In this study, we analyzed the role of the pluripotency factor SOX2 in the in vivo reprogramming of TCam-2 to an EC-like cell fate. Therefore, we generated SOX2 knock out TCam-2 cells by utilizing the CRISPR/ Cas9 system and xenografted these cells into the flank of nude mice. After six weeks of in vivo growth, tumors were isolated and analyzed. Interestingly, TCam-2 cells did not acquire features of an EC, implicating that SOX2 is essential for the transition of TCam-2 cells to an EC-like cell state. Neither upregulation of EC-related pluripotency and epigenetic reprogramming factors, nor induction of NODAL or WNT signaling was detected. Additionally, global 5mC levels remained unaffected and expression of seminoma-associated genes SOX17, PRAME, TFAP2C, PRDM1 and cKIT was maintained. Nevertheless, a small subpopulation initiated differentiation into a mixed non- seminoma, demonstrating that in vivo the seminoma-like cell fate cannot be maintained for longer than 6 weeks. This differentiation was accompanied by upregulation of the pioneer factor FOXA2, which interacts with AFP, ALB, CDX1, DKK1, DLK1, PITX2, TTR, EOMES,
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In Drosophila, PcG proteins function within several distinct complexes: Pc repressive complex 1 (PRC1), Pc repressive complex 2 (PRC2), Pc repressive deubiquitinase (PR-DUB) and Pleiohomeotic (Pho) repressive complex (PhoRC) (Czermin et al., 2002; Klymenko et al., 2006; Müller et al., 2002; Scheuermann et al., 2012; Shao et al., 1999). In order to establish and maintain the transcriptional memory of an inactive gene, PRC2 and PRC1 are recruited to Polycomb response elements (PREs) within the genome by PhoRC, which consists of Scm-related gene containing four mbt domains (Sfmbt) and Pho. Sfmbt establishes a bridge, via protein- protein interactions, between the DNA-binding protein Pho and PRC2/PRC1. After the initial recruitment, Enhancer of zeste [E(z)], a member of PRC2, adds tri-methylation marks to the nucleosomes (at the H3K27 position) both at the PREs and along the gene body. This modification is then recognized by Pc, a member of PRC1, which, in turn, ubiquitylates H2A119 via Sex combs extra (Sce), another PRC1 member, and stabilizes PRC2. The accumulation of PRC1 and PRC2 within gene bodies results in the compaction of local nucleosomes and the further silencing of the inactive genes (Kassis et al., 2017; Simon and Kingston, 2009; Wang et al., 2004). Although PcG proteins have been extensively studied in Drosophila, specifically using changes in imaginal disc development as readouts for their roles in transcriptional silencing (Herz et al., 2014; Janody et al., 2004), we still lack a comprehensive understanding of the mechanisms by which PcG proteins actually regulate fate determination in vivo. For example, the four PcG complexes are thought to cooperate with each other to silence gene targets but distinct phenotypes are elicited when different PcG proteins are removed. The upregulation of cell proliferation pathways is often associated with loss of PRC1 but not with
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Prior to every surgery, mice were deeply anesthetised by intra-peritoneal injection of sleep solution (10 µl per g body weight) that was provided by the Research Unit Comparative Medicine (Helmholtz Zentrum München). After the injection of the anaesthesia, mice were checked for pain reactions by pinching their tail and toes. The fur on top of the head was removed with a small electric razor and Bepanthen (Bayer) was administered to the eyes to prevent their dryness. The animals were then fixed in a stereotaxic frame and the skin of the head was cut following the midline with a scalpel to expose the skull surface. Stab wound injury was performed in the somatosensory cortex, as previously described (Buffo et al., 2008; Heinrich et al., 2014). For the in vivo reprogramming experiments, the injury was performed only in one hemisphere, while for the reactive gliosis culture, both hemispheres were injured in order to obtain a higher amount of reactive glial cells. To perform the injury, bregma was searched using the forceps by pressing gently on the skull bones. Once bregma was found, the stereotactic apparatus was set to coordinate zero. Next, a small circular cranial window (diameter of ~3 mm) was drilled above the cerebral cortex with the high-speed rotary micromotor (Foredom) and the cranial window was collected in a drop of a Ringer solution. Following coordinates below, 1 mm long stab wound was performed in the cortical grey matter parenchyma by moving the V-lance shaped knife (Alcon) back and forth.
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While the analysis of partially reprogrammed cell states has been informative for understanding molecular barriers to reprogramming, a more detailed analysis of the earlier and later stages of reprogramming is crucial for establishing the sequence of transcriptional and epigenetic events that lead to a pluripotent state. In attempts to define such early intermediates, two studies have shown that the reprogramming of murine fibroblasts into iPS cells follows a defined sequence of molecular events that begins with the downregulation of somatic markers, such as Thy1 and collagens, followed by the reactivation of the embryonic marker stage-specific embryonic antigen 1 (SSEA1; Fig. 3) (Brambrink et al., 2008; Stadtfeld et al., 2008b). SSEA1-positive cells then gradually reactivate other markers associated with pluripotency, including Oct4, Sox2, Nanog, telomerase (tert), and the silent X chromosome in female fibroblasts. The reactivation of these late markers correlates with the time window when cells become independent of retroviral transgene expression and enter a self-sustaining pluripotent state. It is possible that the partially reprogrammed cell lines described above are the trapped equivalent of the transient SSEA1-expressing cell population, although direct evidence for this relationship is lacking (Fig. 3). The observation that the somatic markers of a cell become downregulated before it progresses to a pluripotent state supports the notion that the silencing of its differentiation program is an important initial step towards re- establishing pluripotency. It further suggests that the differentiation state of the cell of origin for iPS cells might affect the efficiency and kinetics of the reprogramming process.
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Since the initial postulation of the ‘Warburg Effect’, there have been numerous studies that have concluded that cancer cells switch to aerobic glycolysis over mitochondrial respiration (Schulze et al., 2011, Salminen et al., 2010 and Cai et al., 2010). However, many of these studies do not take into account the complex nature of the cancer microenvironment. Results presented in this thesis support a reverse Warburg type effect in gastric cancer, whereby cancer cells program surrounding cells to provide nutrients, thus enabling cancer cells to maintain an increased rate of mitochondrial respiration. Interestingly, we also found that AGS cells also exhibited some markers of glycolysis, such as, up regulation of GLUT1 and HK1. This suggests that cancer cells may either be utilising both forms of respiration, switching from one form to the other, or that different cells within the total population exhibit different profiles. Further studies utilising quantitative in vitro and in vivo models are required to differentiate between these possibilities.
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A key feature of metabolic pathways is their plasticity. Chang- es in nutrient availability or oxygen levels are the best-character- ized drivers of metabolic reprogramming. For example, hypoxia is a well-known driver of glycolysis, as an oxygen deficit results in limited OXPHOS. Under these circumstances cells must rely on glycolysis to generate ATP. HIF1α is critical for this process, as it induces the expression of glycolytic enzymes such as hexoki- nase and phosphofructokinase, thereby allowing for sustained ATP production (3, 4). Hypoxia and inflammation are inherent- ly linked. Decreasing oxygen levels induce metabolic changes to sustain ATP production. Similarly, quiescent immune cells can be viewed as metabolically inert and require significant metabol- ic reprogramming upon activation to provide sufficient ATP for effector functions. The HIF pathway provides a switch through which metabolic phenotypes can be amended in both of these scenarios and therefore is a critical transcriptional regulator of immunity and inflammation (2).
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Injection of msxb MO only showed a significant reduc- tion of fin outgrowth if injected into a 3 dpa blastema in accordance with the observed increase in msxb expres- sion around that time as detected by qPCR or in situ hybridization . Reasoning that the reprogramming factors pou5f1 and sox2 might be already needed at an earlier time point during fin regeneration we injected morpholino at different times during blastema formation and regrowth. Injection of morpholino into 0 dpa fins had no effect on regeneration, though for technical rea- sons we were only able to inject much lower volumes than into blastemas themselves. Injection of sox2 MO into a 1 dpa regenerating fin also did not lead to a pheno- type (Figure 4F and 4I). However, pou MO injected into 1 dpa blastema inhibited dorsal fin outgrowth by 40%. If morpholinos were injected into 2 dpa fin blastemas again pou MO but not sox2 MO reduced fin outgrowth by a similar extent as at 1 dpa (Figure 4E, 4F and 4I).
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Transcription factors are critical regulators of gene expression, and very likely play the principal role in directing cell-type specific transcription programs. The dominant action of a master tran- scription factor could potentially override the pre-existing cell- specific gene expression profile, and lead to a change in cell identity. This was demonstrated by the induction of a muscle-cell gene expression pattern in a range of different non-muscle cell types with the over-expression of a single transcription factor, MyoD (Weintraub et al., 1989). More recently, ectopic expression of four transcription factors, Oct4, Sox2, Klf4, and c-Myc was found to be sufficient to revert differentiated fibroblasts to pluripo- tent, embryonic stem cell-like cells (Takahashi and Yamanaka, 2006). Further refinement of the induced pluripotent stem cell methodology reveals the requirement for only Oct4 and Sox2 in the reprogramming process (Huangfu et al., 2008). These two transcription factors are maternally expressed in oocytes and zygotes, and should be readily available during SCNT to direct the activation of the embryonic gene expression program in the somatic nuclei. Successful nuclear transfer into zygotes suggest that these and other key embryonic transcription regulators might have been removed during complete enucleation of intact pronu- clei (Egli et al., 2007; Greda et al., 2006). Breakdown of the nuclear envelope during selective enucleation or entry into mito- sis would release these factors into the zygotic cytoplasm, thus conferring reprogramming capacity.
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Development of malignancy is accompanied by a complete metabolic reprogramming closely related to the acquisition of most of cancer hallmarks [2, 28]. Many known genetic and epigenetic alterations converge in a common adaptation of tumor cell metabolism . Indeed, metabolic properties of tumor cells are significantly different from those of non-transformed cells. In addition, tumor metabolic reprogramming is linked to drug resistance in cancer treatment [243, 244]. Accordingly, metabolic adaptations are also involved in different therapeutic approaches for cancer therapy. It is worth noting that some of the first chemotherapeutical agents used in cancer treatment were antimetabolites, such as aminopterin, methotrexate or 5-fluorouracil, that impaired the nucleotide synthesis and DNA replication [245, 246]. From then on, numerous metabolic pathways and enzymes have been successfully tested as anticancer targets . Since aerobic glycolysis is one of the key metabolic features of cancer cells, many studies are focused on inhibiting this pathway by blocking the enzymes that control it . Targeting the PPP with dehydroepiandrosterone (DHEA) and oxythiamine to respectively inhibit G6PD and TKT has also proven to have antitumor effects [50, 244, 249]. Interestingly, promoting pyruvate dehydrogenase (PDH) activity with dichloroacetate (DCA) presents promising results with minor side effects in early phase clinical trials with glioblastoma patients by suppressing angiogenesis, increasing mitochondrial ROS, inducing apoptosis, blocking HIF1 signaling and activating tumor suppressor p53 [250-252]. In fact, DCA inhibits PDHK leading to the metabolic switch from glycolysis to oxidative phosphorylation through PDH reactivation . Moreover, combined therapies with DCA and conventional cancer therapeutics such as omeprazole and tamoxifen show synergistic antitumor effects which can overcome drug resistance . Ongoing clinical trials with DCA as a single agent or in combination with other therapeutics are being conducted for patients with recurrent or metastatic solid tumors and head and neck carcinoma (clinical trials NCT00566410 and NCT01386632).
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ABSTRACT When environmental conditions compromise survival, single celled organisms, such as the budding yeast S. cerevisiae, induce and complete a differentiation program called sporulation. The first step consists of meiosis, which generates genetic diversity within the eventual haploid cells. The post-meiotic maturation stage reinforces protective barriers, such as the spore wall, against deleterious external conditions. In later stages of sporulation, the spore nucleus becomes highly compacted, likely sharing certain characteristics with the metazoan male gamete, the spermatozoon. The sporulation differentiation program involves many chromatin- related events, including execution of a precise transcription program involving more than one thousand genes. Here, we review how chromatin structure and genome reprogramming regulate the sporulation transcription program, and how post-meiotic events reorganize spore chromatin.
However, the low efficiency of iPS cell generation is the major problem of the direct reprogramming system. The reactivation of endogenous Oct4 in somatic cells requires only one to two days of fusion induced reprogramming (Han et al., 2008), but requires at least one week in generating iPS cells. The reprogramming efficiency could be enhanced by treatment with an epigenetic modifier or by additional factors involved in fusion-induced and direct reprogramming. Somatic Oct4 gene can be reactivated within 2 days after fusion with pluripotent stem cells, whereas reprogramming of Xist took about 9 days. Xist reprogramming could be enhanced by histone deacetylase inhibitor (trichostatin A) treatment (Do et al., 2008). Several small molecules also were reported to enhance reprogramming efficiencies in direct repro- gramming. Histone deacetylase inhibitor (valproic acid) and G9a histone methyltranferase inhibitor (BIX-01294) significantly en- hanced the efficiencies of iPS cell generation (Huangfu et al., 2008; Shi et al., 2008a; Shi et al., 2008b).
significantly high biological activities in vivo and in vitro. They can directly lead to DNA mutation, alteration of gene expression, modification of cell signal transduction, cell apoptosis, lipid peroxidation and protein degradation 7 . Oxidation is essential to many living organisms for the production of energy to fuel in biological processes. However, oxygen- centered free radicals and other reactive oxygen species, which are continuously, produced in vivo, result in cell death and tissue damage. Oxidative damage caused by free radicals may be related to aging and diseases, such as atherosclerosis, diabetes, cancer and cirrhosis 8 . Cooperative defence systems that protect the body from free radical damage include the antioxidant nutrients and enzymes. The antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Their role as protective enzymes is well- known and has been investigated extensively with in vivo models 9 .
Despite advances in characterizing mitochondrial factors and their roles in metabolic reprogramming in GC, there have been very few studies that have examined the relation- ship between mitochondria and metabolic reprogramming. However, as described above, the localization of glycolysis- related proteins to mitochondria can affect the functions of mitochondria, while mitochondrial proteins can also affect glycolysis. Thus, it appears that mitochondria are tightly linked with metabolic reprogramming that occurs, and this complex relationship remains to be thoroughly characterized.
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In the last decades, our knowledge about tumor pathogenesis has been growing in a constant manner. Even though many oncogenes and tumor suppressor genes have been identified, it is broadly accepted that they are not enough for governing tumor behavior and that the crosstalk between tumor cells and their microenviron- ment plays a critical role in cancer progression. Indeed, many studies have demonstrated that the malignant phenotype can be reverted by changes in the environmental conditions without altering the tumor cell genotype (Brinster, 1974; Postovit et al., 2007). The epigenetic reprogramming of malignant cells by em- bryonic environments has been suggested to be due to common regulatory signals shared by embryonic and tumor stem cells (Abbott et al., 2008). Supporting this proposal, several factors,
Reciprocal changes in PEPCK-C and PK with age are likely part of a bigger reprogramming of energy metabolism that profoundly affects the physiology of aging organisms, thereby impacting the aging process. It is important to obtain a complete picture of changes in metabolism with age, and their influence on decline in cellular function, cellular senescence, lifespan, and other aging traits. Such investigation should focus on metabolic pathways moving carbons into and out of the TCA cycle, and those affecting the homeostasis of glucose, fats and amino acids. This is because both CR and an optimized ratio of macronutrients (carbohydrates, proteins and fats) without reduction in total calorie intake extend lifespan in mice [123-125], suggesting that alterations in these metabolic pathways have significant but complex impact on aging.
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Cellular reprogramming may be a useful means of allowing the study of cellular processes that take place during the short transi- tional period between two developmental programs. Several genes have been discovered that control embryonic cell identity by estab- lishing or repressing the seed developmental program (Braybrook and Harada, 2008; Zhang and Ogas, 2009; Jia et al., 2013b). In the example presented in this review, overexpression of LEC2 was used to activate the seed developmental program in Arabidop- sis leaves (Santos Mendoza et al., 2005; Stone et al., 2008). This system could then be used to study the cellular and subcellular changes that ensue during the vegetative to embryonic transition (Feeney et al., 2013). The observation that LVs were replaced by PSV-like organelles in leaves presents an opportunity to elucidate the mechanism of LV to PSV transitions in Arabidopsis. Overall, we foresee that the major advantage of cellular reprogramming in vegetative tissues is to provide a convenient and complementary system in which to study cellular processes that normally occur during developmental transitions in developing seeds–tissues that are technically challenging to work with.
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Our work illustrates the richness of epigenetic landscapes in the presence of external drive and delayed feedback. Using a generic model of two self-activating and mutually inhibiting genes, we show that the reprogramming to the common progenitor state requires a balance of the drive and delay timescales, as well as appropriate positive and negative feedback strengths. Apart from the reprogrammed state, the system can ind itself in long-lived oscillatory states or in a transdiferentiated state in diferent parameter regimes. An analysis of the phase diagrams in the delay - drive time plane provides a comprehensive picture of the inal states of the reverse diferentiation process. Additionally, the phase diagrams also provide a signature of chaotic behaviour in appropriate regimes, which have been hypothesized to play an important role in the cell-fate determination process 45 . In the chaotic
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binding sites is important in maintaining pluripotency. To gain insights into the nucleosome occupancy at these sites, we examined the binding sites that were experi- mentally determined using chromatin immunoprecipita- tion (ChIP)-seq in ESCs, including a dozen important pluripotency factors , the p300 histone acetyltrans- ferase  and the chromatin remodeler Chd7 . Then, the nucleosome occupancy surrounding the bind- ing sites was calculated. Four types of nucleosome occu- pancy patterns were observed at the binding sites (Figure 3 and Additional file 6: Figure S3). (1) The bind- ing sites of the core pluripotency factors (Oct4, Sox2 and Nanog), the regulator Smad1 and the chromatin remodeler Chd7 preferentially resided in the linker regions between two adjacent nucleosomes (Figure 3A and Additional file 6: Figure S3A). Smad1 is the key component of the BMP signaling pathway that is linked to the core pluri- potency network. Chromatin remodeling factors provide a means for crosstalk by occupying the target genes of the core pluripotency factors . This setting with nu- cleosome depletion may allow the core pluripotency fac- tors to bind to their target genes relatively easily and construct the core pluripotency networks during repro- gramming. Intriguingly, it was reported that these core pluripotency factors also functioned as ‘pioneer factors’ and bound to closed chromatin at the first 48 hours of reprogramming . The inconsistence between this finding and our own is probably due to the different time point of binding. The reported initial binding of
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evaluation of reprogramming has depended on nonfunctional measures such as flow cytometry or expression of green fluorescent protein (GFP). Using calcium activity, several known and novel combinations of transcription factors were compared in mouse embryonic fibroblasts. It was found that the most efficient combination for generating cardiomyocyte- like cells with cardiomyocyte marker expression, consisted of Hand2, NK2 homeobox 5 (Nkx2.5), GATA4, Mef2c, and Tbx5 (HNGMT) and was .50-fold more efficient than GMT alone. 75 Epigenetics are also of importance when it comes to
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The identification of this intermediate cellular stage, which is devoid of rectal characteristics and in which neural characteristics have only just begun to appear, supports a model whereby successive distinct cellular steps are undertaken by a cell that directly reprograms in vivo (Fig. 2A, model 2), even in the absence of cell division. To determine whether this is consistent with the WT scenario, we next examined the transient expression of Y and PDA markers in N2 worms during the cellular transition event. The Y cell rectal epithelial markers LIN-26, egl-26, egl-5 and cki-1 were expressed in Y during the L1 larval stage, but were then permanently switched off as Y began its migration (Table 1, Fig. 4D). The PDA markers unc-33 and exp-1 switched on towards the end of Y migration, whereas cog-1 expression was apparent earlier during migration (Table 1, Fig. 4D). Co-staining of LIN-26 and cog-1::GFP, the earliest PDA marker, as well as LIN-26 and unc- 33::GFP, revealed that there was never an overlap in expression between the rectal epithelial marker and the neural markers during Y-to-PDA conversion, similar to the sequence of Y identity changes in unc-3(e151) mutants (Table 1). Thus, just after Y retraction from the rectum, the Y cell appeared to transit through a stage that Fig. 3. Initiation of Y-to-PDA reprogramming occurs normally in unc-3 animals and does not involve aberrant cellular changes. (A) Presence of ina-1::GFP in Y [expressed as a percentage of total animals scored (n)] as it migrates to its final position. Schematics beneath illustrate Y migration at the different stages examined. (B) Probing of the cellular identity of the intermediate Y.1 cell. (C) Markers of neighbouring cell identities or different neuron subtypes are not expressed in the intermediate Y.1 cell (50 animals scored for each marker). (D) Neuron DA7 is the lineal sister of Y, DA9 is the lineal contralateral homologue of Y, and DA8 is a morphologically related neuron. (E) Intermediate Y.1 did not adopt DA7, DA8 or DA9 identities (50 animals scored for each marker).
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