ChIP-on-chip
Elucidating transcriptional regulatory elements using chromatin immunoprecipitation-based location analysis
A review of Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning.
Note: This is a review of the published article listed below. All information, quotes, figures, methods, and findings mentioned in this review are from that article, and are the property of its authors and/or the publication in which the article originally appeared.
Guillemette, et al (2005) use chromatin immunoprecipitation-specific microarrays to characterize H2A.Z, an evolutionary conserved histone variant involved in transcriptional regulation, antisilencing, silencing, and genome stability, with respect to regulatory control. The researchers used Agilent’s 60-mer whole genome tiling microarrays to map histone modifications in the model organism, Saccharomyces cerevisiae, at an approximately 300-bp resolution. Using this platform, the group was able to identify 4,862 small regions (typically, one or two nucleosomes wide) decorated with H2A.Z. The variant, H2A.Z, was determined to be involved in regulation of nucleosome positioning, antisilencing functions, and transcriptional activities via different mechanisms. This work presents new perspectives on regulatory mechanisms in eukaryotic cells, particularly in terms of variant physical integration along the chromosome and chromatin structure development.
Figure 1. Genome-Wide Location Analysis of H2A.Z and H2A; a Zoom on Chromosome III
(A) The location of the Z loci along Chromosome III is depicted by gray bars. (B) A zoom in a 120 KB region between position 196611–316611 shows the raw H2A.Z/H2B log2 ratios for each probe in that region (green bars) and the smoothed data resulted from the Gaussian plot analysis of the raw data for H2A.Z/H2B log2 ratios (red line). The position of the Z loci is shown by gray lines. The genes present in that regions are shown in blue. (C) Same as in (B) but for a zoom in region 237700–277700 (40 KB). (D) The size of the Z loci within the promoter of the SRB8 gene as determined by Q-PCR analysis of ChIP experiments. The binding ratios for H2A.Z (red) and TFIIB (blue) are shown relative to the center of the probe that generates the maximum enrichment. The data were smoothed by a sliding median over three probes. The size of the observed H2A.Z domain is about 250 bp larger than that of TFIIB. Since TFIIB covers about 10 bp of DNA, we can infer that H2A.Z covers about 260 bp of DNA at that locus (shaded area).
Figure 2. H2A.Z Is Predominantly Localized within Promoter Regions
(A) A sliding median of the H2A.Z/H2B ratio for all probes on the microarray is plotted against the distance from the 5' boundary of their closest transcribed element (including ORF, tRNA, transposon, etc.) (green). Randomized data (where the H2A.Z/H2B ratios were scrambled prior to calculating the sliding median) are plotted in black. (B) Most Z loci are found within promoters. The fraction of the 4,862 Z loci present within promoters (gold) and nonpromoter region (aqua) is shown. Promoter regions are defined as the -500/+100 interval relative to the 5' boundary of transcribed elements as annotated in the SGD database. (C) The fraction of the 7,571 promoter regions (defined as in [B]) containing (green) or not containing (purple) a Z loci is shown. (D) H2A.Z is preferentially localized downstream of the NFR in yeast promoters. A sliding median of the ratio for H2A.Z/H2B (green) and H2A/H2B (gold) was plotted against the distance from the NFR (defined as a linker of more than 100 bp located less than 500 bp upstream of a 5' gene boundary) for regions where the NFR can be unambiguously assigned to a single gene (i.e., NFR that map to two diverging genes were filtered out). A cartoon representation of the nucleosomal organization of a typical gene is shown at the bottom.
Figure 3. H2A.Z Regulates Nucleosome Positioning
(A) Nucleosome positioning map of genes associated with a Z locus. Peaks represent nucleosomes, and valleys represent linker regions. The vertical thickness of the curves contains 1-SD error bars for the mean log2 ratio. (B) Same as (A) but for genes containing no Z locus. (C) Indirect end-labeling of MNase-digested chromatin from WT and htz1 cells grown in the presence of glucose. (D) High-resolution LM-PCR analysis of MNase digested nucB–C mononucleosomes. Upper part: structure of the GAL1 promoter and PCR probes used; left: nucB analysis probing the right (R), and left (L) boundaries in WT and htz1 () cells; right: same as left part of the figure, but the analysis is with nucC.
Figure 4. H2A.Z Is Unusually Localized in HZAD Genes
(A) The raw (green) and smoothed (red) H2A.Z/H2B log2 ratios, as determined by our microarray experiment, are shown across a 5-KB region around the GIT1 genes. The genes present in that region are shown in blue. (B) Same as (A) but for a 5-KB region around the SRB8 gene. (C) H2A.Z covers wider regions near telomeres. The size of the regions covered by H2A.Z (as determined by distance between the coordinates where the smoothed H2A.Z/H2B log2 ratio reaches “0.1”) is plotted against the distance to telomeres. A moving average (window = 10 KB) was applied to the data.
Title: Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning.
Authors: Guillemette B, Bataille AR, Gévry N, Adam M, Blanchette M, Robert F, Gaudreau L.
Journal: PLoS Biol. 2005 Dec; 3(12): e384.
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