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Query: UNIPROT:P06889 (Mol)
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Expression of Oct4 in embryonic stem cells is controlled by a distal upstream stem cell-specific enhancer that is deactivated during retinoic acid (RA)-induced differentiation by an indirect mechanism not involving binding of RA receptors (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997-3005, 1991). Here we report that in RA-treated P19 embryonal carcinoma cells the Oct4 promoter is also subject to negative regulation by RA. The minimal Oct4 promoter sequence mediating repression consists of a promoter-proximal sequence containing a GC-rich SP1 consensus-like sequence and several hormone response element half-sites that can be arranged into direct repeats with different spacing. The GC box binds a nuclear factor that is invariably present in undifferentiated and RA-treated differentiated P19 cells. By contrast, the hormone response element-containing sequence binds factors that are induced following RA treatment. Mutational analysis and competition experiments show that the functional entity binding the RA-induced factor is a direct repeat sequence with a spacing of one nucleotide, previously shown to be a binding site for COUP transcription factors (COUP-TFs). Cotransfected orphan receptors COUP-TF1, ARP-1, and EAR-2 were able to repress the activity of Oct4 promoter-driven reporters in P19 EC cells, albeit with different efficiencies. Furthermore, the negative transcriptional effect of COUP-TFs is dominant over the activating effect of the Oct4 embryonic stem cell-specific enhancer. These results show that negative regulation of Oct4 expression during RA-induced differentiation of embryonic stem cells is controlled by two different mechanisms, including deactivation of the embryonic stem cell-specific enhancer and promoter silencing by orphan nuclear hormone receptors.
Mol Cell Biol 1994 Feb
PMID:Characterization of a negative retinoic acid response element in the murine Oct4 promoter. 828 93

Csn2 (Trip15/Cops2/Alien) encodes the second subunit of the COP9 signalosome (CSN), an eight-subunit heteromeric complex homologous to the lid subcomplex of the 26S proteasome. CSN is a regulator of SCF (Skp1-cullin-F-box protein)ubiquitin ligases, mostly through the enzymatic activity that deconjugates the ubiquitin-like protein Nedd8 from the SCF Cul1 component. In addition, CSN associates with protein kinase activities targeting p53, c-Jun, and IkappaB for phosphorylation. Csn2 also interacts with and regulates a subset of nuclear hormone receptors and is considered a novel corepressor. We report that targeted disruption of Csn2 in mice caused arrest of embryo development at the peri-implantation stage. Csn2(-/-) blastocysts failed to outgrow in culture and exhibited a cell proliferation defect in inner cell mass, accompanied by a slight decrease in Oct4. In addition, lack of Csn2 disrupted the CSN complex and resulted in a drastic increase in cyclin E, supporting a role for CSN in cooperating with the SCF-ubiquitin-proteasome system to regulate protein turnover. Furthermore, Csn2(-/-) embryos contained elevated levels of p53 and p21, which may contribute to premature cell cycle arrest of the mutant.
Mol Cell Biol 2003 Oct
PMID:Disruption of the COP9 signalosome Csn2 subunit in mice causes deficient cell proliferation, accumulation of p53 and cyclin E, and early embryonic death. 1297 99

To study the mechanisms of mouse peri-implantation development, we explored the in vitro culture of the isolated inner cell mass (ICM) of the blastocyst. As previously reported, individually cultured ICM recapitulated several early embryological events, such as the formation of primitive endoderm, epiblast, and proamniotic cavity. However, we found that the timing and efficiency of these morphogenetic processes significantly varied among the ICM. Due to this unpredictability in developmental potential, individually cultured ICM may be unsuitable for further analysis. By contrast, we found that when five ICM were fused into a single mass, such aggregates (5x ICM) underwent efficient and synchronous morphogenesis. The synchronous nature of 5x ICM development was also demonstrated by the temporal and spatial pattern of apoptotic cell death. TUNEL assay showed that a number of the epiblast cells committed apoptosis in 48 hr of culture, which took place after primitive endoderm differentiation but prior to proamniotic cavity formation. In situ hybridization analysis showed that Oct4 was downregulated and alpha-fetoprotein was upregulated in the primitive endoderm of the cultured 5x ICM. In addition, RT-PCR analysis revealed the expression of various primitive endodermal genes, but not of extraembryonic ectodermal markers in the cultured 5x ICM. Taken together, we propose that the 5x ICM is a useful in vitro tool to study the mechanisms of peri-implantation development of the mouse embryo. Mol. Reprod. Dev. 67: 83-90, 2004.
Mol Reprod Dev 2004 Jan
PMID:Molecular study of mouse peri-implantation development using the in vitro culture of aggregated inner cell mass. 1464 78

Recently, small interfering RNAs (siRNAs) have become a powerful and widely used tool for the analysis of gene function in mammalian cells. Here we report that the microinjection of an siRNA expression vector into the nucleus is an efficient and powerful method of specific gene silencing in pre-implantation mouse embryos. We used this method to examine the expression of two genes EGFP and Oct4. Vectors encoding siRNAs targeted against EGFP or Oct4 were injected into the pronucleus or nucleus of zygotes, which were then cultured until the blastocyst stage. When the effects of RNAi were examined in blastocyst stage eggs, there was robust inhibition of the gene product in a concentration-dependent manner at both the mRNA and the protein level. The expression of other endogenous genes was not affected, showing the specificity of the vector-mediated RNAi. In addition, this method was effective for inhibiting maternally expressed mRNA. To demonstrate that RNAi of Oct4 induced a similar phenotype to that of Oct4-null embryos, the blastocysts were further cultured in ES medium. After the fourth day of culture, the embryos either had outgrown only a layer of trophoblast cells or showed developmental arrest at the blastocyst stage (>90%). Moreover, concomitant with Oct4 suppression at the blastocyst stage, we observed inhibition of Fgf4, a gene that is known to be induced downstream of Oct4 expression. Taken together, these results demonstrate that the use of siRNA expression vector is a powerful way to achieve gene silencing in the pre-implantation stage embryo.
Mol Reprod Dev 2004 May
PMID:Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system. 1503 44

Following hybridization with embryonic stem (ES) cells, somatic genomes are epigenetically reprogrammed and acquire pluripotency. This results in the transcription of somatic genome-derived tissue-specific genes upon differentiation. During nuclear reprogramming, it is expected that DNA and chromatin modifications, believed to function in cell-type-specific epigenotype memory, should be significantly modified. Indeed, current evidence indicates that acetylation and methylation of histone H3 and H4 amino termini play a major role in the regulation of gene activity through the modulation of chromatin conformation. Here, we show that the reprogrammed somatic genome of ES hybrid cells becomes hyperacetylated at H3 and H4, while lysine 4 (K4) of H3 becomes globally hyper-di- and -tri-methylated. In the Oct4 promoter region, histones H3 and H4 are acetylated and H3-K4 is highly tri-methylated on both the ES and reprogrammed somatic genomes, which correlates with gene activation and DNA demethylation. However, H3-K4 is also di- and tri-methylated in the promoter regions of Neurofilament-M (Nfm), Nfl, and Thy-1, which are all silent in both ES and hybrid cells. Thus, H3-K4 di- and tri-methylation of reprogrammed somatic genomes is independent of gene activity and represents one of the major events that occurs during somatic genome reprogramming towards a transcriptional activation-permissive state.
Mol Cell Biol 2004 Jul
PMID:Histone code modifications on pluripotential nuclei of reprogrammed somatic cells. 1521 76

Germ cells in the mouse embryo remain undifferentiated until about 13.5 days post-coitum (dpc), when male germ cells enter mitotic arrest and female germ cells enter meiosis. The molecular signals and transcriptional control mechanisms governing the differential fate of germ cells in males and females remain largely unknown. In order to gain insights into the behavior of germ cells around this period and into likely mechanisms controlling entry into meiosis, we have studied by wholemount in situ hybridization the expression pattern of two germ cell-specific markers, Oct4 and Sycp3, during mouse fetal gonad development. We observed a dynamic wave of expression of both genes in developing ovaries, with Oct4 expression being extinguished in a rostro-caudal wave and Sycp3 being upregulated in a corresponding wave, during the period 13.5-15.5 dpc. These results indicate that entry into meiosis proceeds in a rostro-caudal progression, in turn suggesting that somatically derived signals may contribute to the control of germ cell entry into meiosis in developing ovaries.
Mol Reprod Dev 2004 Aug
PMID:Germ cells enter meiosis in a rostro-caudal wave during development of the mouse ovary. 1523 25

It has been reported that DNA methyltransferase 1-deficient (Dnmt1-/-) embryonic stem (ES) cells are hypomethylated (20% CpG methylation) and die through apoptosis when induced to differentiate. Here, we show that Dnmt[3a-/-,3b-/-] ES cells with just 0.6% of their CpG dinucleotides behave differently: the majority of cells within the culture are partially or completely blocked in their ability to initiate differentiation, remaining viable while retaining the stem cell characteristics of alkaline phosphatase and Oct4 expression. Restoration of DNA methylation levels rescues these defects. Severely hypomethylated Dnmt[3a-/-,3b-/-] ES cells have increased histone acetylation levels, and those cells that can differentiate aberrantly express extraembryonic markers of differentiation. Dnmt[3a-/-,3b-/-] ES cells with >10% CpG methylation are able to terminally differentiate, whereas Dnmt1-/- ES cells with 20% of the CpG methylated cannot differentiate. This demonstrates that successful terminal differentiation is not dependent simply on adequate methylation levels. There is an absolute requirement that the methylation be delivered by the maintenance enzyme Dnmt1.
Mol Cell Biol 2004 Oct
PMID:Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells. 1545 61

Mammalian embryos obtained by somatic nuclear transfer (NT) struggle to survive throughout development, encountering a number of hurdles leading to wrong functional reprogramming of the donor genome. However, despite these obstacles, some of these embryos continue their development, as if the required transcriptional functions are somehow satisfied. The amount of information gathered on the kinetics and quantitative profile of gene expression in NT pre-implantation embryos is still scarce and limited to a handful of genes described in two species, bovine and mouse. Using a single-cell sensitive semi-quantitative RT-PCR, we have compared the onset and profile of abundance of Hprt, Tsx, Bex1, Bax, Cpt2, and Oct4 genes, in in vitro fertilised and NT-derived mouse 1-cell, 2-cell, 4-cell embryos, morulae, and blastocysts. The genes analysed were activated in NT embryos at approximately the correct time compared to control embryos, indicating that the reprogramming phenomenon is developmentally regulated and that the somatic genome is quickly rearranged towards an embryonic-type of expression during the early stages of segmentation. Despite the right timing of genes onset, the high degree of variability in the number of transcripts found in NT embryos at the latest stages of pre-implantation development, suggests that genome reprogramming is incomplete and inaccurate.
Mol Reprod Dev 2005 Feb
PMID:Cloned pre-implantation mouse embryos show correct timing but altered levels of gene expression. 1557 Jun 22

In vitro produced (IVP) bovine embryos were subjected to in vitro culture with or without 1000 U/ml human recombinant leukemia inhibitory factor (LIF) added to the culture medium from Days 5 to 8 post insemination (p.i.). Resulting blastocysts were subsequently plated intact on mouse feeder cells in a medium with or without LIF. Significantly more embryos reached the hatched blastocyst stage, and the number of blastocysts with excellent morphology was significantly higher, when LIF was omitted. At Day 8 p.i., total cell count (TCC) and inner cell mass (ICM) cell count was significantly higher in embryos cultured without LIF. In embryos cultured with LIF, cytoplasmic vesicles and lipid droplets were abundant and a decreased expression of both Oct4 and laminin could be observed. Initial hypoblast formation was revealed in almost 1/3 of the LIF-cultured blastocysts whereas this feature was evident in 2/3 of the blastocysts cultured in the absence of LIF. Overall, almost 60% of the blastocysts cultured without LIF formed outgrowth colonies (OCs) when plated on feeders, whereas this phenomenon was only observed in 30% of the blastocysts cultured in the presence of LIF. A tendency for retaining a tightly packed central growth of putative ICM-derived cells was observed, when attachment to the feeder layer was initiated close to the embryonic pole of the blastocyst. At Day 8 of outgrowth culture, approximately 20% of the colonies contained a central core of putative ICM-derived cells appearing large enough for mechanical isolation and further subculture. Immunohistochemical labeling for Oct4 revealed staining of both trophectodermal and ICM-derived cells. The presence of LIF in the outgrowth culture medium did not have any apparent effect on the plating efficiency or colony type. In conclusion, LIF had an adverse effect on in vitro embryonic development when added to the culture medium in the period from Days 5 to 8 p.i., whereas it had no apparent effect on the OCs subsequently formed from such embryos.
Mol Reprod Dev 2005 Apr
PMID:Effect of leukemia inhibitory factor (LIF) on in vitro produced bovine embryos and their outgrowth colonies. 1568 35

The transcription factor OCT-4 is regarded as a critical factor in controlling mammalian early embryonic development because of its role in toti-/pluripotency. In human preimplantation embryos, OCT-4 studies are limited to RNA analysis of abnormally developing embryos. This study thoroughly investigated the expression pattern of OCT-4 throughout the human preimplantation development. Expression was examined by single-cell RT-PCR or indirect immunocytochemistry in 36 single oocytes of various maturity and 112 normally developing preimplantation embryos at the level of single blastomeres, morulas, blastocysts, or inner cell mass (ICM) and trophectoderm (TE) samples. Oocytes and cleavage stage embryos revealed a variable OCT-4 expression pattern, concomitant with a pure cytoplasmic localization of the protein. During compaction, the variability in expression faded away indicating embryonic OCT-4 expression and the protein appeared in the nucleus implying biological activity. In blastocysts, OCT-4 transcripts and proteins were present in the ICM and the TE. At protein level, blastocysts displayed different spatial expression patterns within a cell for the splice variants of OCT-4, which may endow them with different functional properties. As OCT-4 transcripts were also found in various differentiated cells, the presence of OCT-4 transcripts or proteins may not be sufficient for identifying undifferentiated cell lines in humans. Further, we suggest to examine the localization of OCT-4 proteins within a cell rather than to look for the presence and/or amount of transcripts.
Mol Hum Reprod 2005 Mar
PMID:Oct-4 mRNA and protein expression during human preimplantation development. 1569 70


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