ChIP-on-chip analysis of Ring1B, Ring1A, H2AK119u1 and H3K27me3 in mouse ES cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Takaho Endo
PROVIDER: E-GEOD-38504 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Rybp binding in wild-type, Eed-KO, and Dnmt1-KO ES cells ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent-014716 and Agilent-014717, Palo Alto, Calif., USA). ESCs were subjected to ChIP assay using a Rybp antibody (Santa Cruz; H-115). Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification as described previously (van Bakel et al., 2008). Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions.
2012-04-03 | E-GEOD-32289 | biostudies-arrayexpress
Project description:Human embryonic stem cells (hESCs) are a powerful tool for modeling regenerative therapy. To search for the genes that promote hematopoietic development from human pluripotent stem cell, we overexpressed a list of hematopoietic regulator genes in human pluripotent stem cell-derived CD34+CD43- endothelial cells (ECs) enriched in hemogenic endothelium. Among genes tested, only SOX17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34+CD43+CD45-/low cells expressing a hemogenic endothelial maker VE-cadherin. SOX17 was highly expressed in CD34+CD43- ECs but at a low level in CD34+CD43+CD45- pre-hematopoietic progenitor cells (pre-HPCs) and CD34+CD43+CD45+ HPCs. SOX17-overexpressing cells formed sphere-like colonies and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies upon inactivation of SOX17. Global gene expression analyses revealed that the CD34+CD43+CD45-/low cells expanded upon overexpression of SOX17 are hemogenic endothelium-like cells developmentally placed between ECs and pre-HPCs. Of interest, SOX17 also reprogrammed both pre-HPCs and HPCs into hemogenic endothelium-like cells. Genome-wide mapping of SOX17 revealed that SOX17 directly activates transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation. Depletion of SOX17 in CD34+CD43- ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a critical role in priming hemogenic potential in ECs, thereby regulates hematopoietic development from hESCs. This SuperSeries is composed of the SubSeries listed below. ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent, Palo Alto, Calif., USA). MEFs were subjected to ChIP assay using a Ring1B antibody. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification. Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions. Human ES cells were differentiated for 6 days in EBs, then CD34+CD43-CD45- endothelial cells were isolated, plated onto OP9 cells, and transduced with the 4OH-tamoxifen (4OHT)-inducible 3M-CM-^WFLAG-tagged Sox17-ERT retrovirus. The cells were seeded on OP9 stromal cells and cultured in the presence of 4OH-tamoxifen. At day 27 of the co-culture with OP9 cells, CD34+CD43+CD45low hemogenic endothelium-like cells overexpressing Sox17-ERT were collected by CD34 magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip analysis. ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent, Palo Alto, Calif., USA). MEFs were subjected to ChIP assay using a Ring1B antibody. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification. Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions. Human ES cells were differentiated for 6 days in EBs, then CD34+CD43-CD45- endothelial cells were isolated, plated onto OP9 cells, and transduced with the 4OH-tamoxifen (4OHT)-inducible 3M-CM-^WFLAG-tagged Sox17-ERT retrovirus. The cells were seeded on OP9 stromal cells and cultured in the presence of 4OH-tamoxifen. At day 27 of the co-culture with OP9 cells, CD34+CD43+CD45low hemogenic endothelium-like cells overexpressing Sox17-ERT were collected by CD34 magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip analysis.
2012-12-14 | E-GEOD-38156 | biostudies-arrayexpress
Project description:Polycomb group (PcG) proteins mediate heritable but reversible silencing of developmental regulator genes by modifying their chromatin configuration. Accumulating evidence documents a role for PcG proteins in regulating higher order chromatin structures likely by their clustering, however, underlying mechanisms and its impact on transcriptional regulation remain obscure. In this study, we found that subnuclear clustering of PRC1 at canonical PcG target genes depended on head-to-tail polymerization property of SAM domain of Phc2 and likely Phc1. We show that Phc2-SAM polymerization limits the dynamic nature of PRC1, thereby promotes stable association of PRC1 with PcG target genes and contributes to their robust silencing. Our findings suggest a novel model by which SAM polymerization of Phc2 modulates the structural organization of PcG complexes to enable robust yet reversible PcG-mediated repression during development. ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent, Palo Alto, Calif., USA). MEFs were subjected to ChIP assay using a Ring1B antibody. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification. Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions.
2013-06-01 | E-GEOD-37526 | biostudies-arrayexpress
Project description:Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3) respectively. Compared to H3K27me3, localization and role of H2AK119ub1 is not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation. This SuperSeries is composed of the following subset Series: GSE38224: Expression data from Ring1A(-/-);Ring1B(fl/fl);R26::CreERT2 ES cells expressing either of mock, WT or mutant Ring1B construct before or after OHT treatment GSE38504: ChIP-on-chip analysis of Ring1B, Ring1A, H2AK119u1 and H3K27me3 in mouse ES cells Total RNAs were extracted from the respective ES cells, and were subjected to microarray analysis using Affymetrix GeneChip Mouse Genome 430A 2.0 arrays. ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent, Palo Alto, Calif., USA). MEFs were subjected to ChIP assay using various antibodies. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification. Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions.
2012-06-11 | E-GEOD-38650 | biostudies-arrayexpress
Project description:Polycomb group (PcG) proteins mediate heritable but reversible silencing of developmental regulator genes by modifying their chromatin configuration. Accumulating evidence documents a role for PcG proteins in regulating higher order chromatin structures likely by their clustering, however, underlying mechanisms and its impact on transcriptional regulation remain obscure. In this study, we found that subnuclear clustering of PRC1 at canonical PcG target genes depended on head-to-tail polymerization property of SAM domain of Phc2 and likely Phc1. We show that Phc2-SAM polymerization limits the dynamic nature of PRC1, thereby promotes stable association of PRC1 with PcG target genes and contributes to their robust silencing. Our findings suggest a novel model by which SAM polymerization of Phc2 modulates the structural organization of PcG complexes to enable robust yet reversible PcG-mediated repression during development. This SuperSeries is composed of the SubSeries listed below. Refer to individual Series ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent, Palo Alto, Calif., USA). MEFs were subjected to ChIP assay using a Ring1B antibody. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification. Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions.
2013-06-01 | E-GEOD-38154 | biostudies-arrayexpress
Project description:Overexpression of transcription factor Sox17 in human ES cells-derived endothelial cells enhances expansion of hemogenic endothelium-like cells. Human ES cells were differentiated for 6 days in EBs, then CD34+CD43-CD45- endothelial cells were isolated, plated onto OP9 cells, and transduced with the 4OH-tamoxifen (4OHT)-inducible 3×FLAG-tagged Sox17-ERT retrovirus. The cells were seeded on OP9 stromal cells and cultured in the presence of 4OH-tamoxifen. At day 27 of the co-culture with OP9 cells, CD34+CD43+CD45low hemogenic endothelium-like cells overexpressing Sox17-ERT were collected by CD34 magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip analysis.
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2010-09-13 | E-GEOD-22195 | biostudies-arrayexpress
Project description:Ectopic expression of homeodomain transcription factor HoxB4 in mouse ES cells enhances in vitro development of hematopoietic stem cells (HSCs). Mouse ES cells were differentiated for 6 days in EBs, then c-Kit+CD41+ hematopoietic precursor cells were isolated, plated onto OP9 cells, and transduced with the GFP control or 3M-CM-^WFLAG-tagged HoxB4 retrovirus. At day 12 of the co-culture with OP9 cells, c-Kit+ progenitor cells overexpressing HoxB4 were collected by magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip and microarray analysis. The purity of the recovered c-Kit+ cells was more than 95% as judged by flow cytometry.
2011-05-23 | E-GEOD-26653 | biostudies-arrayexpress
Project description:The Polycomb repressive complex 2 (PRC2) is important for the maintenance of stem cell pluripotency and suppression of cell differentiation by promoting histone H3 lysine 27 trimethylation (H3K27me3) and silencing expression of genes regulating differentiation. Here we show decreased expression of BRCA1 promotes EZH2 re-targeting genome wide and enhances H3K27me3 levels at PRC2 target loci in mouse ES cells. R1 mouse ES cells were transfected with control siRNA and Brca1 specific siRNA. At 48h after transfection, cells were harvested and subjected to ChIP assay with Ezh2 antibody. Brca1 wild-type (AB2.2) and Brca1 deficient (PL2F8) mouse ES cells were harvested and subjected to ChIP assay with Ezh2 antibody. Fragmented DNA obtained from ChIP assay was repaired by DNA Terminator End Repair Kit (Lucigen Corporation, Middleton, WI) and purified by QIAquick PCR purification kit. Then two unidirectional linkers JW102 and JW103 were annealed and ligated to the blunt ends DNA at 16M-BM-0C overnight. The purified products were amplified to 2 M-NM-<g by LM-PCR. NimbleGen Mouse ChIP 3x720K RefSeq Promoter Array (Roche) were used for the hybridization. Relative enrichments were calculated by dividing the normalized level of ChIP DNA to that of input DNA at corresponding locus. Labeling, hybridization and washing were carried out according to manufacturerM-bM-^@M-^Ys instructions. Experiments in AB2.2 and PL2F8 cells were performed in duplicate.
2013-07-01 | E-GEOD-37321 | biostudies-arrayexpress