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ChIP-seq assays for H3K9me2

ABSTRACT: We analyzed the genome-wide chromatin states in Zscan4 positive ES cells (Em+) and Zscan4 negative ES cells (Em-) by using FACS-sorted MC1-ZE7 ES cells. H3K27 hyperacetylation and DNA demethylation were detected in heterochromatic regions of Em+ cells. These results suggested that the heterochromatin is activated in Zscan4 positive state. H3K9me2 in Zscan4 positive and negative cells

ORGANISM(S): Mus musculus

SUBMITTER: Minoru Ko

PROVIDER: E-GEOD-58617 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells.

Akiyama Tomohiko T Xin Li L Oda Mayumi M Sharov Alexei A AA Amano Misa M Piao Yulan Y Cadet J Scotty JS Dudekula Dawood B DB Qian Yong Y Wang Weidong W Ko Shigeru B H SB Ko Minoru S H MS

DNA research : an international journal for rapid publication of reports on genes and genomes 20150831 5

Mouse embryonic stem cells (mESCs) have a remarkable capacity to maintain normal genome stability and karyotype in culture. We previously showed that infrequent bursts of Zscan4 expression (Z4 events) are important for the maintenance of telomere length and genome stability in mESCs. However, the molecular details of Z4 events remain unclear. Here we show that Z4 events involve unexpected transcriptional derepression in heterochromatin regions that usually remain silent. During a Z4 event, we se ...[more]

PMID: 26324425

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ChIP-seq assays for H3K27ac

Project description:By using FACS-sorted MC1-ZE7 ES cells, we analyzed the genome-wide distribution of H3K27ac and DNA methylation in Zscan4 positive ES cells (Em+) and Zscan4 negative ES cells (Em-). H3K27 hyperacetylation and DNA demethylation were detected in heterochromatic regions of Em+ cells, but not Em- cells. These results suggested that the Zscan4 state takes "open" chromatin conformation in ES cells. H3K27ac in Zscan4 positive and negative cells

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Zscan4 mediates transient remodeling and transcriptional burst of heterochromatin in mouse embryonic stem cells

Project description:This SuperSeries is composed of the SubSeries listed below. Repetitive sequences such as telomeres, centromeres, and retrotransposons are packed in permanently-condensed and transcriptionally-silenced heterochromatin, whose maintenance is critical for the genome stability. We have recently found that mouse ES cells occasionally go through a unique cell state marked by the transient expression of Zscan4, which plays a key role in long-term genome stability. Here we report our unexpected and counterintuitive finding that the Zscan4+ state is accompanied by a Zscan4-mediated rapid activation and repression of heterochromatin: the burst of transcription from heterochromatin coincided with its transient histone hyperacetylation and DNA demethylation, the clustering of pericentromeric heterochromatin in the perinucleolar region, and the accumulation of chromatin remodeling complexes for both activation (HATs, SWI/SNF) and repression (HDACs, LSD1/KDM1A, NuRD) on heterochromatin. Overall, these findings suggest that ES cells maintain their extraordinary genome stability and immortality by transiently taking extreme measures – a usually forbidden transition to the activated state of heterochromatin. Refer to individual Series

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Project description:The HELP tagging assay was performed on purified genomic DNAs. The protocol was modified for NEBNext Multiplex Oligos for Illumina (NEB) from the original protocol4 (http://wasp.einstein.yu.edu/index.php/Protocol:HELP_tagging). Briefly, genomic DNA was digested by HpaII or MspI, the former only cutting at CCGG sequences where the central CG dinucleotide is unmethylated. AS and AE adapters were prepared by annealing two oligo DNAs separately. The first Illumina AE adapter was ligated to the compatible cohesive end created, juxtaposing an EcoP15I site beside the HpaII/MspI digestion site and allowing EcoP15I to digest within the flanking genomic DNA sequence. An A-overhang was created, allowing the ligation of the second Illumina AS adapter. This created both AE-insert-AS products and AS-insert-AS molecules. By performing a T7 polymerase-mediated in vitro transcription from a promoter sequence located on the AE adapter, we selectively enriched for the AE-insert-AS product. PCR amplification by NEBNext Multiplex Oligos was performed to generate a single-sized amplicons for Illumina sequencing. DNA methylation in Zscan4 positive and negative cells

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ChIP-seq assays for H3K9me2

Project description:We analyzed the genome-wide chromatin states in Zscan4 positive ES cells (Em+) and Zscan4 negative ES cells (Em-) by using FACS-sorted MC1-ZE7 ES cells. H3K27 hyperacetylation and DNA demethylation were detected in heterochromatic regions of Em+ cells. These results suggested that the heterochromatin is activated in Zscan4 positive state.

2015-09-10 | GSE58617 | GEO

Genome-wide DNA methylation analyses by the MeDIP assay

Project description:We analyzed the genome-wide DNA methylation in Zscan4 overexpressing ES cells. Zscan4 overexpression induced slight DNA demethylation in telomere and major satellite regions. Subsequent genome-wide analysis of non-repeated regions revealed the significant reduction of DNA methylation at Zscan4-dependent hyperacetylation sites. This result indicates that Zscan4 is not only a marker of the Zscan4+ state of ES cells, but also indispensable for the dramatic epigenetic modifications occurring in the Zscan4+ state. DNA methylation in Zscan4 overexresssing ES cells

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Rejuvenation of pluripotent stem cells by frequent activation of Zscan4, Part B

Project description:The gold standard for examining pluripotency of stem cells is to see whether cells can contribute to entire body of animals. Here we show that the increased frequency of Zscan4 activation in mouse ES cells not only enhances, but also maintains their developmental potency in long-term cell culture. As the potency increases, even a whole animal can be produced from a single ES cell injected into 4N blastocyst at unusually high success rate. Although Zscan4-activated cells express genes that are also expressed in 2-cell stage mouse embryos, transiently Zscan4-activated state of ES cells is not associated with the high potency of ES cells. It is thus concluded that ES cells acquire higher potency by going through transient Zscan4 activation state more frequently than the regular state. Taken together, our results indicate that frequent activation of Zscan4 can rejuvenate pluripotent stem cells. Previously, we have shown that constitutive and strong expression of Zscan4 arrest the proliferation of ES cells (refs). We sought for the system to mimic the transient expression of Zscan4 and decided to use ERT2 - Tamoxifen-inducible system (refs). The system allows one to keep a transgene off in the absence of Tmx and turn on in the presence of Tmx at will (refs). We first made a plasmid construct pCAG-Zscan4-ERT2, in which ZSCAN4 ORF fused with ERT2 domain can be driven by a strong ubiquitous promoter CAG. To further investigate this, we transfected pCAG-Zscan4-ERT2 plasmid into an F1 hybrid (C57BL/6J vs. 129) ES cell line V6.5 and isolated multiple cell clones. Based on the qRT-PCR analysis of Zscan4 ORF, we found that cell clones showed a variety of Zscan4 expression levels. We selected the clone that showed the highest Zscan4 expression levels (clone #18) and a clone #2 with the background Zscan4 level.

2013-06-12 | E-GEOD-26162 | biostudies-arrayexpress

Project description:A small number of transcription factors, including Oct-3/4 and Sox2, constitute the transcriptional network that maintains pluripotency in embryonic stem (ES) cells. Previous reports suggested that some of these factors form a complex that binds the Oct-Sox element, a composite sequence consisting of closely juxtaposed Oct-3/4-binding and Sox2-binding sites. However, little is known regarding the components of the complex. In this study, we show that Sall4, a member of the Spalt-like family of proteins, directly interacts with Sox2 and Oct-3/4. Sall4 in combination with Sox2 or Oct-3/4 simultaneously occupies the Oct-Sox elements in mouse ES cells. Sall4 knockdown led to differentiation of ES cells. Overexpression of Sall4 in ES cells increased reporter activities in a luciferase assay when the Pou5f1- or Nanog-derived Oct-Sox element was included in the reporter. Microarray analyses revealed that Sall4 and Sox2 bound to the same genes in ES cells significantly more frequently than expected from random coincidence. These factors appeared to bind the promoter regions of a subset of the Sall4- and Sox2-double-positive genes in precisely similar distribution patterns along the promoter regions, suggesting that Sall4 and Sox2 associate with such Sall4/Sox2-overlapping genes as a complex. Importantly, gene ontology analyses indicated that the Sall4/Sox2-overlapping gene set is enriched for genes involved in maintaining pluripotency. Sall4/Sox2/Oct-3/4-triple-positive genes identified by referring to a previous study identifying Oct-3/4-bound genes in ES cells were further enriched for pluripotency genes than Sall4/Sox2-double-positive genes. These results demonstrate that Sall4 contributes to the transcriptional network operating in pluripotent cells, together with Oct-3/4 and Sox2. ChIP-on-chip experiments using anti-Sall4 or anti-Sox2 antibody were performed.

2012-12-28 | E-GEOD-40072 | biostudies-arrayexpress

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