Project description:The transcription factor Oct3/4 is essential to maintain pluripotency in mouse embryonic stem (ES) cells. It was reported that the Xpc DNA repair complex is involved in this process. Here we examined the role of Xpc on the transcriptional activation of the target genes by Oct3/4 using the inducible knockout strategy. We found that the removal of the C-terminal region of Xpc, including the interaction sites with Rad23 and Cetn2, showed faint impact on the gene expression profile of ES cells and the functional Xpc-ΔC ES cell lines retained proper gene expression profile as well as pluripotency to contribute chimeric embryos. These data indicated that the C-terminal region of Xpc is dispensable for the transcriptional activity of Oct3/4 in mouse ES cells. An inducible knockout ES cell line of Xpc with the Cre-loxP system was generated and gene expression was measured without Xpc deletion, after 4 days of Xpc deletion or constitutive knocked out cells. Each sample was prepared in triplicate.
Project description:The transcription factor Oct3/4 is essential to maintain pluripotency in mouse embryonic stem (ES) cells. It was reported that the Xpc DNA repair complex is involved in this process. Here we examined the role of Xpc on the transcriptional activation of the target genes by Oct3/4 using the inducible knockout strategy. We found that the removal of the C-terminal region of Xpc, including the interaction sites with Rad23 and Cetn2, showed faint impact on the gene expression profile of ES cells and the functional Xpc-ΔC ES cell lines retained proper gene expression profile as well as pluripotency to contribute chimeric embryos. These data indicated that the C-terminal region of Xpc is dispensable for the transcriptional activity of Oct3/4 in mouse ES cells.
Project description:To characterize the transdifferentiation of embryonic stem (ES) cells into trophoblast stem (TS) cells triggered by forced repression of Oct3/4, we performed whole-genome expression analysis after tetracycline (Tet)-induced knockout of Oct3/4. A Tet-inducible Oct3/4 knockout ES cell line ZHBTc4 was treated with Tet for 4 days in the presence of FGF4 and mouse embryonic fibroblasts (MEFs). A total of 15 samples from Day 0 to Day 4 were analyzed in three biological replicates.
Project description:Differentiated cells can be reprogrammed to an embryonic-like state by transfer of their nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about the factors that induce this reprogramming. Here we show that the combination of four factors, Oct3/4, Sox2, c-Myc, and Klf4, can generate pluripotent-like stem cells directly from mouse embryonic or adult fibroblast cultures. Unexpectedly, Nanog was dispensable in this process. These cells, which we designated iPS (induced pluriopotent-like stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent-like cells can be directly generated from fibroblast cultures by the addition of only a few defined factors. Experiment Overall Design: Total RNAs were isolated from indicated cells and labeled with Cy3. Hybridization was performed once for each sample.
Project description:To understand the mechanism underlying the versatility in transcriptional regulation by Sox2, we compared genome-wide binding sites of Sox2 in embryonic stem (ES) cells and trophoblast stem (TS) cells by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). A tetracycline-inducible Oct3/4 knockout ES cell line ZHBTc4 was treated with Tet for 4 days in the presence of FGF4 and mouse embryonic fibroblasts (MEFs).
Project description:Differentiated cells can be reprogrammed to an embryonic-like state by transfer of their nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about the factors that induce this reprogramming. Here we show that the combination of four factors, Oct3/4, Sox2, c-Myc, and Klf4, can generate pluripotent-like stem cells directly from mouse embryonic or adult fibroblast cultures. Unexpectedly, Nanog was dispensable in this process. These cells, which we designated iPS (induced pluriopotent-like stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent-like cells can be directly generated from fibroblast cultures by the addition of only a few defined factors. Keywords: cell type comparison
Project description:We recently identified the DNA repair complex XPC-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4 and SOX2 transcriptional activation. Here we investigate genome-wide the role of SCC in murine ESCs by mapping regions bound by its RAD23B subunit in wild type and Xpc-/- ESCs, and analyzing transcriptional profiles of SCC-depleted ESCs. RAD23B ChIP-seq in wild type murine ESCs was performed in two replicates; normal IgG were used as controls. RAD23B ChIP-seq in Xpc-/- mESCs was compared to normal IgG. Antibodies to immunoprecipitate RAD23B were raised in guinea pigs against the 108-177 peptide of murine RAD23B protein (NP_033037). Anti-sera were affinity-purified. For RNA-seq, we compared wild type JM8.N4 cells with either Rad23b-/- cells, Rad23b-/-Xpc KD1 or Rad23b-/-Xpc KD2 cells. Xpc was knocked down with two independent shRNAs (KD1 and KD2).
Project description:To characterize the transdifferentiation of embryonic stem (ES) cells into trophoblast stem (TS) cells triggered by forced repression of Oct3/4, we performed whole-genome expression analysis after tetracycline (Tet)-induced knockout of Oct3/4.
Project description:CDC14 phosphatases are critical components of the cell cycle machinery that drives exit from mitosis in yeast. However, the two mammalian paralogs, CDC14A and CDC14B, are dispensable for cell cycle progression or exit, and their function remains unclear. By generating a double Cdc14a; Cdc14b-null mouse model, we report here that CDC14 phosphatases control cell differentiation in pluripotent cells and their absence results in deficient development of the neural system. Lack of CDC14 impairs neural differentiation from embryonic stem cells (ESCs) accompanied by deficient induction of genes controlled by bivalent promoters. During ESC differentiation, CDC14 directly dephosphorylates and destabilizes Undifferentiated embryonic Transcription Factor 1 (UTF1), a critical regulator of stemness. In the absence of CDC14, increased UTF1 levels prevent the firing of bivalent promoters, resulting in defective induction of the transcriptional programs required for differentiation. These results suggest that mammalian CDC14 phosphatases function during the terminal exit from the cell cycle by modulating the transition from the pluripotent to the differentiated chromatin state, at least partially by controlling chromatin dynamics and transcription in a UTF1-dependent manner.