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Gene expression analysis of Ash1l mutant mouse embryonic stem cells.

ABSTRACT: Using wild type and Ash1l deltaSET mutant embryonic stem cells, here we report differences of gene expression pattern under undifferentiated state and differentiated state. Interestingly, gene expression changes are frequently observed in a subset of gene group that is regulated by Polycomb group proteins. Examination of 2 cell types in 2 different conditions.

ORGANISM(S): Mus musculus

SUBMITTER: Takaho Endo

PROVIDER: E-GEOD-48419 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Ash1l methylates Lys36 of histone H3 independently of transcriptional elongation to counteract polycomb silencing.

Miyazaki Hitomi H Higashimoto Ken K Yada Yukari Y Endo Takaho A TA Sharif Jafar J Komori Toshiharu T Matsuda Masashi M Koseki Yoko Y Nakayama Manabu M Soejima Hidenobu H Handa Hiroshi H Koseki Haruhiko H Hirose Susumu S Nishioka Kenichi K

PLoS genetics 20131107 11

Molecular mechanisms for the establishment of transcriptional memory are poorly understood. 5,6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) is a P-TEFb kinase inhibitor that artificially induces the poised RNA polymerase II (RNAPII), thereby manifesting intermediate steps for the establishment of transcriptional activation. Here, using genetics and DRB, we show that mammalian Absent, small, or homeotic discs 1-like (Ash1l), a member of the trithorax group proteins, methylates Lys36 of histone ...[more]

PMID: 24244179

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Genome-wide analyses of the DRB response of the histone H3 Lys36 methylation state, as well as Ash1l occupancy in mouse embryonic stem cells.

Project description:Using wild type and Ash1l ?SET mutant embryonic stem cells, against a prevalent notion, here we report di-,tri-methylation of histone H3 Lys36 occur independently of ongoing transcription. Ash1l ?SET mutant shows the impaired methylation levels in broad range of genome. Intriguingly, data implicate that a binding of retinoic acid receptor to a certain genomic region promotes installation of the tri-methylation in the retinoic acid receptor-associated gene independent of its ongoing transcription. Examination of 2 different histone modifications in 2 cell types in 2 different conditions. Examination of Ash1l occupancy in 2 different conditions.

2013-11-20 | E-GEOD-48421 | biostudies-arrayexpress

Mll2 branch of the COMPASS family regulates bivalent promoters in mouse embryonic stem cells

Project description:Promoters of many developmentally regulated genes, in the embryonic stem cell state, have a bivalent mark of H3K27me3 and H3K4me3, proposed to confer precise temporal activation upon differentiation. Although Polycomb repressive complex 2 is known to implement H3K27 trimethylation, the COMPASS family member responsible for H3K4me3 at bivalently marked promoters was previously unknown. Here, we identify Mll2 (KMT2b) as the enzyme catalyzing H3K4 trimethylation at bivalently marked promoters in embryonic stem cells. Although H3K4me3 at bivalent genes is proposed to prime future activation, we detected no substantial defect in rapid transcriptional induction after retinoic acid treatment in Mll2-depleted cells. Our identification of the Mll2 complex as the COMPASS family member responsible for H3K4me3 marking at bivalent promoters provides an opportunity to reevaluate and experimentally test models for the function of bivalency in the embryonic stem cell state and in differentiation. ChIP-Seq in mouse embryonic stem (mES) cells for MLL2. ChIP-seq of H3K4me1, H3K4me3 and H3K27me3 for mES cells with RNAi against MLL2(shMLL2) and control (shGFP). ChIP-seq of H3K4me3 in mES cells with RNAi against MLL3 (shMLL3). RNA-seq of mES cells with RNAi against MLL2 and control (shGFP). RNA-seq of control mES cells (shGFP) or MLL2 RNAi mES cells (shMLL2) induced with RA for 6h and 12h.

2013-08-11 | E-GEOD-48172 | biostudies-arrayexpress

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Project description:Appropriate neural initiation of the pluripotent stem cells in the early embryos is critical for the development of the central nervous system. This process is regulated by the coordination of extrinsic signals and intrinsic programs. However, how the coordination is achieved to ensure proper neural fate commitment is largely unknown. Here, taking advantage of genome-wide ChIP-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) analyses, we demonstrate that the transcriptional factor Pou3f1 is an upstream activator of neural-promoting genes, and it is able to repress neural-inhibitory signals as well. Further studies revealed that Pou3f1 could directly bind neural lineage genes like Sox2 and downstream targets of neural inhibition signaling such as BMP and Wnt. Our results thus identify Pou3f1 as a critical dual-regulator of the intrinsic transcription factors and the extrinsic cellular signals during neural fate commitment. ChIP-seq assay was ultilized to characterize the targets of Pou3f1 on ESC differentiation day 2.

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Histone H3.3 is required for endogenous retroviral element silencing and genome stability [RNA-Seq]

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2015-05-05 | E-GEOD-59104 | biostudies-arrayexpress

E14RNA-seq

Project description:Ctbp2 regulates exit from pluripotency via silencing of embryonic stem cell active genes during differentiation. Gene expression profiles were analyzed for wild-type and Ctbp2-knockdown ESCs at both fferentiated(+LIF) and undifferentiated(-LIF) state.

2014-12-31 | E-MTAB-2030 | biostudies-arrayexpress

Single cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed by reduced representation bisulfite sequencing

Project description:We develop a single cell methylome analysis technique based on RRBS, which works robustly for mouse embryonic stem cells (mESCs), sperm, metaphase II oocytes, and zygotes. In total, 36 samples were analyzed, including 8 single mouse embryonic stem cells (mESCs), pooled-5, 10, 20 mESCs, bulk mESCs, 7 single sperms, 10 single pronuclei from 5 individual zygotes, 2 metaphase II oocytes, 2 the first polar bodies and 3 negative controls.

2013-08-31 | E-GEOD-47343 | biostudies-arrayexpress

m6A RNA Methylation Is Regulated by MicroRNAs and Promotes Reprogramming to Pluripotency

Project description:N6-methyladenosine (m6A) has been recently identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, but its features, regulatory mechanisms, and functions in cell reprogramming are largely unknown. Here, we report m6A modification profiles in the mRNA transcriptomes of four cell types with different degrees of pluripotency. Comparative analysis reveals several features of m6A, especially gene- and cell-type-specific m6A mRNA modifications. We also show that microRNAs (miRNAs) regulate m6A modification via a sequence pairing mechanism. Manipulation of miRNA expression or sequences alters m6A modification levels through modulating the binding of METTL3 methyltransferase to mRNAs containing miRNA targeting sites. Increased m6A abundance promotes the reprogramming of mouse embryonic fibroblasts (MEFs) to pluripotent stem cells; conversely, reduced m6A levels impede reprogramming. Our results therefore uncover a role for miRNAs in regulating m6A formation of mRNAs and provide a foundation for future functional studies of m6A modification in cell reprogramming. m6A-seq in ESC, iPSC, NSC and sertoli cells.

2015-03-10 | E-GEOD-52125 | biostudies-arrayexpress

Serum-based culture conditions provoke gene expression variability in mouse embryonic stem cells as revealed by single cell analysis

Project description:Variation in gene expression is an important feature of mouse embryonic stem cells (ESCs). However, the mechanisms responsible for global gene expression variation in ESCs are not fully understood. We performed single cell mRNA-seq analysis of mouse ESCs and uncovered significant heterogeneity in ESCs cultured in serum. Using novel computational approaches we define highly variable gene clusters; and reveal their distinct epigenetic characteristics. We show that bivalent genes are prone to expression variation. At the same time, we identify an ESC priming pathway that initiates the exit from the naÃ¯ve ESC state. Finally, we provide evidence that a large proportion of intracellular network variability is due to the extracellular culture environment. Serum free culture reduces cellular heterogeneity and transcriptome variation in ESCs. Single cell mRNA-seq analysis of ES cells cultured with serum

2015-12-09 | E-GEOD-75804 | biostudies-arrayexpress

Derivation and differentiation of haploid human embryonic stem cells [RNA-Seq 2]

Project description:Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to insure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but as of yet not from humans. Here we analyzed a large collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics such as self-renewal capacity and a pluripotency-specific molecular signature. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Intriguingly, we found that a haploid genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics, development and evolution. RNA sequencing analysis was performed on a total of 2 samples of in vitro fertilization (IVF) control embryonic stem cell lines.

2016-03-16 | E-GEOD-74953 | biostudies-arrayexpress

The landscape of accessible chromatin in mammalian pre-implantation embryos (RNA-Seq)

Project description:In mammals, extensive chromatin reorganization is essential for reprogramming terminally committed gametes to a totipotent state during preimplantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored. Here we report a genome-wide map of accessible chromatin in mouse preimplantation embryos using an improved assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) approach with CRISPR/Cas9-assisted mitochondrial DNA depletion. We show that despite extensive parental asymmetry in DNA methylomes, the chromatin accessibility between the parental genomes is globally comparable after major zygotic genome activation (ZGA). Accessible chromatin in early embryos is widely shaped by transposable elements and overlaps extensively with putative cis-regulatory sequences. Unexpectedly, accessible chromatin is also found near the transcription end sites of active genes. By integrating the maps of cis-regulatory elements and single-cell transcriptomes, we construct the regulatory network of early development, which helps to identify the key modulators for lineage specification. Finally, we find that the activities of cis-regulatory elements and their associated open chromatin diminished before major ZGA. Surprisingly, we observed many loci showing non-canonical, large open chromatin domains over the entire transcribed units in minor ZGA, supporting the presence of an unusually permissive chromatin state. Together, these data reveal a unique spatiotemporal chromatin configuration that accompanies early mammalian development. Mouse preimplantation embryos were obtained from crosses of C57BL/6N and DBA/2N. RNA-seq was performed in these embryos at various stages in preimplantation development.

2016-06-15 | E-GEOD-66582 | biostudies-arrayexpress

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