Micrococcal nuclease sequencing analysis for nucleosome localization of mouse fetal liver erythroblasts
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE55825 | GEO | 2014/10/01
SECONDARY ACCESSION(S): PRJNA241102
REPOSITORIES: GEO
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Project description:Nucleosomal DNA was prepared using Simple ChIP Enzymatic Chromatin IP Kit according to manufacturer’s instruction. Briefly, Nuclei were isolated from purified Ter119 negative or in vitro cultured erythroblasts. Cross-linked native chromatin was then digested with MNase into mononucleosomal DNA. Sequencing libraries were generated from nucleosomal DNA, and sequencing was carried out using the Illumina system according to the manufacturer’s specification. In this study, we purified chromatin from in vitro cultured mouse fetal liver erythroblasts on day 0, day 1, and day 2. The chromatins were digested by micrococcal nuclease to make mononucleosomal products, which were further analyzed by next generation sequencing analysis. We aim to determine the dynamic changes of nucleosome during terminal erythropoiesis.
2014-10-01 | E-GEOD-55825 | biostudies-arrayexpress
Project description:We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms. MNase has hitherto been very widely used to map nucleosomes, although concerns have been raised over its potential to introduce bias. Having identified the locations and quantified the strength of both the chicken or frog histone octamer binding sites on each DNA, the results obtained with the two enzymes were compared using a variety of criteria. Both enzymes displayed sequence specificity in their preferred cleavage sites, although the nature of this selectivity was distinct for the two enzymes. In addition, nucleosomes produced by CAD nuclease are 8-10 bp longer than those produced with MNase, with the CAD cleavage sites tending to be 4-5 bp further out from the nucleosomal dyad than the corresponding MNase cleavage sites. Despite these notable differences in cleavage behaviour, the two nucleases identified essentially equivalent patterns of nucleosome positioning sites on each of the DNAs tested, an observation that was independent of the histone type. These results indicate that biases in nucleosome positioning data collected using MNase are, under our conditions, not significant.
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2010-10-15 | GSE22514 | GEO
Project description:Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions.
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Project description:For the microarray experiments, MV4-11 and MOLM-14 cells were treated with DMSO control, ABT-869 3 nM, SAHA 6 uM and combination therapy for 24 hours. Cells were then washed in PBS and high-quality total RNA was extracted RNeasy Midi Kit, according to the manufacturer’s instruction (Qiagen, Valencia, USA). RNA quantity, quality, and purity were assessed with the use of the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA, USA). Gene expression profiling was performed using Affymetric U133plus2.0 gene chip (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol.
2011-05-31 | GSE26114 | GEO
Project description:Nucleosome positioning is critical to chromatin accessibility and is associated with gene expression programs in cells1-3. Previous nucleosome mapping methods assemble profiles from cell populations and reveal a cell-averaged pattern: nucleosomes are positioned and form a phased array that surrounds the transcription start sites of active genes3-6 and DNase I hypersensitive sites7. However, even in a homogenous population of cells, cells exhibit heterogeneity in expression in response to active signalling8,9 that may be related to heterogeneity in chromatin accessibility10-12. Here we report a technique, termed single-cell micrococcal nuclease sequencing (scMNase-seq), that can be used to simultaneously measure genome-wide nucleosome positioning and chromatin accessibility in single cells. Application of scMNase-seq to NIH3T3 cells, mouse primary naive CD4 T cells and mouse embryonic stem cells reveals two principles of nucleosome organization: first, nucleosomes in heterochromatin regions, or that surround the transcription start sites of silent genes, show large variation in positioning across different cells but are highly uniformly spaced along the nucleosome array; and second, nucleosomes that surround the transcription start sites of active genes and DNase I hypersensitive sites show little variation in positioning across different cells but are relatively heterogeneously spaced along the nucleosome array. We found a bimodal distribution of nucleosome spacing at DNase I hypersensitive sites, which corresponds to inaccessible and accessible states and is associated with nucleosome variation and variation in accessibility across cells. Nucleosome variation is smaller within single cells than across cells, and smaller within the same cell type than across cell types. A large fraction of naive CD4 T cells and mouse embryonic stem cells shows depleted nucleosome occupancy at the de novo enhancers detected in their respective differentiated lineages, revealing the existence of cells primed for differentiation to specific lineages in undifferentiated cell populations.
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Project description:In the yeast genome, a large proportion of nucleosomes occupy well-defined positions. While the contribution of chromatin remodelers and DNA binding proteins to maintain this organization is well established, the relevance of the DNA sequence to nucleosome positioning in the genomic context remains controversial. Through genome-wide, quantitative analysis of nucleosome positioning and high-resolution mutagenenesis of mononucleosomal DNA, we show that sequence changes distort the nucleosomal pattern at the level of individual nucleosomes. This effect is equally detected in transcribed and non-transcribed regions, suggesting the existence of sequence elements contributing to positioning. To identify such elements, we incorporated information from nucleosomal signatures into artificial synthetic DNA molecules and found that they generated regular nucleosomal arrays indistinguishable from those of endogenous sequences. Strikingly, this information is species-specific and can be combined with coding information through the use of synonymous codons such that genes from one species can be engineered to adopt the nucleosomal organization of another. These findings open up the possibility of designing coding and non-coding DNA molecules capable of directing their own nucleosomal organization.
2016-09-20 | GSE84910 | GEO
Project description:Genome wide DNA methylation profiling of WT and Pml KO mEF cells treated W/O Doxorubicin for 24 hours. TET2 plays critical roles in the regulation of cell identity and inhibition of tumorigenesis through modulating DNA methylation. However, little is known about how TET2 is targeted to specific DNA-binding sites. It was found by us that TET2 is significantly recruited to specific sites by PML. Moreover, TET2 and PML synergistically inhibited cell proliferation. Further study indicated that multi chemotherapies, such as doxorubicin, were able to promote PML-NB, increase 5hmC and result in specific DNA demethylation. Due to the important role of inactivation of tumor suppressors by DNA methylation in cancer, the activity of most therapies depends to an extent on activation of tumor suppressors by demethylation. Consequently, it is not surprising that methylation of tumor suppressors has been reported to diminish in responder patients after end of chemotherapy. Little is known about mechanism of therapy-promoted DNA demethylation, although this therapy-promoted DNA demethylation potentially plays general roles in therapy response, resistance and recurrence. It was also found that knock out of Pml significantly diminished chemotherapy-promoted DNA demethylation. To identify PML-TET2 pathway-regulated genes, WT and Pml knock out MEF cells were treated W/O Doxorubicin. The genomic DNA was extracted and purified from samples using Qiagen DNeasy Kit according to manufacturer’s instruction. The resultant Genome DNA was cut by Restriction Enzyme. After DNA-end repair and 3’-dA overhang, the 40-220bp fragments were selected and treated with ZYMO EZ DNA Methylation-Gold kit according to manufacturer’s instruction. Then the bisulphite converted DNA from the 4 samples (WT, WT Dox, PML KO and Pml KO Dox) were sequenced using standard Illumina protocol. Standard recommended Illumina scanner setting was carried out in study.
2015-11-26 | GSE75408 | GEO
Project description:Staphylococcus aureus is commonly associated with biofilm-related infections and contributes to the large financial loss that accompany nosocomial infections. The micrococcal nuclease Nuc1 enzyme limits biofilm formation via cleavage of eDNA, a structural component of the biofilm matrix. Solid surface hydrophobicity influences bacterial adhesion forces and may as well influence eDNA production. Therefore, it is hypothesized that the impact of Nuc1 activity is dependent on surface characteristics of solid surfaces. For this reason, this study investigated the influence of solid surface hydrophobicity on S. aureus Newman biofilms where Nuc1 is constitutively produced. To this end, biofilms of both a wild-type and a nuc1 knockout mutant strain, grown on glass, salinized glass and Pluronic F-127-coated silanized glass were analysed. Results indicated that biofilms can grow in the presence of Nuc1 activity. Also, Nuc1 and solid surface hydrophobicity significantly affected the biofilm 3D-architecture. In particular, biofilm densities of the wild-type strain on hydrophilic surfaces appeared higher than of the mutant nuc1 knockout strain. Since virulence is related to bacterial cell densities, this suggests that the virulence of S. aureus Newman biofilms is increased by its nuclease production in particular on a hydrophilic surface.
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