RNA methylation destabilizes developmental regulators in murine embryonic stem cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Yue Li
PROVIDER: E-GEOD-46880 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:m6A-seq of mouse embryonic stem cell wildtype or mutant depleted of Mettl3 or Mettl14 m6A-mRNA library for mouse embryonic stem cell each having one biological replicate were generated using HiSeq2000 v3 flowcell (Illumina) and sequenced for 100 bases with separate 7 base indexing read in a single lane.
2014-01-06 | E-GEOD-46878 | biostudies-arrayexpress
Project description:Although internal PolyA RNA modification N6-methyladenosine (m6A) plays essential roles in diverse biological processes, technology to detect precise m6A sites at transcriptome-wide scale is lacking. Here, we discovered that m6A interferes A (Adenine) – U (Uracil) or A-T (Thymidine) pairing. Based on differential hybridization between methylated vs. unmethylated RNAs to a DNA probe, we developed tiling microarray to pinpoint m6A sites in mouse transcriptome. We validated some of the identified sites and provided evidence to suggest that one functional mechanism of m6A is to block small RNA targeting to methylated mRNA. We designed a custom tiling array with to examine the precise location of m6A within meRIP-seq peaks from mouse embryonic stem cells determined in our previous publication (Wang et al., 2014). Each custom two-channel Agilent tiling array harbors 947,952 probes. Each probe is 25 nucleotides (nt), and any two adjacent probes in the genomic coordinate overlap each other by 19 nt. The Cy5 or red channel corresponds to Mettl14 knockout (M14) or DZA mutant mESC cell line, and Cy3 or green channel is associated with wild type cell line treated with scramble hairpin (SCR). Thus, in principle a higher Cy5/Cy3 signal for each probe reflects an increased hybridization to the oligonucleotide due to de-methylation of a particular RNA molecule in M14 or DZA condition relative to the SCR control. Moreover, we employed additional arrays with both channels dedicated for M14 as an external control for technical difference between the Cy5 and Cy3 dye (details below). For each comparison, we have three biological replicates, and therefore there are 9 tiling arrays in total (i.e., 3 arrays for M14 vs SCR, 3 arrays for DZA vs SCR, and 3 arrays for M14 vs M14).
2015-09-08 | E-GEOD-62530 | biostudies-arrayexpress
Project description:Recent methylome studies have located N6-methyladenosine (m6A) RNA modification on thousands of mammalian transcripts. However, its functional mechanism remains unclear. In this study, we examined the role of m6A methylation in mouse embryonic stem cells. To gain an understanding of dynamic changes in cells depleted with (proposed) methyltranferases at molecular level, we examined the time-series gene expression pattern in mESC lines using microarray analysis. After 0, 4 and 8 h incubation with scramble, shRNA for Mettl3 or Mettl14, mESC cells were collected and RNAs were isolated for microarray analysis using Affymetrix Genechip Mouse Gene 2.0 ST array. After 0 h incubation with scramble, shRNA for Mettl3 or Mettl14, mESC cells were collected and RNAs were isolated for microarray analysis using Affymetrix Genechip Mouse Gene 2.0 ST array.
2014-01-06 | E-GEOD-46879 | biostudies-arrayexpress
Project description:Enhancers play a central role in precisely regulating the spatiotemporal expression of developmentally regulated genes. The molecular mechanisms and factors required for controlling the inactive or poised enhancers and their function in future gene activation remain elusive. Here, we have identified that Ell3, a member of the Ell (Eleven-nineteen Lysine-rich Leukemia gene) family of RNA Pol II elongation factors, predominately occupies active and inactive enhancers of many developmentally regulated genes in mouse embryonic stem cells. Ell3 localizes to active enhancers of key stem cell renewal genes, but is not required for maintaining the undifferentiated state. Instead, Ell3 binding to inactive or poised enhancers is essential for marking them for stem cell specification through regulating the activation of key lineage-specific genes throughout differentiation. Ell3M-bM-^@M-^Ys association with enhancers is required for setting up proper Pol II occupancy at the proximal promoter regions of neighboring genes. This functional interaction between Ell3 and proximal promoter Pol II is dependent on cohesin. The depletion of cohesin subunits removes Ell3 from the enhancers and results in reduction in the levels of promoter proximally paused Pol II. Our study demonstrates that Ell3 is required for stem cell fate specification through regulating Pol II pausing on key developmental genes in the undifferentiated state and for their proper expression during differentiation. ChIP-seq of Ell3 in mES cells. ChIP-seq of Pol II in mES cells after Ell3 shRNA and non-targeting shRNA. RNA-seq of mES cells after Ell3 shRNA and non-targeting shRNA. RNA-seq of mouse embryoid bodies at day five after non-targeting shRNA.
2012-12-30 | E-GEOD-38148 | biostudies-arrayexpress
Project description:The tumor suppressor and deubiquitinase (DUB) BAP1 regulates chromatin-associated processes and is frequently mutated in various malignancies. BAP1 and its drosophila orthologue Calypso assemble DUB complexes with ASXL-1, -2, -3 paralogues and ASX respectively, and these cofactors are required for stimulating their DUB activity. However how the DUB activity of BAP1 is regulated remains largely unknown. Here we show that BAP1 promotes monoubiquitination of ASXLs on the ASXM/DEUBAD domain. ASXL2 monoubiquitination promotes its stability or proteasomal degradation, stimulates BAP1 DUB activity and is required for mammalian cell proliferation. Monoubiquitination of ASXL2 is directly catalyzed by UBE2E family of ubiquitin conjugating enzymes and is regulated by deubiquitination. Monoubiquitination of ASX is regulated by Calypso and is required for drosophila development. We further revealed a switch mechanism that tightly regulate BAP1 function, as a monoubiquitination of BAP1 UCH domain is mutually exclusive with ASXL2 monoubiquitination, thus ensuring highly coordinated DUB-mediated signaling.
2018-09-28 | PXD011124 | Pride
Project description:Chromatin modifications have been implicated in the self-renewal and differentiation of embryonic stem cells (ESCs). However, the function of histone variant H2A.Z in ESCs remains unclear. We show that H2A.Z is highly enriched at promoters and enhancers and is required for both efficient self-renewal and differentiation of murine ESCs. H2A.Z deposition leads to an abnormal nucleosome structure, decreased nucleosome occupancy and increased chromatin accessibility. In self-renewing ESCs, knockdown of H2A.Z compromises OCT4 binding to its target genes and leads to decreased binding of MLL complexes to active genes and of PRC2 complex to repressed genes in self-renewal of ESCs. During differentiation of ESCs, inhibition of H2A.Z also compromises RA-induced RARα binding, activation of differentiation markers and the repression of pluripotency genes. We propose that H2A.Z mediates such contrasting activities by acting as a 'general facilitator' that generates access for a variety of complexes both activating and repressive. ChIP-Seq in murine embryonic stem (mES) cells for H2A.Z and acetylated H2A.Z. ChIP-Seq of H3K4me3, H3K27me3, RbBP5, SUZ12 and OCT4 for mES cells of both H2A.Z RNAi knockdown and shLuc control. ChIP-Seq of RARalpha in H2A.Z knockdown (withdraw of LIF and exposure to RA for 3h) and control cells. MNase-Seq and chromatin accessibility assay using Benzonase digestion followed by next-generation sequencing for mES cells of both H2A.Z RNAi knockdown and shLuc control. ChIP-Seq of H2A.Z and H3K4me3 for mES cells of both MLL4 RNAi knockdown and shLuc control. RNA-Seq for mES cells of H2A.Z knockdown and shluc control. RNA-Seq for embryonic bodies derived from mES cells (H2A.Z knockdown and shLuc control) at day 3 and day 7.
2012-12-20 | E-GEOD-34483 | biostudies-arrayexpress
Project description:Embryonic stem (ES) cells were differentiated in culture to midbrain dopaminergic (mDA) progenitors and subjected to ChIP-seq analysis to resolve genome-wide binding sites of forkhead box protein A2 (Foxa2). Foxa2 was found to directly regulate multiple lineage pathways to specify midbrain dopaminergic and floor plate progenitor identity.
2012-07-01 | E-MTAB-1137 | biostudies-arrayexpress
Project description:Network-based analysis of neuroblastoma samples from two large cohorts identified master regulator proteins controlling the transcriptional state of three high-risk molecular subtypes. In particular, a TEAD4-MYCN positive feedback loop emerged as the core regulatory motif of a small protein module presiding over implementation and stability of the subtype associated with MYCN amplification. Specifically, MYCN transcriptionally activates TEAD4, which in turn activates MYCN both transcriptionally and post-translationally. The resulting MYCN-TEAD4 positive feedback loop plays a critical role in maintaining aberrant activity of a 10-protein regulatory module that causally regulates the transcriptional state of this subtype. Consistently, loss of TEAD4 activity induces core module activity collapse and abrogates neuroblastoma cell viability in vitro and in vivo, thus suggesting novel therapeutic strategies for this important childhood cancer. Study of the transcriptional control by TEAD4 and MYCN positive feedback loop using RNA-seq profiles of TEAD4 and MYCN shRNA knockdowns in neuroblastoma BE2 cells. ChIP-Seq analysis using TEAD4 antibody in BE2 cells.
2016-07-18 | E-GEOD-84389 | biostudies-arrayexpress
Project description:We report that TAF3, a TBP-associated core promoter factor, is highly enriched in ES cells. In addition to its role in the core promoter recognition complex TFIID, genome-wide binding studies reveal that TAF3 localizes to chromosomal regions bound by CTCF and cohesin. Enrichment for TAF3/CTCF/cohesin bound regions distinguishes TAF3-activated from TAF3-repressed genes. Our findings support a new role of TAF3 in mediating long-range chromatin regulatory interactions to safeguard the finely-balanced transcriptional programs that give rise to pluripotency. Comparison of genome-wide expression patterns between TAF3-knockdown and WT embryonic stem cells using mRNA-Seq. Significantly differentially expressed protein-coding genes were identified by comparing control and knock-down samples at each timepoint (ES, embryoid body day 3 (EB3), EB6). Single and paired-end samples were combined at each timepoint, resulting in 3 tests for each gene (based on 8, 4, 4 independent measurements at ES ,EB3, EB6, respectively).
2011-09-02 | E-GEOD-31271 | biostudies-arrayexpress
Project description:Although the function of DNA methylation in gene promoter regions is well established in transcriptional repression, the function of the evolutionarily conserved widespread distribution of DNA methylation in gene body regions remains incompletely understood. Here, we show that DNA methylation is enriched in included alternatively spliced exons (ASEs) and inhibiting DNA methylation results in aberrant splicing of ASEs. The methyl-CpG binding protein MeCP2 is enriched in included ASEs, particularly those that are also highly DNA methylated, and inhibition of DNA methylation disrupts specific targeting of MeCP2 to exons. Interestingly, ablation of MeCP2 results in increased nucleosome acetylation and aberrant skipping events of ASEs. We further show that inhibition of histone deacetylases leads to a highly significant overlap of exon skipping events caused by knocking-down MeCP2. Together, our data indicate that intragenic DNA methylation operates in exon definition to modulate alternative splicing and can enhance exon recognition via recruitment of the multifunctional protein MeCP2, which thereby maintains local histone hypoacetylation through its established interaction with HDACs. RNA-Seq in IMR90 and HCT116
2013-08-13 | E-GEOD-47488 | biostudies-arrayexpress