MNase-Seq Analysis for Identifying Stress-Altered Nucleosome Occupancy in Plants.

Nucleosome occupancy plays an important role in chromatin compaction, affecting biological processes by hampering the binding of cis-acting elements such as transcription factors, RNA polymerase machinery, and coregulatory. Accessible regions allow for cis-acting elements to bind DNA and regulate transcription. Here, we detail our protocol to profile nucleosome occupancy and chromatin structure dynamics under drought stress at the genome-wide scale using micrococcal nuclease (MNase) digestion. Combining variable MNase concentration treatments and high-throughput sequencing, we investigate the changes in the overall chromatin state using bread wheat samples from an exemplary drought experiment.

MNase Digestion Protection Patterns of the Linker DNA in Chromatosomes.

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 ß-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.

Chromatin Affinity Purification (ChAP) from Arabidopsis thaliana Rosette Leaves Using in vivo Biotinylation System.

Chromatin Affinity Purification (ChAP) is widely used to study chromatin architecture and protein complexes interacting with DNA. Here we present an efficient method for ChAP from Arabidopsis thaliana rosette leaves, in which in vivo biotinylation system is used. The chromatin is digested by Micrococcal Nuclease (MNase), hence the distribution of nucleosomes is also achieved. The in vivo biotinylation system was initially developed for Drosophila melanogaster ( Mito et al., 2005 ), but the presented protocol has been developed specifically for Arabidopsis thaliana ( Sura et al., 2017 ).

DNA Accessibility by MNase Digestions.

Micrococcal nuclease (MNase) digestion of chromatin cuts linker DNA between neighboring nucleosomes and in this way generates mononucleosomes. The protected fragments can then be analyzed by genome-wide sequencing techniques or by quantitative PCR to obtain information about the positions of nucleosomes in the chromatin. Nucleosomes are differentially sensitive to MNase digestion, which means that titrations of MNase should be performed to obtain a comprehensive map of the nucleosome positions of a chromatin region or genome.

Profiling Nucleosome Occupancy by MNase-seq: Experimental Protocol and Computational Analysis.

Nucleosomes are the basic repeating units of eukaryotic chromatin. They play important roles in chromatin compaction and gene regulation. Therefore, it is important to profile the in vivo locations of nucleosomes in the genome. Here we illustrate how to profile nucleosome occupancy at genome-wide scale using micrococcal nuclease (MNase) digestion combined with high throughput Illumina sequencing (MNase-seq). Nucleosome-associated DNA is relatively insensitive to digestion by micrococcal nuclease (MNase). Upon mild MNase treatment, the undigested nucleosomal DNA can be purified and sequenced allowing a precise localization of in vivo nucleosomes at a genome-wide level.

Establishing nucleosome architecture and stability at promoters: Roles of pioneer transcription factors and the RSC chromatin remodeler.

Improvements in deep sequencing, together with methods to rapidly deplete essential transcription factors (TFs) and chromatin remodelers, have recently led to a more detailed picture of promoter nucleosome architecture in yeast and its relationship to transcriptional regulation. These studies revealed that ∼40% of all budding yeast protein-coding genes possess a unique promoter structure, where we propose that an unusually unstable nucleosome forms immediately upstream of the transcription start site (TSS). This "fragile" nucleosome (FN) promoter architecture relies on the combined action of the essential RSC (Remodels Structure of Chromatin) nucleosome remodeler and pioneer transcription factors (PTFs). FNs are associated with genes whose expression is high, coupled to cell growth, and characterized by low cell-to-cell variability (noise), suggesting that they may promote these features. Recent studies in metazoans suggest that the presence of dynamic nucleosomes upstream of the TSS at highly expressed genes may be conserved throughout evolution.

Assessing HDAC Function in the Regulation of Signal Transducer and Activator of Transcription 5 (STAT5) Activity Using Chromatin Immunoprecipitation (ChIP).

Transcriptional activation by STAT5 is repressed by deacetylase inhibitors. Investigating the role of deacetylases (HDACs) in STAT5-mediated transcription implies the analysis of molecular events taking place at the chromatin level. We describe here two alternative methods of chromatin immunoprecipitation that allow the characterization of chromatin modifications ensuing STAT5 activation and its inhibition by deacetylase inhibitors, in particular changes in histone acetylation, in histone occupancy, and in the association/dissociation of transcription factors and other chromatin-associated factors.