Differentiating i-Mixl1 mESCs cultured in the presence or absence of Doxycycline
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Dmitri Papatsenko
PROVIDER: E-GEOD-40703 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:The local protein composition of chromatin is important for the regulation of transcription and other functions. By integrative analysis of genome-wide binding maps of 53 broadly selected chromatin components in Drosophila cells, we show that the genome is segmented into five principal chromatin types that are defined by unique, yet overlapping combinations of proteins, and form domains that can extend over >100 kb. We identify a novel repressive chromatin type that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two distinct types that differ in molecular organization and H3K36 methylation, and regulate distinct classes of genes. Finally, we provide evidence that the different chromatin types act as guides that help to target DNA-binding factors to specific subsets of their recognition motifs. These results uncover basic principles of chromatin organization in a higher eukaryote. For this study, we generated whole-genome DamID binding profiles of 45 chromatin proteins in Drosophila Kc167 cells. Additionally, we perused published binding data of 8 chromatin proteins and generated a binding profile of one exogenous (yeast) DNA binding factor in Kc167 cells. On the same array platform, we obtained ChIP-on-chip profiles of histone H3, H1, H3K9me2, H3K27me3, H3K4me2, and H3K79me3. See supplementary files below. Gene expression was measured by RNA tag profiling. See GeneCounts supplementary file below. [1] RNA tag sequences were optained on an Illumina GAII with the digital gene expression (DGE) module from duplicate RNA samples. [2] All DamID and ChIP experiments were done in Drosophila Kc167 cells in duplicate. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Every experiment was done in duplicate in the reverse dye orientation, where Dam-fusion material was hybridized over Dam-only material. For ChIP, immunoprecipitated material was hybridized over ChIP input material. 18 previously-submitted Samples were included in this study. 10 of 18 Samples have been renormalized for the GSE22069 study: GSM509087, GSM509088, GSM509089, GSM509090, GSM509091, GSM509092, GSM509093, GSM509094, GSM509095, GSM509096 New GSM accession numbers have been issued for these 10 samples. 8 of 18 Samples are identical in the original studies and in GSE22069: GSM423290, GSM423291, GSM423298, GSM423299, GSM493592, GSM493593, GSM509085, GSM509086 [3] The genomic locations in files GSE22069_norm_aggregated_discretized_tiling_arrays.txt and GSE22069_norm_aggregated_tiling_arrays.txt are relative to FlyBase release 5 (BDGP R5/dm3).
2010-10-01 | E-GEOD-22069 | biostudies-arrayexpress
Project description:In eukaryotes, neighboring genes can be packaged together in specific chromatin structures that ensure their coordinated expression. Examples of such multi-gene chromatin domains are well-documented, but a global view of the chromatin organization of eukaryotic genomes is lacking. To systematically identify multi-gene chromatin domains, we constructed a compendium of genome-scale binding maps for a broad panel of chromatin-associated proteins in Drosophila melanogaster. Next, we computationally analyzed this compendium for evidence of multi-gene chromatin domains using a novel statistical segmentation algorithm. We find that at least 34% of the fly genome is organized into chromatin domains, which often consist of dozens of genes. The domains are characterized by various known and novel combinations of chromatin proteins. The genes in many of the domains are coregulated during development and share biological functions. Furthermore, fewer chromosomal rearrangements occur inside chromatin domains than outside domains during evolution. Our results indicate that a substantial portion of the Drosophila genome is packaged into functionally coherent, multi-gene chromatin domains. This has broad mechanistic implications for gene regulation and genome evolution. Keywords: DamID DamID experiments for multiple chromatin proteins was performed in Drosophila cell cultures. Samples were hybridized to spotted cDNA arrays. Every experiment was repeated at least 2 times, with one sample in the reverse dye orientation. When hybridization was performed 4 times, two samples were hybridized in the reverse dye orientation.
2008-04-07 | E-GEOD-10219 | biostudies-arrayexpress
Project description:Specific interactions of the genome with the nuclear lamina (NL) are thought to assist chromosome folding inside the nucleus and to contribute to the regulation of gene expression. High-resolution mapping has recently identified hundreds of large, sharply defined lamina-associated domains (LADs) in the human genome, and suggested that the insulator protein CTCF may help to demarcate these domains. Here, we report the detailed structure of LADs in Drosophila cells, and investigate the putative roles of five insulator proteins in LAD organization. We found that of these five proteins, only SU(HW) binds preferentially at LAD borders and at specific positions inside LADs, while GAF, CTCF, BEAF-32 and DWG are mostly absent from these regions. By knockdown and overexpression studies we demonstrate that SU(HW) weakens LAD – NL interactions by a local antagonistic effect. Our results provide insights into the evolution of LAD organization and reveal a role for SU(HW) in the regulation of genome – NL interactions. DamID experiments for Lamin, CTCF, SU(HW), GAF, DWG, and BEAF-32, and for Lamin after overexpression and after knockdown of SU(HW), were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Every experiment was done in duplicate in the reverse dye orientation. The supplementary file 'GSE20311_DamID_norm_mean.txt' contains the mean log2(Dam-fusion/Dam-only) values of two replicates.
2011-01-24 | E-GEOD-20311 | biostudies-arrayexpress
Project description:Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem-cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila Kc cells. We found that Pc associates with large genomic regions of up to ~150kb in size, hereafter referred to as âPc-domainsâ. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 lysine 27 and in general transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains. Keywords: DamID, Chromatin immunoprecipitation, ChIP-chip To study PcG binding profiles we used DamID, which is based on the ability of a chromatin protein fused to E.coli DNA adenine methyltransferase (Dam) to methylate the native binding site of the chromatin protein. Dam-fusion proteins are expressed at very low levels to avoid mistargeting. Subsequently, methylated DNA fragments are isolated, labeled and hybridized to a microarray. Methylated DNA fragments from cells transfected with Dam alone served as reference. Genomic binding sites of the protein can then be identified based on the targeted methylation pattern. For detailed background information on DamID, see: van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet 27, 304-8 (2001); van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8 (2000).
2010-06-11 | E-GEOD-4564 | biostudies-arrayexpress
Project description:We attempted to identify candidate genes that are expressed more highly in the ICM than in TE cells. Mouse ES cells are cultured from the ICM, whereas mouse TS cells are cultured from the TE. Although these cells have been cultured in vitro, they represent the in vitro equivalents of the ICM and TE. Therefore, genes which are expressed more highly in ES than TS in microarray studies were good candidates for genes predominantly expressed in the ICM in blastocysts.
2005-01-01 | E-MNIA-73 | biostudies-arrayexpress
Project description:Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome positioning profiles were generated in untreated cells and cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.
2010-06-21 | E-GEOD-16883 | biostudies-arrayexpress
Project description:Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SUUR protein is found at late-replicating intercalary heterochromatin sites and in pericentric heterochromatin. We performed a genome-wide mapping of SUUR target genes in the non-polytenic Drosophila Kc cells by using DamID. This approach is based on the ability of a chromatin protein fused to Escherichia coli DNA adenine methyltransferase (Dam) to methylate the native binding site of the chromatin protein. Dam-fusion proteins are expressed at very low levels to avoid mistargeting. Subsequently, methylated DNA fragments are isolated, labeled (using Cy3 or Cy5) and hybridized to a microarray. Methylated DNA fragments from cells transfected with Dam alone served as reference. Genomic binding sites of the protein can then be identified based on the targeted methylation pattern. For detailed background information on DamID, see: van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet 27, 304-8 (2001); van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8 (2000). We generated both an N- and a C-terminal fusion of the full-length SuUR open reading frame with Dam (Dam-SUUR and SUUR-Dam, respectively). For each SUUR fusion protein we performed four independent replicates. We used for this study a cDNA array developed by the GeneCore facility in EMBL (Heidelberg, Germany), covering the DGC1 and DGC2 cDNA libraries from the Berkeley Drosophila Genome Project, which represents more than 70% of the coding Drosophila genome. We found that SUUR preferentially binds to genes that are transcriptionally silent and late replicated. We compared the SUUR binding profile to the binding profile of three PcG proteins, which are known to bind to many intercalary heterochromatin sites, and found that there is a significant overlap with Pc and esc, but less with Sce. A significant overlap is also detected with two markers of pericentric heterochromatin, the heterochromatin proteins HP1 and SU(VAR)3-9. Finally, we demonstrated that SUUR binding profile negatively correlates with DNA polytenization level in salivary gland polytene chromosomes. Taken together, these results suggest that SUUR modulates the level of underreplication by direct binding to intercalary and pericentric heterochromatin.
2007-07-04 | E-MEXP-863 | biostudies-arrayexpress
Project description:Synchronized C. elegans cultures of three geontypes -- wildype N2, daf-2(e1370) and sma-6(wk7) -- were prepared using standard techniques (http://cmgm.stanford.edu/~kimlab/index_methods.html). Live young adult worms were split between NG plates pre-seeded with the non-pathogenic E. coli strain OP50 or the Pseudomonas aeruginosa clinical isolate PA14 and incubated at 25C for 4 or 24 hours before harvesting. This experiment was repeated at least three times on independent occasions. cDNA probes were prepared from experimental samples and from reference mRNA extracted from mixed stage wild type worms grown at 25C, and were labeled with Cy3 or Cy5, as indicated. A reference experiement design type is where all samples are compared to a common reference. Elapsed Time: Time Infection: Exposure to the non-pathogenic E. coli strain OP50 or to the pathogenic Pseudomonas aeruginosa strain PA14 Strain Name: C. elegans wildtype strain N2 or the immune pathway mutants daf-2(e1370) or sma-6(wk7) Keywords: reference_design Computed
2008-11-05 | E-GEOD-13473 | biostudies-arrayexpress
Project description:We profiled gene expression changes in differentiating i-Mixl1 ES cells(Willey, Ayuso-Sacido et al., 2006, Blood 107(8): 3122-3130) cultured in the presence or absence of Doxycycline (DOX, 0.1 ug/ml, 3 replicates per treatment/time point). Total RNA was isolated from EBs harvested at day 2, 3 and 4 (DOX added 1 day after plating of ES cells). RNA (1 ug) was subjected to one round of linear amplification (RiboAmp System) to yield 10 ug of RNA. The RNA was indirectly labeled using amino allyl-dUTP('t Hoen, de Kort et al., 2003, Nucleic Acids Res. 31: e20), then conjugated with Cy3 or Cy5. The labeled RNAs were used to screen a 15K mouse developmental cDNA microarray(Tanaka, Jaradat et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97: 9127-9132). Pairwise analysis of hybridization results for EBs cultured with or without DOX was performed for samples harvested on each day. Spotfire(R) software was used for data management and filtering. Gene expression ratios were normalized after filtering the data to remove low-intensity and poor quality spots. Data obtained for replicate samples were in excellent agreement.
2015-09-01 | GSE40703 | GEO
Project description:Heterochromatin protein HP1 is thought to play key role in chromatin structure and gene regulation. We performed a genome-wide mapping of HP1 target genes in the non-polytenic Drosophila Kc cells by using DamID. This approach is based on the ability of a chromatin protein fused to Escherichia coli DNA adenine methyltransferase (Dam) to methylate the native binding site of the chromatin protein. Dam-fusion proteins are expressed at very low levels to avoid mistargeting. Subsequently, methylated DNA fragments are isolated, labeled (using Cy3 or Cy5) and hybridized to a microarray. Methylated DNA fragments from cells transfected with Dam alone served as reference. Genomic binding sites of the protein can then be identified based on the targeted methylation pattern. For detailed background information on DamID, see: van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet 27, 304-8 (2001); van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8 (2000). We performed three independent replicates. We used for this study a cDNA array developed by the GeneCore facility in EMBL (Heidelberg, Germany), covering the DGC1 and DGC2 cDNA libraries from the Berkeley Drosophila Genome Project, which represents more than 70% of the coding Drosophila genome.
2007-07-05 | E-MEXP-864 | biostudies-arrayexpress