In Vivo and In Vitro Dynamics of Undifferentiated Embryonic Cell Transcription Factor 1
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE53768 | GEO | 2014/01/03
SECONDARY ACCESSION(S): PRJNA232855
REPOSITORIES: GEO
Similar Datasets
Project description:Pluripotent stem cells retain the ability differentiate into the three germ layers and germ line. As a result, there is a major interest in characterizing regulators that establish and maintain pluripotency. The network of transcription factors continues to expand in complexity, and one factor, Undifferentiated embryonic cell Transcription Factor 1 (Utf1), has recently moved more into the limelight. To facilitate the study of Utf1, we report the generation and characterization of two reporter lines that enable efficient tracking, mapping and purification of endogenous Utf1. In particular, we include a built-in biotinylation system into our targeted locus that allows efficient and reliable pulldown. We also use this reporter to show the dynamic regulation of Utf1 in distinct stem cell conditions and demonstrate its utility for reprogramming studies. The multi-purpose design of the reporter lines enables many directions of future study and should lead to a better understanding of Utf1M-bM-^@M-^Ys diverse roles. ChIP-Seq for endogenous and biotin tagged Utf1 in KH2 mouse embryonic stem cells
2014-01-03 | E-GEOD-53768 | biostudies-arrayexpress
Project description:Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2'-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. *Mouallif M and Guenin S contributed equally to this work
2012-03-21 | GSE36637 | GEO
Project description:[1] Gene expression profiling in Gata4, Mef2a knockdown in HL-1 cells. HL-1 cells infected with adenovirus expressing either control siRNA or Gata4, and Gata4 & Mef2a siRNA. [2] Genome-wide maps of cardiac transcription factors binding region in HL-1 cells. We performed bio-ChIP-seq using streptavidin beads immunoprecipitation of biotinylated 5 cardiac transcription factors (fbio) and P300 antibody ChIP-seq. We used a dox-inducible dual adenovirus system to express biotinylated Core Cardiac TFs in HL1. One adenovirus expressed the dox-activated reverse tet activator protein (rtTA) and the E. coli biotinylating enzyme BirA from the cardiac specific rat troponin T promoter. The second virus expressed a Core Cardiac TF fused to a C-terminal flag-bio epitope tag, where bio is the 15 amino acid substrate for BirA. This in vivo biotinylation approach permits pulldown of different factors to be performed under identical, stringent conditions, and circumvents limitations posed by available antibodies. We titrated adenovirus and dox doses to express GATA4flbio and MEF2Aflbio at near endogenous levels. The same conditions were then used to express SRFflbio, TBX5flbio, and NKX2-5flbio. Reference: 12. de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E et al. (2003) Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 100: 7480-7485. [1] Gene expression profiling: Identify differentially expressed genes after siRNA Gata4 or Gata4_Mef2a double knockdown in HL-1 cells [2] Genome occupancy profiling: Identify cardiac active enhancers by identified 5 cardiac transcription factors and P300 peaks.
2011-03-02 | E-GEOD-21529 | biostudies-arrayexpress
Project description:[1] Gene expression profiling in Gata4, Mef2a knockdown in HL-1 cells. HL-1 cells infected with adenovirus expressing either control siRNA or Gata4, and Gata4 & Mef2a siRNA. [2] Genome-wide maps of cardiac transcription factors binding region in HL-1 cells. We performed bio-ChIP-seq using streptavidin beads immunoprecipitation of biotinylated 5 cardiac transcription factors (fbio) and P300 antibody ChIP-seq. We used a dox-inducible dual adenovirus system to express biotinylated Core Cardiac TFs in HL1. One adenovirus expressed the dox-activated reverse tet activator protein (rtTA) and the E. coli biotinylating enzyme BirA from the cardiac specific rat troponin T promoter. The second virus expressed a Core Cardiac TF fused to a C-terminal flag-bio epitope tag, where bio is the 15 amino acid substrate for BirA. This in vivo biotinylation approach permits pulldown of different factors to be performed under identical, stringent conditions, and circumvents limitations posed by available antibodies. We titrated adenovirus and dox doses to express GATA4flbio and MEF2Aflbio at near endogenous levels. The same conditions were then used to express SRFflbio, TBX5flbio, and NKX2-5flbio. Reference: 12. de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E et al. (2003) Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 100: 7480-7485.
2011-03-02 | GSE21529 | GEO
Project description:The core pluripotency factors (Oct4, Sox2, and Nanog), the Myc network, and the chromatin-modifying complexes such as PRC2 ensure the pluripotency and self-renewal of ES cells (ESC). How these factors coordinate with one another remains poorly understood. We report that Utf1, a target of Oct4 and Sox2, is a new bivalent chromatin component that buffers poised states of bivalent genes. By limiting PRC2 loading and Histone 3 lysine-27 trimethylation, Utf1 sets proper activation thresholds for bivalent genes. It also promotes nuclear tagging of mRNAs transcribed from insufficiently silenced bivalent genes for cytoplasmic degradation through mRNA de-capping. Whereas these opposing functions of Utf1 allow proper execution of ESC pluripotency, the mRNA pruning function also ensures rapid cell proliferation by blocking the Myc-Arf feedback regulation. Thus, Utf1 is an important regulator that couples the core pluripotency factors with Myc and PRC2 networks to promote proliferation and pluripotency execution of ESCs. First we mapped Utf1 binding sites in ESCs using the biotin-mediated and cross-linked ChIP-sequencing. To investigate how Utf1 might regulate gene expression, we did RNA-seq on WT and Utf1-KO ES cells. Then we did ChIP-seq of Suz12 and H3K27me3 on WT and Utf1-KO ES cells to study whether Utf1 affects PRC2 loading and H3K27me3 modofication, using H3 as control. Finally, we did RNAseq on WT and Dcp1a-KD ES cells to confirm Utf1 repress gene expression by recruiting Dcp1a complex.
2012-10-26 | E-GEOD-39513 | biostudies-arrayexpress
Project description:Genome wide DNA methylation profiling of normal and squamous carcinoma of the cervix. The Illumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs in frozen cervical samples. Samples included 4 normal ectocervices (Ecto 1 to 4) and 5 squamous cell carcinoma of the cervix (SCC 1 to 5). In order to identify epigenetic biomarkers for early cervix cancer diagnosis, we performed a methylation screening using Illumina Infinium 27k Human DNA methylation Beadchip v1.2 of stem cell marker promoters during cervical carcinogenesis and demonstrated a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in squamous cell carcinoma (SCC) in comparison with normal ectocervix. Direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples showed that UTF1 promoter methylation increases with lesion severity and is associated with higher expression. By RT-PCR, Western Blot and immunofluorescence, we demonstrated that UTF1 mRNA and protein are expressed in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Methyl-Specific PCR experiments revealed that the inhibition of DNA methyltransferase by 5-aza-2’-deoxycytidine is associated with decreased UTF1 gene methylation and expression. These findings provide evidence that UTF1 promoter methylation profile might be a useful biomarker for cervix cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms involved in the regulation of its expression. Bisulfite converted DNA from the 9 samples were hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2
2012-03-21 | E-GEOD-36637 | biostudies-arrayexpress
Project description:In mammals, female, but not male, germ cells onset meiosis physiologically during embryonic stages. We examined whether germ cell-specific disruption of Max gene leads to the activation of meiosis-related genes in embryonic male germ cells by single cell RNA sequencing.
2024-09-29 | GSE244318 | GEO
Project description:Here, we report the generation of human induced Pluripotent Stem (iPS) cell reporter line in which a venus fluorescent protein have been introduced into the MYOGENIN (MYOG) locus. We use microarrays to compare the transcriptome of MYOG-venus+ cells after 3 weeks of myogenic differentiation to that of undifferentiated iPS Satellite cells (SC) are muscle stem cells which can regenerate adult muscles upon injury. Most SC originate from PAX7-positive myogenic precursors set aside during development. While myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we use microarrays to compare the transcriptome of myogenic cells differentiated in vitro from human and mouse ES and iPS cells reporter cell lines. We generated fluorescent reporter lines in which a fluorescent protein was introduced into the loci coding for Pax7 (mouse ES and human iPS) or MYOG (human iPS). Mouse ES and human iPS cells were differentiated to the myogenic lineage in vitro for three weeks according to the protocols described in Chal et al, Nature Biotech 2015 and to Chal, Al Tanoury et al, Nature Protocols, 2016. Fluorescent Pax7 or MYOG-positive cells were FACS-sorted after 3-weeks of differentiation in vitro and we used Affymetrix microarrays to analyze the transcriptome of the purified mouse and human cell populations and compare it to the transcriptome of undifferentiated mouse ES or human iPS cells.
2020-04-21 | GSE148994 | GEO
Project description:Here, we report the generation of human induced Pluripotent Stem (iPS) cell reporter line in which a venus fluorescent protein have been introduced into the PAX7 locus. We use microarrays to compare the transcriptome of PAX7-venus+ cells after 3 weeks of myogenic differentiation to that of undifferentiated iPS Satellite cells (SC) are muscle stem cells which can regenerate adult muscles upon injury. Most SC originate from PAX7-positive myogenic precursors set aside during development. While myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we use microarrays to compare the transcriptome of myogenic cells differentiated in vitro from human and mouse ES and iPS cells reporter cell lines. We generated fluorescent reporter lines in which a fluorescent protein was introduced into the loci coding for Pax7 (mouse ES and human iPS) or MYOG (human iPS). Mouse ES and human iPS cells were differentiated to the myogenic lineage in vitro for three weeks according to the protocols described in Chal et al, Nature Biotech 2015 and to Chal, Al Tanoury et al, Nature Protocols, 2016. Fluorescent Pax7 or MYOG-positive cells were FACS-sorted after 3-weeks of differentiation in vitro and we used Affymetrix microarrays to analyze the transcriptome of the purified mouse and human cell populations and compare it to the transcriptome of undifferentiated mouse ES or human iPS cells.
2020-04-22 | GSE149055 | GEO
Project description:Here, we use microarrays to compare the transcriptome of mouse Pax7-GFP ES reporter cell line after 3 weeks of myogenic differentiation in vitro to that of undifferentiated ES Satellite cells (SC) are muscle stem cells which can regenerate adult muscles upon injury. Most SC originate from PAX7-positive myogenic precursors set aside during development. While myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we use microarrays to compare the transcriptome of myogenic cells differentiated in vitro from human and mouse ES and iPS cells reporter cell lines. We generated fluorescent reporter lines in which a fluorescent protein was introduced into the loci coding for Pax7 (mouse ES and human iPS) or MYOG (human iPS). Mouse ES and human iPS cells were differentiated to the myogenic lineage in vitro for three weeks according to the protocols described in Chal et al, Nature Biotech 2015 and to Chal, Al Tanoury et al, Nature Protocols, 2016. Fluorescent Pax7 or MYOG-positive cells were FACS-sorted after 3-weeks of differentiation in vitro and we used Affymetrix microarrays to analyze the transcriptome of the purified mouse and human cell populations and compare it to the transcriptome of undifferentiated mouse ES or human iPS cells.
2020-04-21 | GSE148993 | GEO