Chromatin immunoprecipitation of Oct4 in human embyonic stems cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Fran Lewitter
PROVIDER: E-MEXP-1687 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Polycomb group proteins are essential for early development in metazoans but their contributions to human development are not yet well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit Suz12 across the entire non-repeat portion of the genome in human embryonic stem (ES) cells. We found that Suz12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved non-coding elements in the vertebrate genome. We found that preferential activation of PRC2 target genes occurs during differentiation of ES cells into other cell types. The ES cell transcriptional regulators Oct4, Sox2 and Nanog co-occupied a significant subset of these genes, further supporting a link between repression of developmental regulators and stem cell pluripotency. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.
2006-06-14 | E-WMIT-7 | biostudies-arrayexpress
Project description:Understanding the transcriptional regulatory circuitry responsible for pluripotency and self-renewal in embryonic stem (ES) cells is fundamental to understanding human development and realizing the therapeutic potential of these cells. The transcription factor Oct4 and the chromatin-modifying Polycomb complex, key regulators of ES cell pluripotency and self-renewal, contribute to positive and negative control of a known set of protein-coding genes. MicroRNAs (miRNAs), non-coding transcripts that participate in post-transcriptional gene regulation are also important for normal pluripotency and self-renewal in ES cells, but there has been no systematic investigation of how miRNA expression is controlled by transcriptional regulators of ES cell identity. Here we identify promoters for miRNAs in the human and mouse genomes and describe the subset of these genes that are under the control of Oct4 and Polycomb in ES cells. We find that the majority of miRNAs that are uniquely or preferentially expressed in ES cells are bound by and dependent on Oct4. Oct4 also occupies a set of miRNA genes that are co-occupied by the Polycomb Group protein, Suz12. These miRNAs, repressed in ES cells, are later expressed in differentiated cells in a highly tissue-specific fashion, suggesting that they may contribute to cell-fate determinations. These data reveal how the core transcriptional regulatory circuitry of ES cells controls the miRNA expression program that contributes to pluripotency and self-renewal.
2010-12-01 | E-TABM-365 | biostudies-arrayexpress
Project description:The transcription factors Oct4, Sox2, and Nanog have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have identified Oct4, Sox2, and Nanog target genes using genome-scale location analysis. We found, surprisingly, that Oct4, Sox2, and Nanog co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also indicates that Oct4, Sox2, and Nanog collaborate to form regulatory circuitry in ES cells consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how Oct4, Sox2, and Nanog contribute to pluripotency and self-renewal.
2005-09-23 | E-WMIT-5 | biostudies-arrayexpress
Project description:Polycomb group (PcG) proteins are highly conserved from flies to mammals and many of these factors have essential roles in early embryonic development. PcG proteins comprise two multimeric complexes, the Polycomb Repressive complexes 1 and 2 (PRC1 and 2), which have been shown to repress transcription through epigenetic modification of chromatin structure. To gain insight into the role of Polycomb in early development, we have identified PRC1 and PRC2 target genes in mouse embryonic stem (ES) cells using genome-scale location analysis. We found that PRC2 occupies many genes that encode key regulators of development including those encoding transcription factors and components of signaling pathways. These genes are repressed and contain nucleosomes methylated at lysine 27 on histone H3. The majority of PRC2 bound and methylated target genes are co-occupied by PRC1 indicating that these complexes function at a similar set of genes in ES cells. Lack of PRC1 or PRC2 subunits in ES cells results in derepression of target genes and loss of pluripotency. These results provide insight into how PcG proteins contribute to the maintenance of stem cell identity.
2006-05-09 | E-WMIT-12 | biostudies-arrayexpress
Project description:Homologous sets of transcription factors direct conserved tissue-specific transcription, yet transcription factor binding events diverge rapidly between closely related species. We used hepatocytes from a Down syndrome mouse model containing human chromosome 21 to determine whether human genetic sequence or mouse nuclear environment primarily determines tissue-specific transcriptional regulation. Virtually all transcription factor binding locations, transcription initiation events and the resulting gene expression observed in human hepatocytes are recapitulated across the entire human chromosome 21 in the mouse nucleus. Thus, in homologous tissues, genetic sequence is largely responsible for directing transcriptional programs, and interspecies differences in epigenetics, cellular environment, and transcription factors themselves play secondary roles.
2008-06-11 | E-TABM-474 | biostudies-arrayexpress
Project description:Embryonic stem cells have a unique regulatory circuitry, largely controlled by the transcription factors Oct4, Sox2 and Nanog, which generates a gene expression program necessary for pluripotency and self-renewal (Boyer et al. 2005; Loh et al. 2006; Chambers et al. 2003; Mitsui et al. 2003; Nichols et al. 1998). How external signals connect to this regulatory circuitry to influence embryonic stem cell fate is not known. We report here that a terminal component of the canonical Wnt pathway in embryonic stem cells, the transcription factor Tcf3, co-occupies promoters throughout the genome in association with the pluripotency regulators Oct4 and Nanog. Thus Tcf3 is an integral component of the core regulatory circuitry of ES cells, which includes an autoregulatory loop involving the pluripotency regulators. Both Tcf3 depletion and Wnt pathway activation cause increased expression of Oct4, Nanog and other pluripotency factors and enhance pluripotency and self-renewal. Our results reveal that the Wnt pathway, through Tcf3, brings developmental signals directly to the core regulatory circuitry of ES cells to influence the balance between pluripotency and differentiation.
2008-02-28 | E-TABM-409 | biostudies-arrayexpress
Project description:Genome-wide approaches have begun to elucidate the transcriptional and epigenetic regulatory networks responsible for pluripotency in embryonic stem (ES) cells. Chromatin Immunoprecipitation (ChIP) followed either by hybridization to a microarray platform (ChIP-chip), or by DNA sequencing (ChIP-PET), has identified the genomic-binding targets of the ES cell transcription factors Oct4 and Nanog in humans and mice, respectively. A central issue that remains to be resolved is the concordance of the data obtained by these methods, given the differences between the two techniques. Here, we report the identification of the Oct4 and Nanog genomic targets in mouse ES cells by ChIP-Chip and have compared the data with binding data identified previously by ChIP-PET. Binding data have also been combined with Oct4 and Nanog RNAi knockdown expression profiling data in ES cells. Surprisingly, we find a substantial difference between the regions that identified exclusively by one of the two techniques. In both studies, however, targets identified by either technique contain a number of genes that are differentially expressed on Oct4 or Nanog knockdown, and have been implicated in cell-fate determination events. This study provides a comparison between the data obtained by different genomic platform, and offers a more comprehensive picture of the stem cell transcriptional network.
2008-11-04 | E-TABM-410 | biostudies-arrayexpress
Project description:The most widely-used method for detecting and measuring genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Many tiling array platforms, amplification methods, and analysis algorithms exist for ChIP-chip, but a rigorous assessment of the relative performance of these factors has not been reported. In a multi-lab simulation of a ChIP-chip experiment, we conducted the first objective analysis of tiling array platforms and analysis algorithms. We designed a complex mixture of human genomic DNA with a "spike-in" comprised of nearly 100 human sequences at various concentrations. Eight independent groups hybridized these mixtures to four different tiling array platforms. The groups were blind to the composition of the spike-in mix, the range of concentrations covered, or how many sequences it contained. Still blind to the key, each group made predictions of the spike-in locations based on their measurements. The results reveal that all commercial tiling array platforms perform well, although each platform and analysis algorithm has distinct performance characteristics. Simple sequence repeats and genome redundancy tend to result in false positives on oligonucleotide platforms. We also compare genome-wide platforms with regard to performance and cost. The spike-in DNA samples and the resulting array data presented in our study provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Keywords: Spike in Control For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf With the availability of sequenced genomes and whole-genome tiling microarrays, many researchers have conducted experiments using ChIP-chip and related methods to study genome-wide protein-DNA interactions1-9. These are powerful yet challenging techniques, which are comprised of many steps that can introduce variability in the final results. One potentially important factor is the relative performance of different types of tiling arrays. Currently the most popular platforms for performing ChIP-chip experiments are PCR-product based tiling arrays spotted in academic laboratories and commercial oligonucleotide-based tiling arrays from Affymetrix, NimbleGen, and Agilent. A second factor known to introduce variation is the DNA amplification protocol, which is often required because the low DNA yield from a ChIP experiment prevents direct detection on microarrays. A third factor is the algorithm used for detecting regions of enrichment from the tiling array data. A number of algorithms have been developed, but until this report there was no benchmark dataset to systematically evaluate them. In this study, we used a spike-in experiment to systematically evaluate the effects of tiling microarrays, amplification protocols, and data analysis algorithms on ChIP-chip results. There are other potentially important factors that from a practical standpoint are more difficult to systematically control and evaluate, and are not assessed here. These include the experimenter, the amount of starting material used, size of DNA fragments after shearing, DNA labeling method, and hybridization conditions. There have been several studies evaluating the performance of gene expression microarrays and analysis algorithms10-13. However, because tiling arrays must cover large genomic regions, they have different probe properties and high probe densities, and therefore present different biochemical and informatics challenges. Thus the results from the expression array spike-in experiments are not necessarily directly relevant to tiling array experiments. One recent study compared the performance of array-based (ChIP-chip) and sequence-based (ChIP-PET) technologies on a real ChIP experiment14. However, because this was an exploratory experiment, the list of absolute “true positive” targets was and is unknown. Since this experiment was performed without a key, the sensitivity and specificity of each technology had to be estimated retrospectively by qPCR validation of targets predicted from each platform. In our experiment, eight independent research groups at locations worldwide hybridized two different mixtures of DNA to one of four tiling array platforms, and predicted genome location and concentration of the spike-in sequences using a total of 13 different algorithms. Throughout the process, the research groups were entirely blind to the contents of the spike-in mixtures. Using the spike-in key, we analyzed several performance parameters for each platform, algorithm, and amplification method. While all commercial platforms performed well, we found that each had unique performance characteristics. We examined the implications of these results in planning human genome-wide experiments, in which trade-offs between probe density and cost are important.
2008-01-31 | E-GEOD-9842 | biostudies-arrayexpress
Project description:Chromatin immunoprecipitation followed by ultra-high throughput (UHTP) sequencing (ChIP-seq) is a powerful tool to establish protein-DNA interactions genome-wide, and the primary limitation of its broad application at present is the often-limited access to sequencers. Here, we report a protocol, Mab-Seq, to generate preliminary quality evaluations and single-chromosome data for deep-sequencing libraries. We show that commercially available genomic microarrays can be used to maximize the efficiency of library creation, quickly generate preliminary data on a chromosomal scale, and help establish the depth to which novel libraries will require deep sequencing.
2008-06-11 | E-TABM-485 | biostudies-arrayexpress
Project description:We mapped the transcriptional regulatory circuitry for six master regulators in human hepatocytes using chromatin immunoprecipitation and high-resolution promoter microarrays. The results show that these regulators form a highly interconnected core circuitry, and reveal the local regulatory network motifs created by regulator-gene interactions. Auto-regulation was a prominent theme among these regulators. We found that hepatocyte master regulators tend to bind promoter regions combinatorially and that the number of transcription factors bound to a promoter corresponds with observed gene expression. Our studies reveal portions of the core circuitry of human hepatocytes.
2006-05-09 | E-WMIT-9 | biostudies-arrayexpress