The Oct4 gene codes for a transcription factor that plays a critical role in the maintenance of pluripotency in embryonic and cancer stem cells. Its expression thus has to be tightly regulated. We performed biophysical characterization of the promoter region using a combination of UV absorption, CD, and NMR spectroscopies, native PAGE and chemical probing, which was followed by functional studies involving luciferase reporter assays performed in osteosarcoma and human embryonic stem cell lines. We have shown that the evolutionarily conserved G-rich region close to the Oct4 transcription start site in the non-template strand forms a parallel G-quadruplex structure. We characterized its structure and stability upon point mutations in its primary structure. Functional studies then revealed that whereas the wild type quadruplex sequence ensures high reporter gene expression, the expression of mutated variants is significantly decreased proportionally to the destabilizing effect of the mutations on the quadruplex. A ligand, N-methyl mesoporphyrin IX that increases the stability of formed quadruplex rescued the reporter expression of single-mutated variants to the level of wild-type, but it has no effect on a mutated variant that cannot form quadruplex. These data indicate that the quadruplex acts as a strong, positive regulator of Oct4 expression and as such it might serve as a potential target for therapeutic intervention.

• Klíčová slova
• Circular dichroism, Guanine quadruplex, Human embryonic stem cells, Luciferase reporter, Oct4, Transcription regulation,
• MeSH
• cirkulární dichroismus metody   MeSH
• embryonální kmenové buňky účinky léků   metabolismus   MeSH
• G-kvadruplexy účinky léků   MeSH
• lidé MeSH
• magnetická rezonanční tomografie metody   MeSH
• mesoporfyriny farmakologie   MeSH
• mutace genetika   MeSH
• nádorové buněčné linie MeSH
• oktamerní transkripční faktor 3 genetika   MeSH
• osteosarkom genetika   MeSH
• počátek transkripce účinky léků   fyziologie   MeSH
• promotorové oblasti (genetika) účinky léků   genetika   MeSH
• reportérové geny genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mesoporfyriny MeSH
• mesoporphyrin IX MeSH Prohlížeč
• oktamerní transkripční faktor 3 MeSH
• POU5F1 protein, human MeSH Prohlížeč

In the present study, we investigated protein expression of the transcription factors mammalian doublesex and mab-3 related transcription factor 1 (DMRT1), basic helix-loop-helix transcription factor-like 5 (TCLF5), and octamer-binding transcription factor 4 (OCT4) in normal human spermatogenesis, testicular mixed germ cell-sex cord stromal tumor (MGC-SCST), spermatocytic tumor, and seminoma. In normal human spermatogenesis, DMRT1 is expressed in the nuclei of spermatogonia but not in those of more mature germ cells. By way of contrast, TCLF5 is expressed in the nuclei of some clusters of primary spermatocytes that have entered meiosis 1, in secondary spermatocytes, and in round (early) spermatids in the seminiferous tubules of adults during the reproductive years. OCT4 is expressed in primordial germ cells but not in the seminiferous tubules of the normal adult testis during the reproductive years. DMRT1 is expressed in the germ cells of both testicular MGC-SCST and spermatocytic tumor, whereas TCLF5 is not expressed in either neoplasm. These low-grade neoplasms, however, differ histologically in that all the germ cell nuclei of testicular MGC-SCST resemble spermatogonia, whereas in spermatocytic tumor, the nuclei of the medium-sized and large cells resemble those of primary spermatocytes. Both neoplasms lack expression of OCT4. By way of contrast, in seminoma, a fully malignant testicular germ cell tumor, the germ cell nuclei express OCT4 but do not express either DMRT1 or TCLF5.

• Klíčová slova
• DMRT1, Mixed germ cell–sex cord stromal tumor, OCT4, Seminoma, Spermatocytic tumor, Testis,
• MeSH
• biopsie MeSH
• buněčné jádro chemie   patologie   MeSH
• germinální a embryonální nádory chemie   patologie   MeSH
• imunohistochemie MeSH
• lidé MeSH
• nádorové biomarkery analýza   MeSH
• oktamerní transkripční faktor 3 analýza   MeSH
• semenotvorné kanálky chemie   patologie   MeSH
• seminom chemie   patologie   MeSH
• spermatocyty chemie   patologie   MeSH
• spermatogeneze MeSH
• testikulární nádory chemie   patologie   MeSH
• transkripční faktory bHLH analýza   MeSH
• transkripční faktory analýza   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• DMRT1 protein MeSH Prohlížeč
• nádorové biomarkery MeSH
• oktamerní transkripční faktor 3 MeSH
• POU5F1 protein, human MeSH Prohlížeč
• TCFL5 protein, human MeSH Prohlížeč
• transkripční faktory bHLH MeSH
• transkripční faktory MeSH

Protein syntheses at appropriate timings are important for promoting diverse biological processes and are controlled at the levels of transcription and translation. Pou5f1/Oct4 is a transcription factor that is essential for vertebrate embryonic development. However, the precise timings when the mRNA and protein of Pou5f1/Oct4 are expressed during oogenesis and early stages of embryogenesis remain unclear. We analyzed the expression patterns of mRNA and protein of Pou5f1/Oct4 in mouse oocytes and embryos by using a highly sensitive in situ hybridization method and a monoclonal antibody specific to Pou5f1/Oct4, respectively. Pou5f1/Oct4 mRNA was detected in growing oocytes from the primary follicle stage to the fully grown GV stage during oogenesis. In contrast, Pou5f1/Oct4 protein was undetectable during oogenesis, oocyte maturation and the first cleavage stage but subsequently became detectable in the nuclei of early 2-cell-stage embryos. Pou5f1/Oct4 protein at this stage was synthesized from maternal mRNAs stored in oocytes. The amount of Pou5f1/Oct4 mRNA in the polysomal fraction was small in GV-stage oocytes but was significantly increased in fertilized eggs. Taken together, our results indicate that the synthesis of Pou5f1/Oct4 protein during oogenesis and early stages of embryogenesis is controlled at the level of translation and suggest that precise control of the amount of this protein by translational regulation is important for oocyte development and early embryonic development.

• Klíčová slova
• Embryo, Maternal transcript, Mouse, Oocyte, Translational regulation,
• MeSH
• embryonální vývoj genetika   MeSH
• myši inbrední ICR MeSH
• myši MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• oogeneze genetika   MeSH
• těhotenství MeSH
• vývojová regulace genové exprese genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• oktamerní transkripční faktor 3 MeSH
• Pou5f1 protein, mouse MeSH Prohlížeč

BACKGROUND: Oct4 is a specific marker of embryonic stem cell (ESC) pluripotency. However, little is known regarding how Oct4 responds to DNA damage. Here, we investigated whether Oct4 recognizes damaged chromatin in mouse ESCs stably expressing GFP-Oct4. These experiments should contribute to the knowledge of how ESC genomic integrity is maintained, which is crucial for potential application of human ESCs in regenerative medicine. METHODOLOGY/PRINCIPAL FINDINGS: We used time-lapse confocal microscopy, microirradiation by UV laser (355 nm), induction of DNA lesions by specific agents, and GFP technology to study the Oct4 response to DNA damage. We found that Oct4 accumulates in UV-damaged regions immediately after irradiation in an adenosine triphosphate-dependent manner. Intriguingly, this event was not accompanied by pronounced Nanog and c-MYC recruitment to the UV-damaged sites. The accumulation of Oct4 to UV-damaged chromatin occurred simultaneously with H3K9 deacetylation and H2AX phosphorylation (γH2AX). Moreover, we observed an ESC-specific nuclear distribution of γH2AX after interference to cellular processes, including histone acetylation, transcription, and cell metabolism. Inhibition of histone deacetylases mostly prevented pronounced Oct4 accumulation at UV-irradiated chromatin. CONCLUSIONS/SIGNIFICANCE: Our studies demonstrate pluripotency-specific events that accompany DNA damage responses. Here, we discuss how ESCs might respond to DNA damage caused by genotoxic injury that might lead to unwanted genomic instability.

• MeSH
• 53BP1 MeSH
• adenosintrifosfát metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• chromatin metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• embryonální kmenové buňky cytologie   MeSH
• fibroblasty metabolismus   MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• histony chemie   MeSH
• kinetika MeSH
• myši MeSH
• oktamerní transkripční faktor 3 metabolismus   MeSH
• poškození DNA MeSH
• regenerativní lékařství metody   MeSH
• regulace genové exprese * MeSH
• ultrafialové záření MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 53BP1 MeSH
• adenosintrifosfát MeSH
• chromatin MeSH
• chromozomální proteiny, nehistonové MeSH
• DNA vazebné proteiny MeSH
• histony MeSH
• oktamerní transkripční faktor 3 MeSH
• Pou5f1 protein, mouse MeSH Prohlížeč
• Trp53bp1 protein, mouse MeSH Prohlížeč

Oct4-mediated reprogramming has recently become a novel tool for the generation of various cell types from differentiated somatic cells. Although molecular mechanisms underlying this process are unknown, it is well documented that cells over-expressing Oct4 undergo transition from differentiated state into plastic state. This transition is associated with the acquisition of stem cells properties leading to epigenetically "open" state that is permissive to cell fate switch upon external stimuli. In order to contribute to our understanding of molecular mechanisms driving this process, we characterised human fibroblasts over-expressing Oct4 and performed comprehensive small-RNAseq analysis. Our analyses revealed new interesting aspects of Oct4-mediated cell plasticity induction. Cells over-expressing Oct4 lose their cell identity demonstrated by down-regulation of fibroblast-specific genes and up-regulation of epithelial genes. Interestingly, this process is associated with microRNA expression profile that is similar to microRNA profiles typically found in pluripotent stem cells. We also provide extensive network of microRNA families and clusters allowing us to precisely determine the miRNAome associated with the acquisition of Oct4-induced transient plastic state. Our data expands current knowledge of microRNA and their implications in cell fate alterations and contributing to understanding molecular mechanisms underlying it.

• MeSH
• buněčné linie MeSH
• embryo savčí * MeSH
• fibroblasty cytologie   metabolismus   MeSH
• lidé MeSH
• mikro RNA * biosyntéza   genetika   MeSH
• oktamerní transkripční faktor 3 * biosyntéza   genetika   MeSH
• regulace genové exprese * MeSH
• techniky buněčného přeprogramování * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA * MeSH
• oktamerní transkripční faktor 3 * MeSH
• POU5F1 protein, human MeSH Prohlížeč

• Klíčová slova
• CBP, KIX, MED15, Sox9, SoxE, Stem cell, iPSCs,
• MeSH
• geny myc MeSH
• Krüppel-like faktor 4 MeSH
• oktamerní transkripční faktor 3 genetika   MeSH
• transkripční faktory SOXB1 MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Krüppel-like faktor 4 MeSH
• oktamerní transkripční faktor 3 MeSH
• transkripční faktory SOXB1 MeSH

Embryonic stem cells (ESCs) maintain their pluripotency through high expression of pluripotency-related genes. Here, we show that differing levels of Oct4, Nanog, and c-myc proteins among the individual cells of mouse ESC (mESC) colonies and fluctuations in these levels do not disturb mESC pluripotency. Cells with strong expression of Oct4 had low levels of Nanog and c-myc proteins and vice versa. In addition, cells with high levels of Nanog tended to occupy interior regions of mESC colonies. In contrast, peripherally positioned cells within colonies had dense H3K27-trimethylation, especially at the nuclear periphery. We also observed distinct levels of endogenous and exogenous Oct4 in particular cell cycle phases. The highest levels of Oct4 occurred in G2 phase, which correlated with the pKi-67 nuclear pattern. Moreover, the Oct4 protein resided on mitotic chromosomes. We suggest that there must be an endogenous mechanism that prevents the induction of spontaneous differentiation, despite fluctuations in protein levels within an mESC colony. Based on the results presented here, it is likely that cells within a colony support each other in the maintenance of pluripotency.

• MeSH
• antigen Ki-67 metabolismus   MeSH
• buněčná diferenciace * MeSH
• buněčné jádro genetika   metabolismus   MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• epigeneze genetická MeSH
• FRAP MeSH
• G2 fáze MeSH
• histony metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• konfokální mikroskopie MeSH
• kultivované buňky MeSH
• lysin metabolismus   MeSH
• metylace MeSH
• myši MeSH
• nanog MeSH
• nika kmenových buněk MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• protoonkogenní proteiny c-myc genetika   metabolismus   MeSH
• western blotting MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigen Ki-67 MeSH
• histony MeSH
• homeodoménové proteiny MeSH
• lysin MeSH
• Myc protein, mouse MeSH Prohlížeč
• Nanog protein, mouse MeSH Prohlížeč
• nanog MeSH
• oktamerní transkripční faktor 3 MeSH
• protoonkogenní proteiny c-myc MeSH
• zelené fluorescenční proteiny MeSH

Human embryonic stem cells (hES) are unique in their pluripotency and capacity for self-renewal. Therefore, we have studied the differences in the level of chromatin condensation in pluripotent and all-trans retinoic acid-differentiated hES cells. Nuclear patterns of the Oct4 (6p21.33) gene, responsible for hES cell pluripotency, the C-myc (8q24.21) gene, which controls cell cycle progression, and HP1 protein (heterochromatin protein 1) were investigated in these cells. Unlike differentiated hES cells, pluripotent hES cell populations were characterized by a high level of decondensation for the territories of both chromosomes 6 (HSA6) and 8 (HSA8). The Oct4 genes were located on greatly extended chromatin loops in pluripotent hES cell nuclei, outside their respective chromosome territories. However, this phenomenon was not observed for the Oct4 gene in differentiated hES cells, for the C-myc gene in the cell types studied. The high level of chromatin decondensation in hES cells also influenced the nuclear distribution of all the variants of HP1 protein, particularly HP1 alpha, which did not form distinct foci, as usually observed in most other cell types. Our experiments showed that unlike C-myc, the Oct4 gene and HP1 proteins undergo a high level of decondensation in hES cells. Therefore, these structures seem to be primarily responsible for hES cell pluripotency due to their accessibility to regulatory molecules. Differentiated hES cells were characterized by a significantly different nuclear arrangement of the structures studied.

• MeSH
• buněčná diferenciace účinky léků   genetika   MeSH
• buněčné jádro genetika   ultrastruktura   MeSH
• buněčné linie MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• embryonální kmenové buňky metabolismus   ultrastruktura   MeSH
• homolog proteinu s chromoboxem 5 MeSH
• lidé MeSH
• pluripotentní kmenové buňky metabolismus   ultrastruktura   MeSH
• restrukturace chromatinu * MeSH
• signální transdukce genetika   MeSH
• trans-aktivátory účinky léků   metabolismus   MeSH
• tretinoin farmakologie   MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromozomální proteiny, nehistonové MeSH
• homolog proteinu s chromoboxem 5 MeSH
• trans-aktivátory MeSH
• tretinoin MeSH

MicroRNA (miRNAs) are short noncoding RNA molecules involved in many cellular processes and shown to play a key role in somatic cell induced reprogramming. We performed an array based screening to identify candidates that are differentially expressed between dermal skin fibroblasts (DFs) and induced pluripotent stem cells (iPSCs). We focused our investigations on miR-145 and showed that this candidate is highly expressed in DFs relative to iPSCs and significantly downregulated during reprogramming process. Inhibition of miR-145 in DFs led to the induction of "cellular plasticity" demonstrated by: (a) alteration of cell morphology associated with downregulation of mesenchymal and upregulation of epithelial markers; (b) upregulation of pluripotency-associated genes including SOX2, KLF4, C-MYC; (c) downregulation of miRNA let-7b known to inhibit reprogramming; and (iv) increased efficiency of reprogramming to iPSCs in the presence of reprogramming factors. Together, our results indicate a direct functional link between miR-145 and molecular pathways underlying reprogramming of somatic cells to iPSCs.

• Klíčová slova
• Induced pluripotent stem cells, KLF4, Mesenchymal-to-epithelial transition, OCT4, Reprogramming, SOX2, c-MYC, miR-145, microRNA,
• MeSH
• fibroblasty cytologie   metabolismus   MeSH
• indukované pluripotentní kmenové buňky cytologie   MeSH
• Krüppel-like faktor 4 MeSH
• lidé MeSH
• mikro RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• přeprogramování buněk * genetika   MeSH
• regulace genové exprese MeSH
• reprodukovatelnost výsledků MeSH
• sekvence nukleotidů MeSH
• škára cytologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• KLF4 protein, human MeSH Prohlížeč
• Krüppel-like faktor 4 MeSH
• mikro RNA MeSH
• MIRN145 microRNA, human MeSH Prohlížeč

Human induced pluripotent stem cell (iPSC) lines were generated from peripheral blood mononuclear cells (PBMCs) isolated from a patient diagnosed with spontaneous late-onset Alzheimer's disease (AD) carrying ApoE3/3 gene and one age-, sex-, and ApoE-matched healthy control. Reprogramming was done using a commercially available Epi5 Reprogramming Kit containing OCT4, SOX2, KLF4, LIN28, and L-MYC as reprogramming factors. The pluripotency of the iPSC lines was verified by the expression of pluripotency markers and by their capacity to differentiate into all three embryonic germ layers in vitro. These newly established iPSC lines offer a valuable platform for in vitro modeling of AD.

• MeSH
• Alzheimerova nemoc * genetika   metabolismus   MeSH
• apolipoprotein E3 genetika   MeSH
• buněčná diferenciace MeSH
• genotyp MeSH
• indukované pluripotentní kmenové buňky * metabolismus   MeSH
• Krüppel-like faktor 4 MeSH
• leukocyty mononukleární metabolismus   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• apolipoprotein E3 MeSH
• Krüppel-like faktor 4 MeSH