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.
Project description:The aim of this analysis was to analyze nucleosome distribution in the filamentous ascomycete Sordaria macrospora by micrococcal nuclease digestion and sequencing.
Project description:The nucleosome plays a central role in genome regulation. Traditional methods for mapping nucleosomes depend on the resistance of the nucleosome core to micrococcal nuclease (MNase). However, the lengths of the protected DNA fragments are heterogeneous, limiting the accuracy of nucleosome position information. To resolve this problem, we removed residual linker DNA by simultaneous digestion of yeast chromatin with MNase and exonuclease III (ExoIII). Paired-end sequencing of mono-nucleosomes revealed not only core particles (145-147 bp), but also intermediate particles in which ~8 bp project from one side (154 bp) or both sides (161 bp) of the nucleosome core. We term these particles "pseudo-chromatosomes" because they are present in yeast lacking linker histone. They are also observed after MNase-ExoIII digestion of chromatin reconstituted using recombinant core histones. We propose that the pseudo-chromatosome provides a DNA framework to facilitate H1 binding. Comparison of budding yeast nucleosome sequences obtained using micrococcal nuclease (MNase-seq) and MNase + exonuclease III (ExoIII) (MNase-ExoIII-seq): wild type cells and hho1-null cells. Nucleosome sequences from native chromatin and H1-depleted chromatin from mouse liver. Nucleosome sequences from a plasmid reconstituted into nucleosomes using recombinant yeast histones or native chicken erythrocyte histones.
Project description:Functional interactions between gene regulatory factors and chromatin architecture have been difficult to directly assess. Here, we use micrococcal nuclease (MNase) footprinting to probe the functions of two chromatin remodeling complexes. By simultaneously quantifying alterations in small MNase footprints over the binding sites of 30 regulatory factors in mouse embryonic stem cells (ESCs), we provide evidence that esBAF and Mbd3/NuRD modulate the binding of several regulatory proteins. In addition, we find that nucleosome occupancy is reduced at specific loci in favor of subnucleosomes upon depletion of esBAF, including sites of histone H2A.Z localization. Consistent with these data, we demonstrate that esBAF is required for normal H2A.Z localization in ESCs, suggesting esBAF either stabilizes H2A.Z-containing nucleosomes or promotes subnucleosome to nucleosome conversion by facilitating H2A.Z deposition. Therefore, integrative examination of MNase footprints reveals insights into nucleosome dynamics and functional interactions between chromatin structure and key gene regulatory factors. Examine three read size footprints from MNase-Seq in EGFP KD, Mbd3 KD, and Smarca4 KD mESCs using ChIP-Seq datasets.
Project description:Functional interactions between gene regulatory factors and chromatin architecture have been difficult to directly assess. Here, we use micrococcal nuclease (MNase) footprinting to probe the functions of two chromatin remodeling complexes. By simultaneously quantifying alterations in small MNase footprints over the binding sites of 30 regulatory factors in mouse embryonic stem cells (ESCs), we provide evidence that esBAF and Mbd3/NuRD modulate the binding of several regulatory proteins. In addition, we find that nucleosome occupancy is reduced at specific loci in favor of subnucleosomes upon depletion of esBAF, including sites of histone H2A.Z localization. Consistent with these data, we demonstrate that esBAF is required for normal H2A.Z localization in ESCs, suggesting esBAF either stabilizes H2A.Z-containing nucleosomes or promotes subnucleosome to nucleosome conversion by facilitating H2A.Z deposition. Therefore, integrative examination of MNase footprints reveals insights into nucleosome dynamics and functional interactions between chromatin structure and key gene regulatory factors.
Project description:We analyed the nucleosome positions by using 2 concentrations of micrococcal nuclease of yeast strains that were grown in raffinose and galactose containing media (synthetic complete).
Project description:Open chromatin provides access to a wide spectrum of DNA binding proteins for DNA metabolism processes such as transcription, repair, recombination, and replication. In this regard, open chromatin profiling has been widely used to identify the location of regulatory regions, including promoters, enhancers, insulators, silencers, replication origins, and recombination hotspots. Regulatory DNA elements are made accessible by nucleosome-depeleted states. Thus, nucleosome remodelling and modification should be intimately coupled with open chromatin formation and regulation. However, our knowledge of nucleosome regulation is largely limited to promoter regions, which comprise only a subset of all regulatory loci in the genome. In order to examine nucleosome patterns in open chromatin regions, we performed micrococcal nuclease (MNase) sequencing for a laboratory strain of yeast. Nucleosome occupancy profiled by Micrococcal nuclease (MNase) digestion
Project description:We have used micrococcal nuclease (MNase) digestion followed by deep sequencing in order to obtain a higher-resolution map than previously available of nucleosome positions in the fission yeast, Schizosaccharomyces pombe. Our data confirm an unusually short average nucleosome repeat length, ~152 bp, in fission yeast and that transcriptional start sites (TSSs) are associated with nucleosome-depleted regions (NDRs), ordered nucleosome arrays downstream and less regularly spaced upstream nucleosomes. In addition, we found enrichments for associated function in four of eight groups of genes clustered according to chromatin configurations near TSSs. At replication origins, our data revealed asymmetric localization of Pre-Replication Complex (pre-RC) proteins within large NDRsM-bM-^@M-^Ta feature that is conserved in fission and budding yeast and is therefore likely to be conserved in other eukaryotic organisms. Examination of nucleosome positioning in Schizosaccharomyces pombe