ABSTRACT: We produced a map of nucleosome positions in IMR90 by sequencing the ends of MNase-digested chromatin fragments. Overall design: IMR90 cells were grown in culture, about 1E6 cells were isolated and digested using micrococcal nuclease (MNase). Mononucleosomes were gel-selected and fragment ends were sequenced using the Illumina GAIIx sequencing platform.
INSTRUMENT(S): Illumina Genome Analyzer II (Homo sapiens)
Project description:We produced a map of nucleosome positions in IMR90 by sequencing the ends of MNase-digested chromatin fragments. IMR90 cells were grown in culture, about 1E6 cells were isolated and digested using micrococcal nuclease (MNase). Mononucleosomes were gel-selected and fragment ends were sequenced using the Illumina GAIIx sequencing platform.
Project description:Histone variants (H3.1, H3.3, H2A, and macroH2A) were studied about their proteomic and genomic distribution in Hela cells HeLa S3 cells stably expressing either FLAG-tagged H3.1, and H3.3 were grown in suspension in Joklik media containing 10% newborn calf serum (Hyclone), 1% GlutaMAX (Invitrogen), and 1% penicillin-streptomycin, and cells were harvested at log phase. Nuclei were isolated, mononucleosomes were subsequently obtained from these cell lines. Cells were lysed in hypotonic TMSD buffer to isolate nuclei, which were then digested with micrococcal nuclease. The resulting mononucleosomes were immunoprecipitated with anti-FLAG M2-agarose beads (Sigma) and eluted with FLAG peptide (Sigma)
Project description:The nuclear lamina interacts with the genome through megabase-size lamina-associated domains (LADs). LADs have been identified in proximity labeling assays and recently by chromatin immunoprecipitation-sequencing (ChIP-seq) of A- and B-type lamins. LADs localize mainly to the nuclear periphery, they are gene-poor and largely heterochromatic. Here, we show that the mode of chromatin fragmentation for ChIP, namely either bath sonication (used to date for ChIP of nuclear lamins) or digestion with micrococcal nuclease (MNase) leads to the discovery of distinct sets of lamin-interacting domains (which we refer to as LiDs) with distinct gene content, histone composition enrichment and relationship to lamin B1-interacting domains. We find that total genome coverage by lamin A/C ('LMNA') LiDs identified in sonicated or MNase-digested chromatin is similar (~730 megabases). Over half of these domains, however, are uniquely detected in sonicated or MNase-digested chromatin. Whereas sonication-specific LMNA LiDs are gene-poor and devoid of a broad panel of histone modifications, MNase-specific LMNA LiDs are of higher gene density and are enriched in H3K9me3, H3K27me3 and in histone variant H2A.Z. Analysis of published LMNB1 LADs and of LMNB1 LiDs identified by ChIP-seq further shows that LMNA can associate with 'open' chromatin domains displaying euchromatin features which are not associated with LMNB1. The differential genetic and epigenetic properties of lamin-interacting chromatin domains indicate the existence of distinct LiD populations identifiable in different chromatin contexts, including nuclease-accessible 'open' regions presumably localized in the nuclear interior. Overall design: There are two experiments of two samples each for LMNA ChIPs (ChIP and Input), and one LMNB1 ChIP sample, so 5 samples in total. The difference between the experiments are the chromatin fragmentation method (see extract protocols 1 and 2). One uses sonication while the other uses micrococcal nuclease (MNase).
Project description: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:Analysis of histone H3 turnover in wild-type and mutant fission yeast cells by using MNase-ChIP-chip Exponentially growing cultures of fission yeast cells expressing an ectopic copy of FLAG tagged histone H3 under the control of inv1 promoter were synchronized and expression of H3-FLAG was induced by changing the carbon source of the medium to Sucrose. Cells were crosslinked with 1% Formaldehyde and chromatin was fragmented into mononucleosomes by using micrococcal nuclease (MNase). Chromatin immunoprecipitated DNA recovered with anti-FLAG antibody from wild-type and mutant fission yeast cells and whole cell extracts DNA were amplified by random priming and respectively labeled with Cy5 and Cy3. Labeled DNA samples were mixed and hybridized onto 44k pombe Agilent oligo arrays. Data were processed using Agilent scanner and Feature Extraction software.
Project description:The eukaryotic nuclear genome is organized into the fundamental units of chromatin, nucleosomes. The positions and biochemical states of nucleosomes on DNA can regulate protein-DNA interactions, and in turn influence DNA-templated events. Despite the increasing number of genome-wide maps of nucleosome position, how global changes in nucleosome position relate to changes in gene expression is poorly understood. Using micrococcal nuclease (MNase) to map nucleosome positions, we show that in the maize genome, nucleosome occupancy signals are remarkably uniform between different tissues, whereas particular genomic regions are highly susceptible to variation. We demonstrate that much of this variation is associated with the degree to which chromatin is digested with MNase. Using high-density DNA microarrays, we exploited this digestion-linked variation to identify protein footprints in the maize genome that are hypersensitive to MNase digestion, a method we term Differential Nuclease-Sensitivity profiling (DNS-chip). Hypersensitive footprints were enriched at the 5’ and 3’ ends of genes, associated with gene-expression levels, and significantly overlapped with conserved noncoding sequences and the binding sites of the homeobox transcription-factor Knotted1, suggesting a functional role in genome regulation. We also found that the tissue-specific regulation of gene expression was linked to tissue-specific hypersensitive footprints in gene promoters. These results demonstrate the value of DNS-chip for revealing biochemical features of nucleosome organization that correlate with gene expression levels and colocalize with functional DNA elements. This approach to chromatin profiling should be broadly applicable to other species and should shed light on how chromatin organization might influence protein-DNA interactions and genome regulation. Overall design: Comparison of buffers and concentrations of Mnase in immature ears and seedlings
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.