MPE-seq, a New Method for the Genome-wide Analysis of Chromatin Structure
ABSTRACT: In this study we developed MPE-seq, a method for the genome-wide characterization of chromatin that involves the digestion of nuclei with methidiumpropyl-EDTA-Fe(II) [MPE-Fe(II)] followed by massively parallel sequencing. Like micrococcal nuclease (MNase), MPE-Fe(II) preferentially cleaves the linker DNA between nucleosomes. We also performed MNase-seq as a comparison. We further performed ChIP-seq using chromatin samples obtained by MPE-Fe(II) or MNase digestion of nuclei. Nuclei from J1 mouse embryonic stem cells were treated with MPE-Fe(II) or MNase. The isolated DNA was sequenced by Illumina HiSeq sequencers. Some of the digested chromatin was studied by performing ChIP-seq using antibodies against histone H2B or H3.
Project description:MNase-seq was performed in order to analyze changes in nucleosomal occupancy after depletion of CTCF/P190 and ISWI from Drosophila S2 cells MNase-seq from Drosophila S2 nuclei after CTCF/CP190 or ISWI-specific RNAi treatment
Project description:Distinctive SWI/SNF-like ATP-dependent chromatin remodeling esBAF complexes are indispensable for the maintenance and pluripotency of mouse embryonic stem (ES) cells. To understand the mechanism underlying the roles of these complexes in ES cells, we performed high-resolution genome-wide mapping of the core ATPase subunit, Brg, using ChIP-Seq technology. We find that that esBAF, as represented by Brg, binds to genes encoding components of the core ES transcriptional circuitry, including Polycomb group proteins. esBAF colocalizes extensively with Oct4, Sox2 and Nanog genome-wide, and shows distinct functional interactions with Oct4 and Sox2 at its target genes. Surprisingly, no significant colocalization of esBAF with PRC2 complexes, represented by Suz12, is observed. Lastly, esBAF co-binds with Stat3 and Smad1 genome-wide, consistent with a direct and critical role in LIF and BMP signaling essential to maintain pluripotency. Taken together, our studies indicate that esBAF is both an essential component of the core pluripotency transcriptional network, and might also be a critical component of the LIF and BMP signaling pathways essential for maintenance of self-renewal and pluripotency. Brg knockdown effect on expression, Brg ChIP-Seq
Project description:DNA are packaged into nucleosomes and chromatin. We performed incomplete MNase digestion of chromatin to identify nucleosome-free regions that may indicate active promoters and regulatory regions. Additionally, HpaII digestion was performed which cleaves CCGG sites when the internal C remains unmodified. The hypomethylation state and nuclease sensitivity of the chromatin are indicators of transcription regulatory regions. Four other data sets from this study were also deposited at ArrayExpress under accession numbers E-MTAB-1935, E-MTAB-1952, E-MTAB-1961 and E-MTAB-2014.
Project description:We have combined standard micrococcal (MNase) digestion of nuclei with a modified protocol for construction paired-end DNA sequencing libraries to map both nucleosomes and subnucleosome-sized particles at single base-pair resolution throughout the budding yeast genome. We found that partially unwrapped nucleosomes and subnucleosome-sized particles can occupy the same position within a cell population, suggesting dynamic behavior. By varying the time of MNase digestion, we have been able to observe changes that reflect differential sensitivity of particles, including eviction of nucleosomes. Our protocol and mapping method provide a general strategy for characterizing full epigenomes. We used micrococcal nuclease mapping, chromatin immunoprecipitation and paired-end sequencing to determine the structure of yeast centromeres at single base-pair resolution.
Project description:We addressed the roles of three nucleosome spacing enzymes (ISW1, ISW2 and CHD1) in specifying chromatin organization in S. cerevisiae. Comparison of budding yeast nucleosome positions obtained using micrococcal nuclease digestion followed by paired-end sequencing (MNase-seq). We sequenced nucleosomes from isogenic strains with the isw1Δ, isw2Δ or chd1Δ mutations in all combinations. We measured gene activity by ChIP-seq for the Rpb3 subunit of RNA polymerase II for some of these mutants.
Project description:Micrococcal nuclease (MNase) is commonly used to map nucleosomes genome-wide, but nucleosome maps are affected by the degree of digestion. It has been proposed that many yeast promoters are not nucleosome-free but occupied by easily digested, unstable, “fragile” nucleosomes. We analyzed the histone content of all MNase-sensitive complexes by MNase-ChIP-seq and Sonication-ChIP-seq. We find that yeast promoters are predominantly bound by non-histone protein complexes, with little evidence for fragile nucleosomes. We do detect MNase-sensitive nucleosomes elsewhere in the genome, including transcription termination sites. However, they have high A/T-content, suggesting that MNase sensitivity does not indicate instability, but the preference of MNase for A/T-rich DNA, such that A/T-rich nucleosomes are digested faster than G/C-rich nucleosomes. We confirm our observations by analyzing ChIP-exo, chemical mapping and ATAC-seq data from other laboratories. Thus, histone ChIP-seq experiments are essential to distinguish nucleosomes from other DNA-binding proteins that protect against MNase. Overall design: We mapped MNase-sensitive complexes in budding yeast by comparing protected DNA fragments at different levels of digestion (MNase titration). We used MNase-ChIP-seq and Sonication-ChIP-seq to detect histones H4 and H2B in order to distinguish between MNase-sensitive nucleosomes and non-histone complexes.
Project description:Nucleosome structure and positioning play pivotal roles in gene regulation, DNA repair and other essential processes in eukaryotic cells. Nucleosomal DNA is thought to be uniformly inaccessible to DNA binding and processing factors, such as MNase. Here, we show, however, that nucleosome accessibility and sensitivity to MNase varies. Digestion of Drosophila chromatin with two distinct concentrations of MNase revealed two types of nucleosomes: sensitive and resistant. MNase-resistant nucleosome arrays are less accessible to low concentrations of MNase, whereas MNase-sensitive arrays are degraded by high concentrations. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C containing dinucleotides. In contrast, MNase-sensitive nucleosomes form on A/T rich sequences represented by transcription start and termination sites, enhancers and DNase hypersensitive sites. Lowering of cell growth temperature to ~10°C stabilizes MNase-sensitive nucleosomes suggesting that variations in sensitivity to MNase are related to either thermal fluctuations in chromatin fiber or the activity of enzymatic machinery. In the vicinity of active genes and DNase hypersensitive sites nucleosomes are organized into synchronous, periodic arrays. These patterns are likely to be caused by “phasing” nucleosomes off a potential barrier formed by DNA-bound factors and we provide an extensive biophysical framework to explain this effect. RNA-seq S2 cells Drosophila melanogaster
Project description:To determine whether P. aeruginosa strain PA14 exhibits a specific transcriptional response to extracellular Fe(II), a microarray experiment was performed using Affymetrix GeneChips. The transcriptional response to Fe(II) or Fe(III) shock was measured and compared to a no-Fe control.
Project description:Paired-end sequencing study of (1) nucleosome core particles and under-digested chromatin from MNase-treated nuclei; (2) ChIP samples for HA-tagged histone H4 and H2B; (3) ChIP for the Rpb3 subunit of Pol II. Nucleosomal DNA and immunopurified sonicated DNA fragments were subjected to paired-end sequencing.
Project description:MNase-Seq and ChIP-Seq have evolved as popular techniques to study chromatin and histone modification. Although many tools have been developed to identify enriched regions, software tools for nucleosome positioning are still limited. We introduce a flexible and powerful open-source R package, PING 2.0, for nucleosome positioning using MNase-Seq data or MNase- or sonicated- ChIP-Seq data combined with either single-end or paired-end sequencing. PING uses a model-based approach, which enables nucleosome predictions even in the presence of low read counts. We illustrate PING using two paired-end datasets from Saccharomyces cerevisiae and compare its performance to nucleR and ChIPseqR. Identification of nucleosomes from two different mononucleosomes data. A yeast strain (W303 background) with the HTZ1 gene expressed a fusion with a myc epitope was used to map total and Htz1-containign nucleosome by MNase-ChIP-Seq. Cells were grown to mid-log phase and monomucleosomes were generated using MNase treatment of isolated nuclei. Especially for the sample of SC0017_61YDGAAXX_8_TCATTC, the Htz1-containing nucleosomes were enriched by immunoprecipitation using an anti-Myc antibody (3E10). DNA from both total nucleosomes and Htz1-enriched nucleosomes were purified and sequenced on an Illumina GA IIx using the by paired-end protocol.