Abstract
Nucleosomes regulate the transcription output of the genome by occluding the underlying DNA sequences from DNA-binding proteins that must act on it. Knowledge of the precise locations of nucleosomes in the genome is thus essential towards understanding how transcription is regulated. Current nucleosome-mapping strategies involve digesting chromatin with nucleases or chemical cleavage followed by high-throughput sequencing. In this review, we compare the traditional micrococcal nuclease (MNase)-based approach with a chemical cleavage strategy, with discussion on the important insights each has uncovered about the role of nucleosomes in shaping transcriptional processes. Genome-wide nucleosome positioning maps provide deep insight into the regulatory role of nucleosomes in transcriptional processes. Micrococcal nuclease (MNase) digests linker DNA in between nucleosomes while nucleosome-protected DNA remains intact. Sequencing the protected DNA allows for the determination of nucleosome positions genome-wide. The chemical mapping method relies on site-directed hydroxyl radical cleavage of nucleosomes carrying modified histones to determine the positions of nucleosomes in the genome. MNase-defined NDRs of cis regulatory elements are nucleosome enriched in the chemical map of mouse ES cells. Emerging evidence shows that such regions are occupied by ‘fragile nucleosomes’, which are lost due to overdigestion by MNase. Results in mouse ES cells illustrate that fragile nucleosomes exist in the mouse genome and chemical mapping is capable of detecting them.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 495-507 |
| Number of pages | 13 |
| Journal | Trends in Genetics |
| Volume | 33 |
| Issue number | 8 |
| DOIs | |
| State | Published - Aug 2017 |
Funding
We thank K. Durbin and A. Sebeson for assisting with this manuscript. L.N.V. was funded by the CMBD training grant NIH T32 GM08061 and the NSF Graduate Research Fellowship J-P.W. and X.W. were supported by a grant from NIGMSR01GM107177. We thank K. Durbin and A. Sebeson for assisting with this manuscript. L.N.V. was funded by the CMBD training grant NIH T32 GM08061 and the NSF Graduate Research Fellowship J-P.W. and X.W. were supported by a grant from NIGMS R01GM107177 . Fenton reaction a reaction in which transition metal ions, such as iron or copper, are oxidized by hydrogen peroxide, forming a hydroxyl radical and a hydroxide ion in the process. In the case of chemical mapping, cuprous ions are oxidized as follows: Cu OH + OH + + H 2 O 2 → Cu 2+ + − . Global run-on sequencing (GRO-seq) measures elongating RNAPII activity genome-wide. Nucleosome dyad the center of the nucleosomal DNA fragment. Nucleosome occupancy the fraction of cells from a population in which a given base pair is wrapped in a nucleosome. Unlike nucleosome positioning, occupancy is not concerned with where the nucleosome is positioned so long as the base pair is covered by one. Nucleosome positioning commonly refers to the exact base pair position of a nucleosome on DNA with respect to a reference point, such as the start location or the dyad. Phasing (with regard to nucleosomes) refers to an array of nucleosomes that are approximately aligned and show rhythmic patterning in nucleosome occupancy when the DNA is aligned at a genomic landmark (e.g., TSS, TTS, etc.) RNA polymerase II (RNAPII) an enzyme that transcribes DNA into mRNA. Well-positioned nucleosome a nucleosome that is dominantly positioned in the exact same location in all cells, shown in the data as a sharp and well-separated nucleosome occupancy peak. Conversely, in the literature, the term fuzzy nucleosomes describes poorly positioned nucleosomes or nucleosomes that have many alternative positions nearby, shown as a very wide and noisy occupancy peak in the data.
Keywords
- MNase
- chemical mapping
- chromatin
- nucleosomes
- transcriptional regulation
ASJC Scopus subject areas
- Genetics