lentiviral transduction further recapitulates saRNA results, providing proof-of-concept that RNAa may be utilized to activate development-related genes and manipulate cell fate.
Published: 2012

34. Transgenic mouse analysis of Sry expression during the pre- and peri-implantation stage

Author: Silversides, David W., Raiwet, Diana L., Souchkova, Ouliana, Viger, Robert S., Pilon, Nicolas, Silversides, David W., Raiwet, Diana L., Souchkova, Ouliana, Viger, Robert S., and Pilon, Nicolas
Abstract: Background: The SRY/Sry gene is expressed in pre-Sertoli cells of the male genital ridge and functions as the mammalian testis determining factor (TDF). In addition, expression of SRY/Sry outside the genital ridge has been reported, including preimplantation embryos, although the functional significance of this is not well understood. Results: Using Cre-mediated lineage studies and transgenic reporter mouse models, we now show that promoter sequences of human, pig and mouse SRY drive robust reporter gene expression in epiblast cells of peri-implantation embryos between embryonic day (E) 4.5 and E6.5. Analysis of endogenous Sry expression revealed that linear transcripts are produced by means of multiple polyadenylation sites in E4.5 embryos. Within the epiblast, SRY reporter expression mimics the expression seen using a Gata4 reporter model, but is dissimilar to that seen using an Oct4 reporter model. In addition, we report that overexpression of mouse Sry in embryonic stem cells leads to down-regulation of the core pluripotency markers Sox2 and Nanog. Conclusion: We propose that SRY/Sry may function as a male-specific maturation factor in the peri-implantation mammalian embryo, providing a genetic mechanism to help explain the observation that male embryos are developmentally more advanced compared with female embryos, and suggesting a role for SRY beyond that of TDF.
Published: 2012

35. Establishment and initial characterization of SOX2-overexpressing NT2/D1 cell clones

Author: Drakulić, Danijela, Krstić, A., Stevanović, Milena, Drakulić, Danijela, Krstić, A., and Stevanović, Milena
Abstract: SOX2, a universal marker of pluripotent stem cells, is a transcription factor that helps control embryonic development in vertebrates; its expression persists in neural stem/progenitor cells into adulthood. Considering the critical role of the SOX2 transcription factor in the regulation of genes required for self-renewal and pluripotency of stem cells, we developed and characterized SOX2-overexpressing NT2/D1 cell clones. Using Southern blot and semi-quantitative RT-PCR, we confirmed integration and expression of exogenous SOX2 in three NT2/D1 cell clones. Overexpression of the SOX2 gene was detected in two of these clones. SOX2 overexpression in NT2/D1 cell clones resulted in altered expression of key pluripotency genes OCT4 and NANOG. Furthermore, SOX2-overexpressing NT2/D1 cell clones entered into retinoic acid-dependent neural differentiation, even when there was elevated SOX2 expression. After 21 days of induction by retinoic acid, expression of neural markers (neuroD1 and synaptophysin) was higher in induced cell clones than in induced parental cells. The cell clone with SOX2 overexpression had an approximately 1.3-fold higher growth rate compared to parental cells. SOX2 overexpression did not increase the population of cells undergoing apoptosis. Taken together, we developed two SOX2-overexpressing cell clones, with constitutive SOX2 expression after three weeks of retinoic acid treatment. SOX2 overexpression resulted in altered expression of pluripotency-related genes, increased proliferation, and altered expression of neural markers after three weeks of retinoic acid treatment.
Published: 2012

36. Enclosed Microfluidic Platform Realizing Enhanced Stem-cell Survival

Author: Zhang, Yolanda Yue, Tirrell, Matthew V1, Zhang, Yolanda Yue, Zhang, Yolanda Yue, Tirrell, Matthew V1, and Zhang, Yolanda Yue
Abstract: Here we present a microfluidic culture platform for enhancing single stem cell survival. Traditional plate culture is inadequate for large scale single cell studies because of (1) less than 40% single stem cell survival leading to (2) possible inaccuracies in the biostatistics in single cell studies from lack of sufficient data. This platform mitigates these issues by doubling overall survival rates as well as increasing the available data points by two orders of magnitude, both factors which improve statistical certainty. The platform is fabricated using widely accepted biocompatible polymers and incorporates the novel integration of an enclosed microwell array with an upstream linear gradient generator. The device can selectively trap single cells, tightly control intercell spacing, and change the chemical microenvironment around the stem cells in real-time. In this dissertation, we first characterize the performance of the platform in improving single cell survivability. Subsequently, the system was tested using two difference stem cell types: mouse embryonic stem cells (mES) and induced pluripotent stem cells (iPS). Using this platform we are able to highlight the thresholds of leukemia inhibitory factor concentrations which lead to metastable gene expression of Nanog, a key stem cell pluripotency regulator in mES cells. The enhanced survival of single cells has also enabled the statistically significant observation of Nanog+, non-proliferative, single mES cells which are previously unreported in literature.
Published: 2011

37. Three-dimensional localisation of NANOG, OCT4, and E-CADHERIN in porcine pre- and peri-implantation embryos

Author: Wolf, Xenia Asbæk, Serup, Palle, Hyttel, Poul, Wolf, Xenia Asbæk, Serup, Palle, and Hyttel, Poul
Abstract: The expression patterns of NANOG and OCT4 have previously been reported to differ markedly between mammalian species indicating distinct species-specific roles during development. We investigate the three-dimensional expression pattern of NANOG and OCT4 in porcine pre- and peri-implantation embryos. The expression of NANOG differed remarkably from that reported in other species. NANOG was not detected in the inner cell mass of hatched porcine blastocysts, but later appeared in the epiblast and hypoblast of spherical blastocysts where Rauber's layer had disintegrated. In pre-gastrulating, filamentous embryos NANOG was localised to nuclei in a minor portion of the epiblast cells in which E-CADHERIN seemed to be up-regulated and OCT4 down-regulated. Later NANOG was restricted to the potential PGCs. OCT4 was detected in inner cell mass, epiblast, and mesoderm, and we found that OCT4 expression, in contrast to earlier speculations, at least in hatched blastocysts, resembles the expression pattern in the mouse embryo.
Published: 2011

38. The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells

Author: Région Auvergne-Rhône-Alpes, Biotechnology and Biological Sciences Research Council (UK), National Institutes of Health (US), European Commission, Lavial, Fabrice, Acloque, Hervé, Bertocchini, Federica, MacLeod, David J., Boast, Sharon, Bachelard, Élodie, Montillet, Guillaume, Thenot, Sandrine, Sang, Helen M., Stern, Claudio D., Samarut, Jacques, Pain, Bertrand, Région Auvergne-Rhône-Alpes, Biotechnology and Biological Sciences Research Council (UK), National Institutes of Health (US), European Commission, Lavial, Fabrice, Acloque, Hervé, Bertocchini, Federica, MacLeod, David J., Boast, Sharon, Bachelard, Élodie, Montillet, Guillaume, Thenot, Sandrine, Sang, Helen M., Stern, Claudio D., Samarut, Jacques, and Pain, Bertrand
Abstract: Embryonic stem cells (ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self-renew has been shown to be governed by the transcription factors Oct4 (Pou5f1) and Nanog. Oct4 appears to control cell-fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In non-mammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 (spg; pou5f1) and Xenopus Pou91 (XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC (cESC), which display similar properties of pluripotency and long-term self-renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV (cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self-renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self-renewal are not exclusive to mammals.
Published: 2007

39. Functional analysis of Sox15 and Sox2

Author: Maruyama, Masayoshi and Maruyama, Masayoshi
Abstract: http://library.naist.jp/mylimedio/dllimedio/show.cgi?bookid=83902
Published: 2006

40. Transgenic mouse analysis of Sry expression during the pre- and peri-implantation stage

Author: Silversides, David W., Raiwet, Diana L., Souchkova, Ouliana, Viger, Robert S., Pilon, Nicolas, Silversides, David W., Raiwet, Diana L., Souchkova, Ouliana, Viger, Robert S., and Pilon, Nicolas
Abstract: Background: The SRY/Sry gene is expressed in pre-Sertoli cells of the male genital ridge and functions as the mammalian testis determining factor (TDF). In addition, expression of SRY/Sry outside the genital ridge has been reported, including preimplantation embryos, although the functional significance of this is not well understood. Results: Using Cre-mediated lineage studies and transgenic reporter mouse models, we now show that promoter sequences of human, pig and mouse SRY drive robust reporter gene expression in epiblast cells of peri-implantation embryos between embryonic day (E) 4.5 and E6.5. Analysis of endogenous Sry expression revealed that linear transcripts are produced by means of multiple polyadenylation sites in E4.5 embryos. Within the epiblast, SRY reporter expression mimics the expression seen using a Gata4 reporter model, but is dissimilar to that seen using an Oct4 reporter model. In addition, we report that overexpression of mouse Sry in embryonic stem cells leads to down-regulation of the core pluripotency markers Sox2 and Nanog. Conclusion: We propose that SRY/Sry may function as a male-specific maturation factor in the peri-implantation mammalian embryo, providing a genetic mechanism to help explain the observation that male embryos are developmentally more advanced compared with female embryos, and suggesting a role for SRY beyond that of TDF.

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