Unique cell cycle-dependent variations in the pluripotent epigenetic landscape define novel cohorts of temporal expressed bivalent genes during hESCs differentiation
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Troy Whitfield
PROVIDER: E-GEOD-55502 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation.
Grandy Rodrigo A RA Whitfield Troy W TW Wu Hai H Fitzgerald Mark P MP VanOudenhove Jennifer J JJ Zaidi Sayyed K SK Montecino Martin A MA Lian Jane B JB van Wijnen André J AJ Stein Janet L JL Stein Gary S GS
Molecular and cellular biology 20151207 4
Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By ...[more]
Similar Datasets
Project description:genomic localization of the budding yeast cohesin complex was mapped in mitotically arrested cells by Mcd1 ChIP followed by hybridization to high-density tiled microarrays Cells were synchronized first in G1 and then released into media containing the microtubule poison nocodazole. Cells were fixed and processed for ChIP once arrested as large-budded cells. Immunoprecipitated DNA and total genomic DNA not subjected to immunoprecipitation were competitively hybridized to Nimblegen whole genome arrays.
2009-09-30 | E-GEOD-18021 | biostudies-arrayexpress
Project description:While it has been clearly established that well positioned H2A.Z-containing nucleosomes flank the nucleosome depleted region (NDR) at the transcriptional start site (TSS) of active mammalian genes 1,2, how this chromatin-based information is transmitted through the cell cycle is unknown. We show here that in trophoblast stem (TS) cells, the level of H2A.Z at promoters decreases during S phase coinciding with homotypic (H2A.Z/H2A.Z) nucleosomes flanking the TSS becoming heterotypic (H2A.Z/H2A). Surprisingly, these nucleosomes remain heterotypic at M phase. At the TSS, we identify an unstable heterotypic H2A.Z-containing nucleosome in G1 which, strikingly, is lost following DNA replication. These dynamic changes in H2A.Z at the TSS mirror a global expansion of the NDR at S and M which, unexpectedly, is unrelated to transcriptional activity. Coincident with the loss of H2A.Z at promoters, it is targeted to the centromere when mitosis begins. We surveyed whole genome expression using microarrays, in combination with H2A.Z ChIP-Seq (Illumina) data, to study the H2A.Z distribution on promoters of coding genes in different stages of the cell cycle in Trophoblast Stem (TS) cells. These data are published in Nekrasov et al., H2AZ inheritance during the cell cycle and it's impact on promoter organization and dynamics, Nature Structural and Molecular Biology (in press: accepted for publication on 24 Sep 2012). Mouse Trophoblast stem (TS) cells were grown in cell culture medium. By adding specific drugs cells were arrested at particular stages of the cell cycle: G1; S and M. Total RNA was extracted from TS cells arrested at G1; S and M stages of cell cycle using TRizol (Invitrogen) extraction protocol and purified further the by RNAeasy Extraction Kit (Qiagen). RNA quality was assesed on Bioanalyzer total RNA chip. RNA samples were reverse transcribed and resulted cDNA samples were subjected to DNA microarray analysis (in triplicate) using the GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Robust Multichip Average (RMA) correction and probe set summaries were obtained using Affymetrix Power Tools software annotation (Affymetrix) based on the mm9 mouse genome. Unless otherwise stated, all subsequent expression analyses (see Supplementary Methods) were performed in the R statistical computing environment (version 2.11.1).
2012-11-23 | E-GEOD-41370 | biostudies-arrayexpress
Project description:The SMC protein complexes safeguard genomic integrity through their functions in chromosome segregation and repair. The chromosomal localization of the budding yeast Smc5/6 complex here determined reveals that the complex works specifically on the duplicated genome in differently regulated pathways. One controls the association to centromeres and chromosome arms in unchallenged cells, the second regulates the association to DNA breaks, and the third directs the complex to the chromosome arm that harbors the ribosomal DNA arrays. The chromosomal interaction pattern predicts a function that becomes more important with increasing chromosome length, and that the complex's role in unchallenged cells is independent of DNA damage. Additionally, localization of Smc6 to collapsed replication forks indicates an involvement in their rescue. Altogether this shows that the complex maintains genomic integrity in multiple ways, and evidence is presented that the Smc5/6 complex is needed during replication to prevent the accumulation of branched chromosome structures. Keywords: ChIP-chip analysis ⢠The goal of the experiment - Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways. ⢠Keywords, for example, time course, cell type comparison, array CGH (the use of MGED ontology terms is recommended). Cell cycle, Double Strand Break Induction, time course after break induction, Saccharomyces cerevisiae, Chromosome VI array, Whole Genome Tiling Array, Smc5, Smc6, Nse1, Nse4, Nse5, Scc1, Scc2, Mre11, Rad53 ⢠Experimental factors Distribution of the Smc5/6 complex in the cell cycle, distribution of Smc5/6 complex before and after induction of a DNA double strand break on chromosome VI in G2/metaphase cells, distribution of Smc6 in the absence of functional Scc2, Mre11 or Rad53, distribution of Smc6 at collapsed replication forks, and distribution of Cohesin subunit Scc1 in G2/metaphase. Distribution was analyzed on Chromosome VI and/or whole genome arrays. Cells were grown in rich yeast cell extract media. All experiments were performed in cells with the same genetic background (Saccharomyces cerevisiae W303; ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3 RAD5) ⢠Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Cerevisiae chromosome VI array or Whole genome arrays were used. Total number of hybridization was 69. All description about hybridized samples is attached as the separate sheet. ⢠Quality control steps taken Duplication, confirmation using different tags, different subunits of the same complex, and time course after double-strand break induction. Checking of the ChIP fraction by Western blotting. Mock hybridisation of samples immunoprecipitated from cells containing no tag recognized by antibody used. ⢠Links to the publication, any supplemental websites or database accession numbers. http://tilingarray.bio.titech.ac.jp/smc56dsb Samples used, extract preparation and labelling: ⢠The origin of each biological sample Saccharomyces cerevisiae W303 (ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3 RAD5). ⢠Manipulation of biological samples and protocols used Strains containing flag-tagged versions of Smc6, Smc5, Nse1, Nse4, or Nse5 proteins were used. For synchronization in G1, alpha factor was added (Zymo research and Innovagen AB) at 3 µg/ml final concentrations to logarithmically growing cells, and G1 arrest was obtained at indicated temperatures for 2.5 h. For S-phase arrest, cells were grown to log phase and arrested in G1 with alpha factor for 2 h, and then incubated in the presence of 200 mM hydroxyurea (HU) (Sigma) for 30 min. Cells were then washed twice with rich medium containing 100µg/ml pronase (Calbiochem). Finally cells were resuspended in medium containing HU and pronase, and growth continued for indicated time periods. G2/M arrest was achieved 2.5 h after addition of benomyl (Sigma, 80µg/ml), to logarithmically growing cells. G1, S and G2/M arrests were checked microscopically and by FACS. DSBs were induced by activation of the HO endonuclease from the GAL1-10-promoter by addition of 2% galactose to cell cultures initially grown in 2% raffinose. ⢠Technical protocols for preparing the hybridization extract Extract preparation was processed following the Shirahige lab protocol (http://chromosomedynamics.bio.titech.ac.jp ). 5x108 cells were disrupted using Multi-Beads Shocker (MB400U, YASUI KIKAI, Osaka). Whole cell extract was sonicated to obtain 400-600 bp genomic DNA fragments. Anti-Flag monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Dynal, protein A Dynabeads) were used for chromatin immunoprecipitation. The immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. ⢠Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://chromosomedynamics.bio.titech.ac.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3'biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix). ⢠Measurement data and specifications: S. cerevisiae arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the cel files data and processed data files can be downloaded from GEO database or from our Web sites. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site at http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf. The analysis is available from K.S. on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp and for whole genome arrays to 100bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-value<=0.01 for chromosome VI and pvalue?0.0025 for whole genome array). Secondly, reliability of binding ratio was judged by change p-value (p-value<=0.01 for chromosome VI and p-value<=0.0025 for whole genome array). Thirdly, clusters consisting of at least 500bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. Under these conditions both types of arrays give essentially the same results for the binding profile. Repeated sequences were masked out and not included in the calculation of protein binding profile. The correlation coefficient of Smc6 binding profiles in G2/M from two independent experiments was 0.955, showing that the whole genome ChIP on chip data was reproducible. For comparison of the localization of Smc6 in different experiments and that of Smc6 and Scc1 on whole genome maps, peaks with signal to log ratio higher than 0.6 were chosen, and peaks which were distributed within a 5kb region was recognized as one peak. In the analysis of the number of Smc6 localization sites on each chromosome a 40 kb region spanning all centromeres were excluded. This region in addition to that downstream the rDNA repeats was also excluded from the analysis when comparing Smc6 localization in wild type, scc2-4 and scc1-73 cells. ⢠Array Design: General array design: in situ synthesized arrays by Affymetrix Chromosome VI S.cerevisiae: rikDACF, P/N# 510636 Whole Genome Array S.cerevisiae: S.cerevisiae Tiling1.0F Arrays, Part#520286 and Part#520280
2010-06-11 | E-GEOD-4808 | biostudies-arrayexpress
Project description:The transcription factor RUNX1 exhibits recurrent loss-of-function mutations in estrogen receptor-positive (ER+) breast cancer (BCa). Its knockdown in vitro decreased AXIN1 expression in estrogen-dependent manner. Consistently, RUNX1 and AXIN1 mRNA levels are strongly correlated in ER+, not ER- tumors. RUNX1 occupies AXIN1â??s second intron in living cells, abutting an ERa-binding site. Potentially promoting BCa progression, decreased AXIN1 expression after RUNX1 knockdown associated with upregulation of b-catenin, and this was preventable by AXIN stabilizers. Unlike in colon cancer, however, deregulation of b-catenin in BCa cells affect neither c-Myc, nor CCND1, nor G1/S cell cycle phase transition. Instead, cyclin B1 was decreased and the G2/M checkpoint was compromised as indicated by mitotic slippage in the presence of microtubule disruptors. Thus, combined analysis of the RUNX1 transcriptome, its cistrome, and differential mRNA expression in tumors with wild type versus mutant RUNX1, altogether highlight a role for the RUNX1/AXIN1/b-catenin axis in ER+ BCa. Significance: Three recent exome sequencing studies assigned to RUNX1 a BCa suppressor role. The present study begins to uncover the underlying molecular mechanisms, offers an explanation for the specificity to ER+ tumors, and marks AXIN1 as a therapeutic target for ER+/RUNX1- BCa. Examination of RUNX1 binding in MCF7 cells
2016-02-22 | E-GEOD-65313 | biostudies-arrayexpress
Project description:The budding yeast genome is marked by 250-350 origins of DNA replication. These origins are bound by the origin recognition complex (ORC) throughout the cell cycle. ORC has known DNA binding sequence preferences which, though necessary for binding, are not sufficient to fully specify a genomic locus as being bound by ORC, indicating that the cell must use additional chromosomal cues to specify ORC binding sites and origins of replication. Using high-throughput sequencing to precisely locate both ORC binding sites and nucleosome locations genome-wide, we find that a nucleosome depleted region (NDR) and precisely positioned nucleosomes are a ubiquitous feature of yeast replication origins. The ARS consensus sequence (ACS) and adjacent sequences are sufficient to maintain the nucleosome-free properties of the NDR. We use a temperature sensitive ORC1 mutant to demonstrate that ORC is required to maintain precisely positioned nucleosomes at origins of replication. These findings demonstrate the importance of local nucleosome positioning at replication origins, and that chromatin organization is an important determinant of origin selection. Examination of nucleosome positioning in wild-type and orc1-161ts mutant S. cerevisiae at room temperature and heatshock temperatures. Examination of ORC binding locations by ChIP-seq. All reported coordinates are based on the SGD genome build released 12/16/2005.
2010-03-19 | E-GEOD-16926 | biostudies-arrayexpress
Project description:Here we analysed the role of yeast Senataxin (Sen1) in coordinating replication with transcription and in protecting genome integrity. Senataxin is mutated in the two severe neurodegenerative diseases AOA2 and ALS4. We show that a fraction of Sen1/Senataxin DNA/RNA helicase associates with replication forks and protects the integrity of those fork encountering highly expressed RNAPII genes. sen1 mutants accumulate aberrant DNA structures and RNA-DNA hybrids while forks clash head-on with RNAPII transcription units and counteract recombinogenic events and accumulation of checkpoint signals. Nrd1, which acts togheter with Sen1 in trascription temination, is not recruited at replication forks. nrd1 mutants does not display replication defects, high genome instability and checkpoint activation observed in sen1 mutants The Sen1 function in replication can be therefore separable from its role in RNA processing. We propose a role for Sen1/Senataxin during chromosome replication in facilitating replisome progression across RNAPII transcribed genes thus preventing DNA-RNA hybrids accumulation when forks encounter nascent transcripts on the lagging strand template. Chip on chip analysis was carried out as described (Bermejo et al., 2011), employing anti-Flag monoclonal antibody M2 (Sigma-Aldrich) Labelled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.
2012-11-09 | E-GEOD-39139 | biostudies-arrayexpress
Project description:Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these functional defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly during DNA replication and analyse effects n histone modifications by mass spectrometry. Immediately upon CAF-1 depletion from human TERT-RPE-1 cells, we observe an acute cellular response affecting histone repertoire, global chromatin composition and transcriptional fidelity resulting in a p53-dependent cell cycle arrest. Our work reveals the immediate local and global consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered.
2024-11-14 | PXD050618 | Pride
Project description:The essential functions of the conserved Smc5/6 complex remain elusive. To uncover its roles in genome maintenance, we established Saccharomyces cerevisiae cell cycle-regulated alleles that enable restriction of Smc5/6 components to S or G2/M. Unexpectedly, the essential functions of Smc5/6 segregated fully and selectively to G2/M. Genetic screens that became possible with generated alleles identified processes that crucially rely on Smc5/6 specifically in G2/M: metabolism of DNA recombination structures triggered by endogenous replication stress, and replication through natural pausing sites located in late-replicating regions. In the first process, Smc5/6 modulates remodeling of recombination intermediates, cooperating with dissolution activities. In the second, Smc5/6 prevents chromosome fragility and toxic recombination instigated by prolonged pausing and the fork protection complex, Tof1-Csm3. Our results thus dissect Smc5/6 essential roles, and reveal that combined defects in DNA damage tolerance and pausing site-replication cause recombination-mediated DNA lesions, which we propose to drive developmental and cancer-prone disorders. BrdU incorporation profiles by ssDNA-BrdU IP on chip have been generated as described (Katou et al., 2003). Protein binding profiles by ChIP-chip analysis were generated as described (Bermejo et al., 2009). Labeled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.
2015-11-19 | E-GEOD-72241 | biostudies-arrayexpress
Project description:Chromatin organization must be maintained during cell proliferation to preserve cellular identity and genome integrity. However, DNA replication results in transient displacement of DNA bound proteins, and it is unclear how they regain access to newly replicated DNA. Using quantitative MS-based proteomics coupled to Nascent Chromatin Capture, we provide time resolved binding kinetics for thousands of proteins behind replisomes of mid-S replicated regions. This shows that most proteins regain access within the first 15 minutes after the passage of the fork. In contrast, 25% of the identified proteins do not, and this delay cannot be inferred from their known function, physicochemical properties, or nuclear abundance. A sample treated with nocodazole to block the cell cycle progression. The comparison between a normal cell cycle progression and a nocodazole treated sample shows that the reassociation onto chromatin of proteins with a delayed restoration can be regulated by the cell cycle progression and/or the post-replication time. In addition, we provide evidence that DNA replication not only disrupts but also promotes recruitment of transcription factors and chromatin remodellers, providing a significant advance in understanding how DNA replication could contribute to programmed changes of cell memory. Data available in this entry are related with the supplementary table 5 from our manuscript.
2023-02-02 | PXD038113 | Pride
Project description:au13-11_gravity - gravity - Cell cycle and cell proliferation are decoupled under altered gravity conditions. We have previously shown that semisolid cell cultures of Arabidopsis suffer overall genome changes in response to altered gravity and also that cell cycle stages duration is altered. By using synchronized cell cultures we will demonstrate the precise alterations in cell cycle duration and also the transcriptional signature in any of them. - Experiments consists on exposures of Arabidopsis cell cultures to 1g control/simulated microgravity (RPM) conditions. Asynchronous cells exposed for 14 h + Syncronous populations choosen to have an enrichment of cell cycle phases were used (being T7/T10 samples on G2 phase, T14/T16 samples on G1 phase). 6 dye-swap - time course,treated vs untreated comparison
2014-08-23 | E-GEOD-60473 | biostudies-arrayexpress