Identification of Ccr4-Not complex as a regulator of transition from partial to genuine iPS cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE44339 | GEO | 2014/01/07
SECONDARY ACCESSION(S): PRJNA189503
REPOSITORIES: GEO
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Project description:Partial induced pluripotent cells (iPSCs) are cell lines strayed from normal route from somatic cells to iPSCs and are immortalized. Mouse partial iPSCs are able to convert to real iPSCs by the exposure to 2i condition using MAPK and GSK3? inhibitors. However, the molecular mechanisms of this conversion are totally not known. Our piggyback vector mediated genome-wide screen revealed that Cnot2, one of core components of Ccr4-Not complex participates in this conversion. Subsequent analyses revealed other core components, i.e., Cnot1 and Cnot3 and Trim28 which is known to extensively share genomic binding sites with Cnot3 contribute to this conversion as well. Our bioinformatics analyses indicate that the major role of these factors in the conversion is the down-regulation of developmental genes in partial iPSCs. Two partial iPSC clones (2B1 and 5C5) cultured under conventional culture condition with leukemia inhibitory factor (LIF) and serum and those converted to iPSCs by the exposure to 2i condition were used for RNA source. Partial iPSCs (2B1) cultured under conventional condition in which either one of Cnot1, Cnot2, Cnot3 and Trim28 cDNA or these factors were combinatorially incorporated after retrovirus infection and those in which empty vector or Nanog were stably integrated were also used for RNA source. In addition, embryonic fibroblasts and embryonic stem cells (ESCs) from 13.5 dpc embryos and blastocysts, respectively, from Nanog-GFP transgenic mice and wild-type ESC line (EBRTcH3) were used for RNA source.
2014-01-07 | E-GEOD-44339 | biostudies-arrayexpress
Project description:The multi-subunit CCR4 (carbon catabolite repressor 4)-NOT (Negative on TATA) complex serves as a central coordinator of all different steps of eukaryotic gene expression. Accordingly, members of the CCR4-NOT complex have been implicated in a variety of biological functions. Here we performed a systematic and comparative analysis of cells where the CCR4-NOT subunits CNOT1, CNOT2 or CNOT3 were individually downregulated using doxycycline-inducible shRNAs. Microarray experiments showed that downregulation of either CNOT subunit resulted in elevated expression of major histocompatibility complex class II (MHC II) target genes which are found in a gene cluster on chromosome 6. Increased expression of MHC II genes after knock-down or knock-out of CNOT subunits was seen in a variety of cell systems and also in naïve macrophages from CNOT3 conditional knock-out mice. CNOT2-mediated repression of MHC II genes occurred independent from the master regulator class II transactivator (CIITA) and detectable changes of the chromatin structure at the chromosomal MHC II locus. CNOT2 downregulation resulted in an increased de novo transcription of mRNAs and tethering of CNOT2 to a regulatory region governing MHC II expression resulted in dimished transcription. These results expand the known repertoire of CCR4-NOT members for immune regulation and identify CNOT proteins as a novel group of corepressors serving to restrict inappropriate or exaggerated class II expression, which can be causative for various diseases.
2017-03-15 | GSE90474 | GEO
Project description:mRNA degradation critically contributes to tissue development and function as well as transcription. The CCR4-NOT complex serves as a major deadenylase that initiates mRNA degradation. We used microarrays to identify deregulated genes in the white adipose tissues lacking CNOT3, a core subunit of the CCR4-NOT complex.
2017-01-01 | GSE86930 | GEO
Project description:The CNOT3 protein is a subunit of the CCR4-Not complex, a cytoplasmic protein complex mediating mRNA deadenylation as the first step in mRNA degradation. We recently identified CNOT3 loss-of-function mutations in patients with T-cell acute lymphoblastic leukemia (T-ALL). However, it is not clear yet how loss of this gene is involved in cancer development. Here we use different Drosophila melanogaster eye cancer models to study the potential tumor suppressor function of Not3, the CNOT3 orthologue, as well as other members of the CCR4-NOT complex. We show that not only Not3 but also the structural components Not1 and Not2, as well as the deadenylases Twin and Pop2 all behave as tumor suppressor genes. Moreover, RNA-sequencing analyses revealed that Not3 loss leads to increased expression levels of genes involved in DNA replication and ribosome biogenesis pathways, and that CycB is a Not3 target gene in our cancer models. Furthermore, we were able to confirm those results on human T-ALL cell lines, pointing out the conserved function of Not3 in both model systems and in tumor development. Together, our data establish a critical role for Not3 and the entire CCR4-NOT complex as a tumor suppressor.
2017-09-15 | GSE103872 | GEO
Project description:Somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) by overexpression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc. Bmi1 is responsible for conversion of adult mouse astrocytes and fibroblasts into neural stem cell-like cells and conversion of fibroblasts into iPSCs under enforced expression of Oct4. Here, in the first step of generating iPSCs, stable intermediate cells were generated from mouse astrocytes by Bmi1 in the absence of other transcription factors. These cells [called induced epiblast stem cell (EpiSC)-like cells (iEpiSCLCs)] are similar to EpiSCs in terms of expression of specific markers, epigenetic state, and ability to differentiate into three germ layers. Treatment with MEK/ERK and GSK3 pathway inhibitors in the presence of leukemia inhibitory factor resulted in conversion of iEpiSCLCs into iPSCs that were similar to mouse embryonic stem cells (mESCs), suggesting that Bmi1 is sufficient to reprogram astrocytes to pluripotency. Next, Bmi1 function was replaced with Shh activators (oxysterol and purmorphamine), which demonstrated that specific combinations of small molecules alone can reprogram mouse fibroblasts into iPSCs. These iPSCs resembled mESCs in terms of global gene expression profile, epigenetic status, and developmental potential, demonstrating that combinations of small molecules can compensate for reprogramming factors and are sufficient to directly reprogram mouse somatic cells into iPSCs. 4 Affymetrix and 4 Agilent samples
2013-06-29 | E-GEOD-48411 | biostudies-arrayexpress
Project description:Somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) by overexpression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc. Bmi1 is responsible for conversion of adult mouse astrocytes and fibroblasts into neural stem cell-like cells and conversion of fibroblasts into iPSCs under enforced expression of Oct4. Here, in the first step of generating iPSCs, stable intermediate cells were generated from mouse astrocytes by Bmi1 in the absence of other transcription factors. These cells [called induced epiblast stem cell (EpiSC)-like cells (iEpiSCLCs)] are similar to EpiSCs in terms of expression of specific markers, epigenetic state, and ability to differentiate into three germ layers. Treatment with MEK/ERK and GSK3 pathway inhibitors in the presence of leukemia inhibitory factor resulted in conversion of iEpiSCLCs into iPSCs that were similar to mouse embryonic stem cells (mESCs), suggesting that Bmi1 is sufficient to reprogram astrocytes to pluripotency. Next, Bmi1 function was replaced with Shh activators (oxysterol and purmorphamine), which demonstrated that specific combinations of small molecules alone can reprogram mouse fibroblasts into iPSCs. These iPSCs resembled mESCs in terms of global gene expression profile, epigenetic status, and developmental potential, demonstrating that combinations of small molecules can compensate for reprogramming factors and are sufficient to directly reprogram mouse somatic cells into iPSCs.
2013-06-29 | GSE48411 | GEO
Project description:Shortening of polyadenosine (poly(A)) tails (deadenylation) is the initial step in the decay of most mRNAs. The CCR4-NOT complex is the major deadenylase in mammals. Interaction of Cnot1 with RNA binding proteins, including the miRNA-induced silencing complex (miRISC), which includes Argonaute (Ago) and GW182 as core proteins, and the TTP family of AU-rich element (ARE)-binding proteins (TTP and BRF1/2), recruits the CCR4-NOT complex to the 3’-untranslated regions (3’-UTR) of mRNAs, leading to their degradation. This experiment aimed to determine the binding affinity of the CCR4-NOT complex, Ago2, and BRF1 with their target mRNAs in liver. Liver isolated from C57BL/6J wild-type mice at 8 weeks of age was solubilized in TNE buffer for 30 min at 4ºC. Lysate was incubated with antibodies against Cnot3, Ago2, and BRF1 for 1 hr at 4ºC, and then incubated with Dynabeads (Invitrogen) for 2 hr at 4ºC. mRNAs in immune complexes were isolated using Isogen II. 100 ng of total RNA was used for RNA-seq library preparation with TruSeq Stranded mRNA Library Prep Kit for NeoPrep. 150 base-pair pair-end read RNA-seq was performed with Hiseq 3000/4000 PE Cluster Kit and Hiseq 3000/4000 SBS Kit on Hiseq4000.
2020-03-30 | E-MTAB-6941 | biostudies-arrayexpress
Project description:Induced pluripotent stem (iPS) cell reprogramming is a gradual epigenetic process that reactivates the pluripotent transcriptional network by erasing and establishing heterochromatin marks. Here, we characterize the physical structure of heterochromatin domains in full and partial mouse iPS cells by correlative Electron Spectroscopic Imaging (ESI). In somatic and partial iPS cells, constitutive heterochromatin marked by H3K9me3 is highly compartmentalized into chromocenter structures of densely packed 10 nm chromatin fibers. In contrast, chromocenter boundaries are poorly defined in pluripotent ES and full iPS cells, and are characterized by unusually dispersed 10 nm heterochromatin fibers in high Nanog-expressing cells, including pluripotent cells of the mouse blastocyst prior to differentiation. This heterochromatin reorganization accompanies retroviral silencing during conversion of partial iPS cells by Mek/Gsk3 2i inhibitor treatment. Thus, constitutive heterochromatin reorganization serves as a novel biomarker with retroviral silencing for identifying iPS cells in the very late stages of reprogramming.
2011-09-22 | GSE21595 | GEO
Project description:Induced pluripotent stem (iPS) cell reprogramming is a gradual epigenetic process that reactivates the pluripotent transcriptional network by erasing and establishing heterochromatin marks. Here, we characterize the physical structure of heterochromatin domains in full and partial mouse iPS cells by correlative Electron Spectroscopic Imaging (ESI). In somatic and partial iPS cells, constitutive heterochromatin marked by H3K9me3 is highly compartmentalized into chromocenter structures of densely packed 10 nm chromatin fibers. In contrast, chromocenter boundaries are poorly defined in pluripotent ES and full iPS cells, and are characterized by unusually dispersed 10 nm heterochromatin fibers in high Nanog-expressing cells, including pluripotent cells of the mouse blastocyst prior to differentiation. This heterochromatin reorganization accompanies retroviral silencing during conversion of partial iPS cells by Mek/Gsk3 2i inhibitor treatment. Thus, constitutive heterochromatin reorganization serves as a novel biomarker with retroviral silencing for identifying iPS cells in the very late stages of reprogramming. We compared the expression profiles of partially and fully reprogrammed iPS cell lines derived from CD1 mouse embryonic fibroblasts (MEFSs) by retroviral reprogramming (pMX-Oct4, pMX-Klf4 and pMX-Sox2). to the differentiated MEFS and the J1 embryonic stem cell line. We also studied the effect of a 2i cocktail treatment in partially reprogrammed iPS cells.
2011-09-21 | E-GEOD-21595 | biostudies-arrayexpress
Project description:In addition to transcriptional regulation, mRNA degradation critically contributes to gene expression as shown by various biological analysis. The CCR4-NOT complex serves as a major deadenylase that initiates mRNA degradation. We used microarrays to identify CCR4-NOT targets by searching for genes specifically stablized in the absence of CNOT3, a core subunit of the CCR4-NOT complex. Primary mouse embryonic fibroblasts (MEFs) were used for RNA extraction and hybridization on Affymetrix microarrays. CNOT3-knockdown MEFs were generated by Cre-mediated somatic recombination. We treated both control and CNOT-knockdown (CNOT3KD) MEFs with a transcriptional inhibitor, actinomycin D to focus on a stability of mRNAs.
2015-09-01 | E-GEOD-67418 | biostudies-arrayexpress