MNase digestion buffer

2007 ◽  
Vol 2007 (11) ◽  
pp. pdb.rec11036-pdb.rec11036
Keyword(s):  
Mnase Digestion

scholarly journals Quantitative MNase-seq accurately maps nucleosome occupancy levels

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Răzvan V. Chereji ◽  
Terri D. Bryson ◽  
Steven Henikoff
Keyword(s):  
Time Course ◽  
Nucleosome Occupancy ◽  
Theoretical Framework ◽  
Human Cells ◽  
Micrococcal Nuclease ◽  
Experimental Protocol ◽  
Digestion Time ◽  
Mnase Digestion

Abstract Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono-nucleosomes, whereas cleavages within nucleosomes destroy them. Here, we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time course. As with human cells, we observe no overall differences in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.

MNase digestion buffer for ChIP

2009 ◽  
Vol 2009 (6) ◽  
pp. pdb.rec11852-pdb.rec11852
Keyword(s):  
Mnase Digestion

scholarly journals A computational approach to map nucleosome positions and alternative chromatin states with base pair resolution

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Xu Zhou ◽  
Alexander W Blocker ◽  
Edoardo M Airoldi ◽  
Erin K O'Shea
Keyword(s):  
Base Pair ◽  
Transcription Initiation ◽  
Computational Approach ◽  
Significant Heterogeneity ◽  
Transcription Start Sites ◽  
Mnase Digestion ◽  
Genome Wide ◽  
A Cell ◽  
Simple Error

Understanding chromatin function requires knowing the precise location of nucleosomes. MNase-seq methods have been widely applied to characterize nucleosome organization in vivo, but generally lack the accuracy to determine the precise nucleosome positions. Here we develop a computational approach leveraging digestion variability to determine nucleosome positions at a base-pair resolution from MNase-seq data. We generate a variability template as a simple error model for how MNase digestion affects the mapping of individual nucleosomes. Applied to both yeast and human cells, this analysis reveals that alternatively positioned nucleosomes are prevalent and create significant heterogeneity in a cell population. We show that the periodic occurrences of dinucleotide sequences relative to nucleosome dyads can be directly determined from genome-wide nucleosome positions from MNase-seq. Alternatively positioned nucleosomes near transcription start sites likely represent different states of promoter nucleosomes during transcription initiation. Our method can be applied to map nucleosome positions in diverse organisms at base-pair resolution.

scholarly journals MNase Digestion Protection Patterns of the Linker DNA in Chromatosomes

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2239
Author(s):  
Chang-Hui Shen ◽  
James Allan
Keyword(s):  
Globin Gene ◽  
Linker Histone ◽  
Computational Studies ◽  
Core Particle ◽  
Linker Histones ◽  
Dna Accessibility ◽  
The Core ◽  
Mnase Digestion ◽  
Recent Developments ◽  
Nucleosomal Structure

The compact nucleosomal structure limits DNA accessibility and regulates DNA-dependent cellular activities. Linker histones bind to nucleosomes and compact nucleosomal arrays into a higher-order chromatin structure. Recent developments in high throughput technologies and structural computational studies provide nucleosome positioning at a high resolution and contribute to the information of linker histone location within a chromatosome. However, the precise linker histone location within the chromatin fibre remains unclear. Using monomer extension, we mapped core particle and chromatosomal positions over a core histone-reconstituted, 1.5 kb stretch of DNA from the chicken adult β-globin gene, after titration with linker histones and linker histone globular domains. Our results show that, although linker histone globular domains and linker histones display a wide variation in their binding affinity for different positioned nucleosomes, they do not alter nucleosome positions or generate new nucleosome positions. Furthermore, the extra ~20 bp of DNA protected in a chromatosome is usually symmetrically distributed at each end of the core particle, suggesting linker histones or linker histone globular domains are located close to the nucleosomal dyad axis.

scholarly journals Nucleosome fragility is associated with future transcriptional response to developmental cues and stress in C. elegans

2016 ◽  
Author(s):  
Tess E. Jeffers ◽  
Jason D. Lieb
Keyword(s):  
Time Course ◽  
Neuronal Response ◽  
Transcriptional Response ◽  
Relative Sensitivity ◽  
Histone Variants ◽  
Micrococcal Nuclease ◽  
Gene Promoters ◽  
C Elegans ◽  
Mnase Digestion ◽  
Context Specific

ABSTRACTNucleosomes have structural and regulatory functions in all eukaryotic DNA-templated processes. The position of nucleosomes on DNA and the stability of the underlying histone-DNA interactions affect the access of regulatory proteins to DNA. Both stability and position are regulated through DNA sequence, histone post-translational modifications, histone variants, chromatin remodelers, and transcription factors. Here, we explored the functional implications of nucleosome properties on gene expression and development in C. elegans embryos. We performed a time-course of micrococcal nuclease (MNase) digestion, and measured the relative sensitivity or resistance of nucleosomes throughout the genome. Fragile nucleosomes were defined by nucleosomal DNA fragments recoverable preferentially in early MNase-digestion time points. We found fragile nucleosomes at locations where we expected to find destabilized nucleosomes, like transcription factor binding sites where nucleosomes compete with DNA-binding factors. Contrary to our expectation, the presence of fragile nucleosomes in gene promoters was anti-correlated with transcriptional activity. Instead, genes with fragile nucleosomes in their promoters tended to be expressed in a context-specific way, operating in neuronal response, the immune system, and stress response. Nucleosome fragility at these promoters was strongly and positively correlated with the AT content of the underlying DNA. There was not a strong correlation between promoter nucleosome fragility and the levels of histone modifications or histone variants. Our data suggest that in C. elegans promoters, nucleosome fragility is primarily a DNA-encoded feature that poises genes for future context-specific activation in response to environmental stress and developmental cues.

scholarly journals c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts display more decondensed nucleosomal organization than normal fibroblasts.

1990 ◽  
Vol 111 (1) ◽  
pp. 9-17 ◽  
Author(s):  
J Laitinen ◽  
L Sistonen ◽  
K Alitalo ◽  
E Hölttä
Keyword(s):  
Cell Cycle ◽  
Micrococcal Nuclease ◽  
Normal Fibroblast ◽  
Transformed Cells ◽  
Serum Starvation ◽  
Quiescent State ◽  
Normal Cells ◽  
3T3 Fibroblasts ◽  
Mnase Digestion ◽  
Nih 3T3

We have compared the nucleosomal organization of c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts with that of normal fibroblasts by using micrococcal nuclease (MNase) as a probe for the chromatin structure. The bulk chromatin from asynchronously and exponentially growing ras-transformed cells was much more sensitive to MNase digestion than chromatin from the normal cells. Southern hybridization analyses of the MNase digests with probes specific for the ornithine decarboxylase (odc) and c-myc genes showed that the coding and/or 3' end regions of these growth-inducible genes carry a nucleosomal organization both in ras-transformed and normal cells. Studies with cells synchronized by serum starvation showed that in both cell lines the nucleosomal organization of chromatin is relatively condensed at the quiescent state, becomes highly decondensed during the late G1 phase of the cell cycle, and starts again to condense during the S phase. However, in ras-transformed cells the decondensation state stayed much longer than in normal cells. Moreover, irrespective of the phase of the cell cycle the bulk chromatin as well as that of the odc and c-myc genes was more sensitive to MNase digestion in the ras-transformed cell than in the normal fibroblast. Decondensation of the chromatin was also observed in the normal c-Ha-ras protooncogene-transfected cells, but to a lesser extent than in the mutant ras-transformed cells. Whether the increased degree of chromatin decondensation plays a regulatory role in the increased expression of many growth-related genes in the ras-transformed cells remains an interesting object of further study.

scholarly journals CENP-A, -B, and -C Chromatin Complex That Contains the I-Type α-Satellite Array Constitutes the Prekinetochore in HeLa Cells

2002 ◽  
Vol 22 (7) ◽  
pp. 2229-2241 ◽  
Author(s):  
Satoshi Ando ◽  
Hua Yang ◽  
Naohito Nozaki ◽  
Tuneko Okazaki ◽  
Kinya Yoda
Keyword(s):  
Quantitative Analysis ◽  
Hela Cells ◽  
Soluble Fraction ◽  
Micrococcal Nuclease ◽  
Mass Analysis ◽  
Centromeric Chromatin ◽  
Mnase Digestion ◽  
Hypersensitive Sites ◽  
C Complex ◽  
Active Centromere

ABSTRACT CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes. We have isolated centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies and shown that the CENP-A/B/C complex is predominantly formed on α-satellite DNA that contains the CENP-B box (αI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the αI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex. Molecular mass analysis by glycerol gradient sedimentation showed that MNase digestion released a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex. Quantitative analysis by immunodepletion of CENP-A nucleosomes showed that most of the CENP-C and approximately half the CENP-B took part in formation of the CENP-A/B/C chromatin complex. A kinetic study of the solubilization of CENPs showed that MNase digestion first released the CENP-A/B/C chromatin complex into the soluble fraction, and later removed CENP-B and CENP-C from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA. On the basis of these results, we propose that the CENP-A/B/C chromatin complex is selectively formed on the I-type α-satellite array and constitutes the prekinetochore in HeLa cells.

MNase Digestion for Nucleosome Mapping in Neurospora

BIO-PROTOCOL ◽  
2016 ◽  
Vol 6 (11) ◽  
Author(s):  
Cigdem Sancar ◽  
Gencer Sancar ◽  
Michael Brunner
Keyword(s):  
Mnase Digestion ◽  
Nucleosome Mapping
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